Increased Brain Glutamine Research Articles

Neurometabolic changes in a rat pup model of type C hepatic encephalopathy depend on age at liver disease onset

Chronic liver disease (CLD) is a serious condition where various toxins present in the blood affect the brain leading to type C hepatic encephalopathy (HE). Both adults and children are impacted, while children may display unique vulnerabilities depending on the affected window of brain development.We aimed to use the advantages of high field proton Magnetic Resonance Spectroscopy (1H MRS) to study longitudinally the neurometabolic and behavioural effects of Bile Duct Ligation (animal model of CLD-induced type C HE) on rats at post-natal day 15 (p15) to get closer to neonatal onset liver disease. Furthermore, we compared two sets of animals (p15 and p21-previously published) to evaluate whether the brain responds differently to CLD according to age onset.We showed for the first time that when CLD was acquired at p15, the rats presented the typical signs of CLD, i.e. rise in plasma bilirubin and ammonium, and developed the characteristic brain metabolic changes associated with type C HE (e.g. glutamine increase and osmolytes decrease). When compared to rats that acquired CLD at p21, p15 rats did not show any significant difference in plasma biochemistry, but displayed a delayed increase in brain glutamine and decrease in total-choline. The changes in neurotransmitters were milder than in p21 rats. Moreover, p15 rats showed an earlier increase in brain lactate and a different antioxidant response. These findings offer tentative pointers as to which neurodevelopmental processes may be impacted and raise the question of whether similar changes might exist in humans but are missed owing to 1H MRS methodological limitations in field strength of clinical magnet.

Dysregulation of Astrocytic Glutamine Transport in Acute Hyperammonemic Brain Edema.

Acute liver failure (ALF) impairs ammonia clearance from blood, which gives rise to acute hyperammonemia and increased ammonia accumulation in the brain. Since in brain glutamine synthesis is the only route of ammonia detoxification, hyperammonemia is as a rule associated with increased brain glutamine content (glutaminosis) which correlates with and contributes along with ammonia itself to hyperammonemic brain edema-associated with ALF. This review focuses on the effects of hyperammonemia on the two glutamine carriers located in the astrocytic membrane: Slc38a3 (SN1, SNAT3) and Slc7a6 (y + LAT2). We emphasize the contribution of the dysfunction of either of the two carriers to glutaminosis- related aspects of brain edema: retention of osmotically obligated water (Slc38a3) and induction of oxidative/nitrosative stress (Slc7a6). The changes in glutamine transport link glutaminosis- evoked mitochondrial dysfunction to oxidative-nitrosative stress as formulated in the “Trojan Horse” hypothesis.

PET CMRglc mapping and 1H-MRS show altered glucose uptake and neurometabolic profiles in BDL rats

Type C hepatic encephalopathy (HE) is a complex neuropsychiatric disorder occurring as a consequence of chronic liver disease. Alterations in energy metabolism have been suggested in type C HE, but in vivo studies on this matter remain sparse and have reported conflicting results. Here, we propose a novel preclinical 18F-FDG PET methodology to compute quantitative 3D maps of the regional cerebral metabolic rate of glucose (CMRglc) from a labelling steady-state PET image of the brain and an image-derived input function. This quantitative approach shows its strength when comparing groups of animals with divergent physiology, such as HE animals. PET CMRglc maps were registered to an atlas and the mean CMRglc from the hippocampus and the cerebellum were associated to the corresponding localized 1H MR spectroscopy acquisitions. This study provides for the first time local and quantitative information on both brain glucose uptake and neurometabolic profile alterations in a rat model of type C HE. A 2-fold lower brain glucose uptake, concomitant with an increase in brain glutamine and a decrease in the main osmolytes, was observed in the hippocampus and in the cerebellum. These novel findings are an important step towards new insights into energy metabolism in the pathophysiology of HE.

L-Ornithine L-Aspartate for Prevention and Treatment of Hepatic Encephalopathy in Patients with Cirrhosis

In cirrhosis, the loss of metabolic capacity of periportal and perivenous hepatocytes coupled with the development of portal-systemic shunting frequently results in severe hyperammonemia. Molecular Imaging and Spectroscopic techniques reveal increases in the cerebral metabolic rate for ammonia and increased brain glutamine as a function of the severity of hepatic encephalopathy [HE]. L-Ornithine L-Aspartate [LOLA] is effective for the lowering of blood, muscle and brain ammonia and does so via three independent mechanisms namely [i] the stimulation of ammonia removal via the urea cycle in residual hepatocytes, [ii] prevention of hepatocellular damage due to the production of antioxidants [glutathione, glutamine] and [iii] the prevention of muscle wasting [sarcopenia] thus protecting the ammonia-detoxification pathway via muscle glutamine synthesis. Results of RCTs, systematic reviews and meta-analyses provide evidence for the efficacy of LOLA for the prevention and treatment of HE in all its forms [MHE, OHE, episodic HE, post-TIPSS HE] and for primary, secondary and post-TIPSS prophylaxis. Keywords: L-ornithine L-aspartate; LOLA; Hepatic encephalopathy; Cirrhosis; Treatment; Prevention; Prophylaxis; Ammonia; Hepatoprotection; Sarcopenia; Combination therapy

