Cerebrospinal Fluid Glutamine Research Articles

Elevation of brain gamma‐aminobutyric acid levels is associated with vigabatrin‐associated brain abnormalities on magnetic resonance imaging

ObjectiveVigabatrin (VGB) is an effective antiseizure medication for West syndrome. It works by irreversibly inhibiting gamma-aminobutyric acid (GABA) transaminase and increasing central GABA levels. Vigabatrin-associated brain abnormalities on magnetic resonance imaging (VABAM) are an adverse effect of VGB that has only been reported in children, but the pathophysiology of this effect is unknown. In this study, we evaluated the relationship of VGB and brain GABA levels as well as the association between VABAM and GABA. MethodsFor the 15 consecutive pediatric patients treated with VGB, free GABA, glutamine, and glutamate in cerebrospinal fluid (CSF) and plasma, GABA to creatine and phosphocreatine (Cr) peak ratio (GABA/Cr), glutamine (Gln)/Cr, and glutamate (Glu)/Cr as quantified by proton MR spectroscopy (1H-MRS) were retrospectively examined. GABA/Cr was compared with in-house normal pediatric controls. Differences in the levels of each metabolite in VABAM and non-VABAM cases were also examined. ResultsThirteen of the included subjects underwent magnetic resonance imaging (MRI) and 1H-MRS, and two of them presented VABAM; both cases were very-low-birth-weight preterm infants with post-hemorrhagic hydrocephalus. CSF levels of free GABA were significantly higher in VABAM (5.26 ± 1.95 μmol/L) than in non-VABAM (0.59 ± 0.63 μmol/L) cases (P = 0.03). The GABA/Cr was significantly higher in patients with VGB (0.34 ± 0.16) than in pediatric controls (0.20 ± 0.05) (P = 0.02). The GABA/Cr tended to be higher in VABAM (0.48 ± 0.10) than in non-VABAM cases (0.31 ± 0.15) (P = 0.31). SignificanceElevated brain GABA levels were observed in VABAM patients, suggesting its involvement in the pathogenesis of this condition. In particular, a marked increase in CSF-free GABA was characteristic. Although the elevation of the GABA/Cr is mild, it may be useful for early identification of patients with risk of VABAM.

β1-Integrin- and KV1.3 channel-dependent signaling stimulates glutamate release from Th17 cells.

Although the impact of Th17 cells on autoimmunity is undisputable, their pathogenic effector mechanism is still enigmatic. We discovered soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) complex proteins in Th17 cells that enable a vesicular glutamate release pathway that induces local intracytoplasmic calcium release and subsequent damage in neurons. This pathway is glutamine dependent and triggered by binding of β1-integrin to vascular cell adhesion molecule 1 (VCAM-1) on neurons in the inflammatory context. Glutamate secretion could be blocked by inhibiting either glutaminase or KV1.3 channels, which are known to be linked to integrin expression and highly expressed on stimulated T cells. Although KV1.3 is not expressed in CNS tissue, intrathecal administration of a KV1.3 channel blocker or a glutaminase inhibitor ameliorated disability in experimental neuroinflammation. In humans, T cells from patients with multiple sclerosis secreted higher levels of glutamate, and cerebrospinal fluid glutamine levels were increased. Altogether, our findings demonstrate that β1-integrin- and KV1.3 channel-dependent signaling stimulates glutamate release from Th17 cells upon direct cell-cell contact between Th17 cells and neurons.

Efficacy of Asparaginase Erwinia chrysanthemi With and Without Temozolomide Against Glioma Cells and Intracranial Mouse Medulloblastoma.