P: 37 Probiotics Combined With Rifaximin for the Treatment of Chronic Hepatic Encephalopathy: A Longitudinal In Vivo 1H-MRS Study of Brain Metabolism Using BDL Rats

BACKGROUND: Chronic hepatic encephalopathy (HE) is a severe complication of chronic liver disease (CLD), and finding the right treatment to reduce HE episodes before liver transplant remains a challenge. Both rifaximin (non-absorbable antibiotic) and probiotics are currently used to reduce HE symptoms, but their precise effect on brain metabolites has never been studied. Our aims were: 1) to assess in vivo and longitudinally the effect of the combination of probiotics and rifaximin on bile duct ligated (BDL) rats in different brain regions; and 2) to compare these results with both non-treated (n = 17) and rifaximin-only treated rats (n = 12).1,2 METHODS: In vivo 1H-MRS at 9.4 Tesla combined with biochemical tests (plasma NH4+, bilirubin) and microbiota analysis were performed on adult Wistar rats (n = 9) before BDL3,4 (week 0) and at weeks 2, 4, 6 and 8 after surgery. Evolution of metabolites was studied using the SPECIAL sequence (TE = 2.8 ms) in the hippocampus (2 × 2.8 × 2 mm3) and cerebellum (2.5 × 2.5 × 2.5 mm3). Metabolite concentrations were estimated using LCModel and water as internal reference. Probiotics administration (VIVOMIXX®, 60 billion bacteria/kg of rat) started two weeks before BDL-surgery until the end of the study. Rifaximin (15.7 mg/kg/day = 'human-dose') was administered twice daily starting two weeks after BDL-surgery. RESULTS: All rats displayed the characteristic rise in plasma bilirubin, regardless of treatment group, as well as a similar ammonium increase (Figure 1a). The characteristic pattern of chronic HE was observed (Figure 1e): a gradual increase of brain glutamine followed by a gradual decrease in the other brain osmolytes (myo-inositol, taurine, total choline) and a later decrease of glutamate and creatine. The combination of probiotics and rifaximin improved some of the neuro-metabolic changes associated with CLD at early stages of HE (week 4) in the cerebellum: the 'probiotics + rifaximin' group showed a lower rise of brain glutamine (+33% vs +66%, Figure 1c) and a smaller decrease of creatine (−4% vs −14%). In the hippocampus, rats receiving both probiotics and rifaximin exhibited a smaller increase in brain glutamine even at week 8 after BDL compared to non-treated rats (+99% vs +136%, Figure 1d) and a smaller decrease in brain myo-inositol and glutamate (−20% vs −30% and −7% vs −13%, respectively). Also, bifidobacteria concentration was slightly higher in the 'probiotics+rifaximin' group at week 8 (Figure 1b). Finally, the administration of rifaximin associated with this probiotic showed more beneficial effects than rifaximin only, and both could be used to maintain a balanced microbiota and may provide opportunities for reducing the spread of antibiotic resistances. CONCLUSIONS: To conclude, some promising changes were induced in the neurometabolic profile of BDL-rats who were treated with this specific probiotic and rifaximin (glutamine, myo-inositol, creatine and glutamate).

P: 68 Brain Regional Susceptibility to Oxidative Stress in a Rat Model of Chronic Hepatic Encephalopathy: In-Vivo 1H MRS, Ex-Vivo ESR Spectroscopy and Histology Findings