Anti-metabolites are less-myelosuppressive than DNA-damaging anticancer drugs and may be useful against brain tumors. We evaluated the asparagine/glutamine-deaminating agent Erwinaze with/without temozolomide against brain tumor cells and mouse medulloblastomas. Erwinaze treatment of cell lines and neurospheres led to dose-dependent reductions of cells (reversible by L-glutamine), with half maximal inhibitory concentrations (IC50s) of 0.12->10 IU/ml. Erwinaze at <1 IU/ml reduced temozolomide IC50s by 3.6- to 13-fold (300-1,200 μM to 40-330 μM). Seven-week-old SMO/SMO mice treated with Erwinaze (regardless of temozolomide treatment) had better survival 11 weeks post-therapy, compared to those not treated with Erwinaze (81.25% vs. 46.15, p=0.08). Temozolomide-treated mice developed 10% weight loss, impairing survival. All 16 mice treated with temozolomide (regardless of Erwinaze treatment) succumbed by 40-weeks of age, whereas 5/8 animals treated with Erwinaze alone and 2/6 controls survived (p=0.035). Erwinaze enhances cytotoxicity of temozolomide in vitro, and improves survival in SMO/SMO mice, likely by reducing cerebrospinal fluid glutamine. Temozolomide-associated toxicity prevented demonstration of any potential combinatorial advantage with Erwinaze in vivo.

Maternal Cerebrospinal Fluid Glutamate in Response to Variable Foraging Demand: Relationship to Cerebrospinal Fluid Serotonin Metabolites in Grown Offspring

Background Maternal response to allostatic overload during infant rearing may alter neurobiological measures in grown offspring, potentially increasing susceptibility to mood and anxiety disorders. We examined maternal cerebrospinal fluid (CSF) glutamate response during exposure to variable foraging demand (VFD), a bonnet macaque model of allostatic overload, testing whether activation relative to baseline predicted concomitant CSF elevations of the stress neuropeptide, corticotropin-releasing factor. We investigated whether VFD-induced activation of maternal CSF glutamate affects maternal–infant attachment patterns and offspring CSF 5-hydroxyindoleacetic acid concentrations. Methods Mother–infant dyads were exposed to the “VFD stressor,” a paradigm in which mothers experience 16 weeks of foraging uncertainty while rearing their infant offspring. Through staggering the infant age of VFD onset, both a cross-sectional design and a longitudinal design were used. Maternal CSF glutamate and glutamine concentrations post-VFD exposure were cross-sectionally compared to maternal VFD naive controls. Proportional change in concentrations of maternal glutamate (and glutamine), a longitudinal measure, was evaluated in relation to VFD-induced elevations of CSF corticotropin-releasing factor. The former measure was related to maternal–infant proximity scores obtained during the final phases of VFD exposure. Maternal glutamatergic response to VFD exposure was used as a predictor variable for young adolescent offspring CSF metabolites of serotonin, dopamine, and norepinephrine. Results Following VFD exposure, maternal CSF glutamate concentrations correlated positively with maternal CSF CRF concentrations. Activation relative to baseline of maternal CSF glutamate concentrations following VFD exposure correlated directly with a) increased maternal-infant proximity during the final phases of VFD and b) offspring CSF concentrations of monoamine metabolites including 5-hydroxyindoleacetic acid, which was elevated relative to controls. Conclusions Activation of maternal CSF glutamate in response to VFD-induced allostasis is directly associated with elevations of maternal CSF corticotropin-releasing factor. Maternal CSF glutamate alterations induced by VFD potentially compromise serotonin neurotransmission in grown offspring, conceivably modeling human vulnerability to treatment-resistant mood and anxiety disorders.

Secondary NAD+ deficiency in the inherited defect of glutamine synthetase.

Glutamine synthetase (GS) deficiency is an ultra-rare inborn error of amino acid metabolism that has been described in only three patients so far. The disease is characterized by neonatal onset of severe encephalopathy, low levels of glutamine in blood and cerebrospinal fluid, chronic moderate hyperammonemia, and an overall poor prognosis in the absence of an effective treatment. Recently, enteral glutamine supplementation was shown to be a safe and effective therapy for this disease but there are no data available on the long-term effects of this intervention. The amino acid glutamine, severely lacking in this disorder, is central to many metabolic pathways in the human organism and is involved in the synthesis of nicotinamide adenine dinucleotide (NAD(+)) starting from tryptophan or niacin as nicotinate, but not nicotinamide. Using fibroblasts, leukocytes, and immortalized peripheral blood stem cells (PBSC) from a patient carrying a GLUL gene point mutation associated with impaired GS activity, we tested whether glutamine deficiency in this patient results in NAD(+) depletion and whether it can be rescued by supplementation with glutamine, nicotinamide or nicotinate. The present study shows that congenital GS deficiency is associated with NAD(+) depletion in fibroblasts, leukocytes and PBSC, which may contribute to the severe clinical phenotype of the disease. Furthermore, it shows that NAD(+) depletion can be rescued by nicotinamide supplementation in fibroblasts and leukocytes, which may open up potential therapeutic options for the treatment of this disorder.

CSF and Serum Biomarkers Focusing on Cerebral Vasospasm and Ischemia after Subarachnoid Hemorrhage

Delayed cerebral vasospasm (CVS) and delayed cerebral ischemia (DCI) remain severe complications after subarachnoid hemorrhage (SAH). Although focal changes in cerebral metabolism indicating ischemia are detectable by microdialysis, routinely used biomarkers are missing. We therefore sought to evaluate a panel of possible global markers in serum and cerebrospinal fluid (CSF) of patients after SAH. CSF and serum of SAH patients were analyzed retrospectively. In CSF, levels of inhibitory, excitatory, and structural amino acids were detected by high-performance liquid chromatography (HPLC). In serum, neuron-specific enolase (NSE) and S100B level were measured and examined in conjunction with CVS and DCI. CVS was detected by arteriography, and ischemic lesions were assessed by computed tomography (CT) scans. All CSF amino acids were altered after SAH. CSF glutamate, glutamine, glycine, and histidine were significantly correlated with arteriographic CVS. CSF glutamate and serum S100B were significantly correlated with ischemic events after SAH; however, NSE did not correlate neither with ischemia nor with vasospasm. Glutamate, glutamine, glycine, and histidine might be used in CSF as markers for CVS. Glutamate also indicates ischemia. Serum S100B, but not NSE, is a suitable marker for ischemia. These results need to be validated in larger prospective cohorts.

Analysis of glutamine, glutamate, pyroglutamate, and GABA in cerebrospinal fluid using ion pairing HPLC with positive electrospray LC/MS/MS

A simple and sensitive method for the separation and quantitation of glutamine, glutamate, pyroglutamate, and γ-aminobutyric acid (GABA) in cerebrospinal fluid (CSF) is presented. The method utilizes ion pairing with heptafluorobutyric acid (HFBA) to achieve HPLC separation with detection by positive ESI LC/MS/MS. The method does not require extraction or derivatization, utilizes a heavy labeled internal standard for each analyte, and allows for rapid throughput with a 5 min run time. The method was developed with particular attention taken to prevent conversion between analytes known to occur under certain conditions. The lower limit of quantitation is 7.8 ng/ml for all analytes, and the intra-day and inter-day accuracy (%RE) and precision (%R.S.D.) are defined for all analytes. The method was developed as a sensitive, selective, and robust method to investigate the excitatory and inhibitory neurotransmitters (glutamate and GABA) as biomarkers in drug development.

Amino acid concentrations in cerebrospinal fluid in children with acute lymphoblastic leukemia undergoing chemotherapy

Cerebrospinal fluid (CSF) amino acid concentrations were measured in 45 children with acute lymphoblastic leukemia (ALL). Central nervous system (CNS) disease was absent in 34 and present in 11 (Groups L and M, respectively) at diagnosis. Thirty-two otherwise healthy children with febrile convulsions were studied for comparison. Results from this study show that glutamine levels at Day 0 were significantly higher in patients than in controls. Patients in Group M had elevated glutamine levels compared to Group L. In comparison, at Day 14, concentrations of glutamine and asparagine decreased, while glutamic acid amounts increased significantly in Group L. Glutamine levels fell at Day 42 in Group M, which may have resulted from more intensive treatment. From this study we hypothesise that higher baseline glutamine levels are indicative of a greater risk for CNS leukemia. Large-scale prospective trials are required to confirm increased baseline CSF glutamine levels in ALL patients, to identify glutamine as a marker for CNS disease and to clarify underlying mechanisms regulating glutamine in ALL.

Brain glutamine by MRS in a patient with urea cycle disorder and coma

In patients who undergo metabolic decompensation from urea cycle disorders, cerebrospinal fluid glutamine level may be a better marker of cerebral dysfunction than blood ammonia or glutamine levels. However, obtaining cerebrospinal fluid by lumbar puncture carries risk in these acutely ill patients with cerebral edema. Using magnetic resonance single voxel spectroscopy as an alternative to cerebrospinal fluid analysis, elevated brain glutamine/glutamate complex levels were detected in a patient with carbamyl phosphate synthetase deficiency, who had been comatose for many days after normalization of blood ammonia and improvement in blood glutamine levels. Brain glutamine by single voxel spectroscopy decreased toward normal with neurologic recovery. We conclude that brain glutamine may be a better marker than serum ammonia for the management of urea cycle disorders, particularly in patients with prolonged mental status changes.

Cerebrospinal fluid from patients with dementia contains increased amounts of an unknown factor.

Increased levels of an unidentified peak have been found in cerebrospinal fluid (CSF) from patients with Alzheimer's disease or vascular dementia compared to the level in healthy controls using proton magnetic resonance spectroscopy. No increase was found in patients with amyotrophic lateral sclerosis. Reexamination of spectra from a study published previously (Gårseth et al. [2000] J. Neurosci. Res. 60:779-782), however, shows that this peak was also elevated significantly in CSF from patients with Huntington's disease compared to that in controls. The level in patients with Parkinson's disease, where dementia develops in up to 40% of patients, was not elevated significantly compared to that in controls. To the best of our knowledge, this peak has not yet been identified and we therefore find it appropriate to temporarily designate the name "dementia associated factor" (DAF), although there is as yet no certainty that this substance is specific for these conditions. Apart from a significantly increased level of glutamine in CSF from patients with vascular dementia compared to that in controls, no other significant difference was found for any other metabolite measured in the patient groups using proton magnetic resonance spectroscopy.

Liquid Chromatographic Determination of Glutamine in Cerebrospinal Fluid Using 2-Hydroxynaphthaldehyde Derivatizing Reagent

An analytical procedure has been developed for the selective determination of glutamine from cerebrospinal fluid (CSF) using 2-hydroxynaphthaldehyde derivatizing reagent. Arginine and tyramine could also be determined simultaneously. Separation was on a Phenomenex C-18, (150 × 4.6 mm i.d.) column with methanol: water (63:38 v/v) mobile phase at 1mL min−1 and UV detection at 330nm. Detection limits for glutamine, arginine, and tyramine were 2.8 ng, 17.4 ng and 3.45 ng injection−1 (5 μL), respectively. A large number of amines and amino acids eluted did not affect the determination of glutamine. The analysis of CSF of four patients suffering from hydrocephalus for glutamine indicated concentrations within range 37.4–11.24 μg mL−1 with coefficient of variation 3.0–6.2%.

Determination of glutamine and serine in rat cerebrospinal fluid using capillary electrochromatography with a modified photopolymerized sol–gel monolithic column

Capillary electrochromatographic separations of amino acid mixtures were studied using two modified porous photopolymerized sol–gel monolithic columns. One was modified with dimethyloctadecylchlorosilane (DMOS), and the other was modified with DMOS, followed by chlorotrimethylsilane to end-cap residual silanol groups. Prior to separation, amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole using as a mobile phase 50 m M phosphate (pH 2.5), water, and acetonitrile in the ratio of 1:1:8. Five derivatized amino acids (Asn, Phe, Ala, Ile, and Leu) were separated within 7 min. Theoretical plate numbers varied between 58 700 and 105 000/m. This separation method with the end-capped monolithic column was applied to rat cerebrospinal fluid. The dominant amino acid found was Gln at a concentration of 420 μ M along with small quantities of Ser (54 μ M).

Pyruvate carboxylase deficiency—insights from liver transplantation

Pyruvate carboxylase deficiency, complex form, presents in early infancy with lethal metabolic acidosis, resulting from ketoacidosis and lactic acidemia. Renal tubular acidosis, hyperammonemia, and citrullinemia complete the picture. In an infant with this disease, large amounts of glucose ameliorated the ketoacidosis, but worsened the lactic acidosis. Orthotopic hepatic transplantation completely reversed the ketoacidosis and the renal tubular abnormality and ameliorated the lactic acidemia. Concentrations of glutamine in cerebrospinal fluid were low and did not improve with liver transplantation.

Serum and CSF glutamine levels in valproate-related hyperammonemic encephalopathy.

To investigate ammonia and glutamine levels in valproate (VPA)-related hyperammonemic encephalopathy (VHE). We reviewed the medical records and EEG recordings of seven adults diagnosed with VHE. Venous ammonia levels were elevated in five (71%) of the seven patients. Elevated serum or cerebrospinal fluid (CSF) glutamine levels were found in four (80%) of five cases tested, including two who had normal ammonia levels. Initial behavioral signs included violent outbursts in three patients, paranoid ideation severe enough to require restraint in two cases, and milder abnormalities in two instances. The severity of encephalopathy was not related to any particular serum VPA level. In four patients serum VPA levels did not exceed 100 microg/ml, and in one case, VHE developed after taking only one 250-mg dose. Symptoms eventually cleared after reducing the dose of, or discontinuing, VPA. Liver-function tests were normal. Each of six patients tested had EEG findings that supported the diagnosis of VHE and excluded nonconvulsive status epilepticus. The rate of normalization of one patient's serum glutamine level and the EEGs of two cases correlated better with the timing of their delayed clinical recovery than did the more rapid rate of decline of the serum ammonia levels. Serum or CSF glutamine levels are initially elevated in a majority of patients with suspected VHE, sometimes in the absence of hyperammonemia. Glutamine levels may be useful adjunctive laboratory tests for the diagnosis of VHE.

Increased cerebrospinal fluid glutamine levels in depressed patients

Background: There is increasing evidence for an association between alterations of brain glutamatergic neurotransmission and the pathophysiology of affective disorders. Methods: We studied the association between cerebrospinal fluid (CSF) metabolites, including glutamine, in unipolar and bipolar depressed patients versus control subjects using a proton magnetic resonance spectroscopy technique. Cerebrospinal fluid samples were obtained from 18 hospitalized patients with acute unmedicated severe depression without medical problems and compared with those of 22 control subjects. Results: Compared with the control group, the depressed patient group had significantly higher CSF glutamine concentrations, which correlated positively with CSF magnesium levels. Conclusions: These findings suggest an abnormality of the brain glial–neuronal glutamine/glutamate cycle associated with N-methyl- d-aspartate receptor systems in patients with depression.

Cerebral blood flow and the development of ammonia-induced brain edema in rats after portacaval anastomosis.

Two mechanisms may account for brain edema in fulminant hepatic failure: the osmotic effects of brain glutamine, a product of ammonia detoxification, and a change of cerebral blood flow (CBF). We have shown brain edema, a marked increase in brain glutamine, and a selective rise in CBF in rats after portacaval anastomosis receiving an ammonia infusion. In this study, we inhibited the activity of glutamine synthetase with methionine-sulfoximine (MSO) and examined ammonia levels, brain water and CBF. Four groups received either a continuous ammonium acetate or control infusion; half of the animals had been pretreated with MSO or vehicle. The ammonia group exhibited brain edema (79.97 +/- 0.04 vs. 81.11 +/- 0. 13% water), an increase in cerebrospinal fluid (CSF) glutamine (1.29 +/- 0.21 vs. 2.84 +/- 0.39 mmol/L) and CBF (63 +/- 11 vs. 266 +/- 45 mL/min/100 g brain). When MSO was added to the ammonia infusion, ammonia levels rose further (928 +/- 51 vs. 1,293 +/- 145 mmol/L, P <.05) but CSF glutamine decreased (2.84 +/- 0.39 vs. 1.61 +/- 0.2 mmol/L, P <.01). Brain edema (80.48 +/- 0.11%) and cerebral hyperemia (140 +/- 25 mL/min/100 g brain) were significantly ameliorated in the ammonia plus MSO group. Brain output of circulating nitric oxide (NO(x)) was increased in the ammonia-infused group but normalized in the ammonia plus MSO group. In this model, the rise of CBF reflects intracranial events that occur after glutamine synthesis. Activation of nitric oxide synthase in the brain could account for these findings.

Abnormal Substrate Levels That Depend upon Mitochondrial Function in Cerebrospinal Fluid from Alzheimer Patients

Background: Impaired oxidative and energy metabolism are important features in Alzheimer’s disease. These metabolic abnormalities may induce functional disturbances and are associated with significant cognitive impairment. Objective: To determine whether mitochondrial function is altered by Alzheimer’s disease, a quantitative analysis of substrates that enter the tricarboxylic acid cycle was carried out in cerebrospinal fluid (CSF) from Alzheimer patients. Methods: Organic acid levels related to carbohydrate oxidation were measured in CSF from patients affected by dementia of Alzheimer type (n = 17) and from nondemented elderly controls (n = 17) using a gas chromatography/mass spectrometry system. CSF glucose and glutamine concentrations were determined by a quantitative enzymatic method and by ion exchange chromatography, respectively. Results: Compared to age-matched controls, patients had a higher CSF level of lactate (p = 0.002) and a lower mean level of succinate (p = 0.002), fumarate (p = 0.003) and glutamine (p = 0.0005). The CSF glucose level was not modified. Conclusion: Our results suggest an impairment of mitochondrial oxidative metabolism in brain cells of patients with Alzheimer’s disease.

Measurement of glutamine and glutamate by capillary electrophoresis and laser induced fluorescence detection in cerebrospinal fluid of meningitis sick children.

Cerebrospinal fluid glutamine and glutamate concentrations were measured by CZE-LIFD in normal and meningitis sick children. A derivatization procedure for glutamine was optimized using CZE-LIFD. Cytochemical and bacteriological analysis were made to 152 CSF from children suspicious of suffering meningitis. CSF-glutamine concentration was lower in children with viral and bacterial meningitis. In addition, the group infected with Haemophilus influenzae had the lowest concentrations. By contrast, glutamate concentration was higher in children with meningitis. Glutamine concentration increased as the clinical condition of the patient improved. Lower concentrations of glutamine in children with bacterial meningitis might be due to the use of this amino acid for bacterial growth while higher concentrations of glutamate might be due to cell destruction. The results indicate that CZE-LIFD is an alternative tool for the determination of amino acids in CSF.

The effects of ammonia and portal-systemic shunting on brain metabolism, neurotransmission and intracranial hypertension in hyperammonaemia-induced encephalopathy

Background/Aims: The pathogenetic factors contributing to encephalopathy in portracaval shunted rats with hyperammonaemia were studied. Methods: Hyperammonaemia was induced by ammonium-acetate infusions in portacaval shunted rats (2.8 mmol·kg bw −1·h −1; AI-portacaval shunted rats) and in sham-portacaval shunted rats (6.5 mmol·kg bw −1; AI-NORM rats). Severity of encephalopathy was qualified by clinical grading and EEG spectral analysis. Changes in brain metabolites were assessed by amino acid analysis of brain cortex homogenates, whereas changes in amino acids with neurotransmitter activity were assessed in cerebrospinal fluid; brain water content was measured by subtracting dry from wet brain weights and intracranial pressure was measured by a pressure transducer connected to a cisterna magna cannula. Results: Although similar increased blood and brain ammonia concentrations were obtained in both experimental groups, only AI-portacaval shunted rats developed encephalopathy, associated with a significant increase in intracranial pressure. Other significant differences were: higher concentrations of brain glutamine and aromatic amino acids, higher concentrations of cerebrospinal fluid glutamine, aromatic amino acids, glutamate and aspartate in AI-portacaval shunted rats than in AI-NORM rats. Conclusions: These results indicate that hyperammonaemia alone does not induce encephalopathy, whereas portal-systemic shunting adds an essential contribution to the pathogenesis of encephalopathy. It is hypothesised that the larger increase in brain glutamine in AI-portacaval shunted rats than in AI-NORM rats is responsible for increased brain concentrations of aromatic amino acids, for cell swelling and for extracellular release of glutamate and aspartate. This might promote encephalopathy. If cell swelling is not restricted, intracranial hypertension will develop

Rapid determination of glutamine in biological samples by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of glutamine in biological samples is presented. Glutamine was derivatized with ortho-phthalaldehyde reagent containing 3-mercaptopropionic acid and separated by reversed phase chromatography on a C18 column containing 3-μm particles. No interference from other amino acids was observed. The assay was linear over a range from 1 to 2,000 μmol/l. Analytical recovery of plasma samples spiked with glutamine was 98.6 ± 3.8%. Within- and between-batch imprecision were 1.5% and 2.2%, respectively. The derivatization step was fully automated. Total analysis time, including derivatization and chromatography, amounted to 6 min. The method can be used for the determination of glutamine in plasma, urine, cerebrospinal fluid and tissue homogenates.