BACKGROUND: Oxidative stress (OS) is believed to be an important feature in the pathogenesis of chronic hepatic encephalopathy (CHE).1 In CHE impaired ammonium clearance by the diseased liver leads to brain glutamine accumulation. In-vitro, affected ammonium detoxification together with glutamine induces reactive-oxygen-species (ROS) generation associated with astrocyte impairment.2,3 ROS play an important role in cellular signalling, synaptic-plasticity, learning and memory. When in excess, they cause cellular damage.4,5 Systemic oxidative stress was previously shown in bile-duct-ligated rats (BDL). Using in-vivo-longitudinal 1H-MRS we previously observed the indirect presence of OS as a decrease of brain Asc in the hippocampus and cerebellum of BDL rats (model of CHE).6 We aimed to validate these findings using for the first time ex-vivo electron spin (ESR) spectroscopy and histological measures to assess OS levels. METHODS: In-vivo-1H-MRS: Cerebellum/hippocampus of adult rats were scanned before BDL and after every 2-weeks up to week 6 (n = 18) at 9.4T-MR (Varian/Magnex-Scientific) using SPECIAL-sequence 7 (TE = 2.8 ms). Ex-vivo-ESR: ESP300E (Bruker-BioSpin) was used for intracellular superoxide anion detection. Hippocampus/cerebellum were extracted at 6weeks post-BDL/sham-surgery (n = 9), incubated in medium with 10 mM-CMH-cell-permeable spin-trap (Noxygen GmbH). Immunohistochemistry (ICH): GPX1 (anti-oxidative-enzyme) staining was performed (n = 6). RESULTS: The increase in plasma NH4+ and bilirubin confirmed the presence of chronic liver disease. A ∼80% increase in brain glutamine was measured at week6, while Asc showed a stronger decrease in cerebellum (−32% at week 6, P < 0.001) (Figure 1a). ESR revealed differences in redox-state between the two brain regions (∼31% higher in cerebellum, P < 0.004) (Figure 1b). However, the relative change in both regions between BDL and shams was similar ∼42%. The significant increase of hippocampal/cerebellar OS in BDL (P < 0.01, P < 0.001) corroborate the 1H-MRS findings of decreased Asc concentrations (Figure 1a). ICH with GPX1-enzyme,8,9 revealed an increase activity in Purkinje and granular-cell-layer of BDL cerebellum (Figure 1c). Purkinje-cells also showed shrinking soma (BDL: 8.04 ± 1.8 µm2, sham: 12.5 ± 1.2 µm2, P < 0.00001). Elevated GPX1 in cerebellum suggests that GSH-synthesis may increase (confirmed by increased GSH in 1H MRS, Figure 1a) in response to OS-related injury. Elevated OS might suggest that ROS could lead to brain functions disruption.10–12 CONCLUSIONS: Our results showed for the first time the presence of central OS in BDL rats at 6-weeks post-surgery. Changes varied according to brain region and proved a different susceptibility of cerebellum and hippocampus to CHE, reflected by a relationship between increased OS and interrupted antioxidant-defence-system. OS is involved in the propagation of cellular injury and may be an important factor in the aetiology of the CHE.

P: 33 In Vivo Longitudinal 1H MRS Study of Hippocampal, Cereberal and Striatal Metabolic Changes in the Adult Brain Using an Animal Model of Chronic Hepatic Encephalopathy

BACKGROUND: Chronic hepatic encephalopathy (CHE) is a severe complication of chronic liver disease (CLD) characterized by cognitive and motor deficits. The diseased liver fails to metabolize toxins from the blood (ammonium, bilirubin etc.) which accumulate in the blood and brain.1 There is evidence that ammonium uptake rate differs among the brain regions.2 Since the CHE patients present various symptoms with different severity, the susceptibility to CHE and the mechanisms causing the damage may depend on the brain region. The aim of this study was to investigate, for the first time, potential metabolic differences between hippocampus, cerebellum and striatum as key brain regions implicated in manifestation of CHE. METHODS: Hippocampus (n = 7), cerebellum (n = 8) and striatum (n = 4) of adult male Wistar rats were scanned longitudinally using in-vivo 1H-MRS (SPECIAL sequence-TE = 2.8 ms,3 quantification with LCModel) at 9.4T before (week 0) and after bile duct ligation (BDL-CHE model4). Scans and blood tests were performed every two-weeks till week 8. RESULTS: All BDL rats showed an increase in plasma bilirubin and blood ammonia validating the presence of CLD. Increase in brain Glutamine (Gln) was observed for all brain regions being the most pronounced in cerebellum (+134%-week 8) (Figure 1c). Furthermore, this increase showed a strong correlation with blood ammonia for all three brain regions (Figure 1b). The main brain organic osmolytes (Inositol, Taurine, Creatine and total-Choline) displayed a similar decreasing trend in concentration as a response to Gln increase (osmoregulation) for both hippocampus and cerebellum, always having a stronger change for cerebellum. Interestingly, despite the smallest Gln increase, striatum showed more pronounced decrease in concentration of osmolytes than hippocampus (Figure 1d,e). Also, trend towards a decrease in NAA and PE was observed uniquely for striatum (data not shown). A tendency of increase in Lactate was observed being the strongest for cerebellum (+84%-cerebellum, +8%-hippocampus, +5%-striatum) indicating a possible energy metabolism perturbation (Figure 1f). Brain regions displayed different antioxidant response with a decrease in Ascorbate being stronger in cerebellum (data not shown). CONCLUSIONS: This is the first study showing in-vivo longitudinal analysis of neurometabolism in three different brain regions in a model of CHE. Hippocampus and cerebellum displayed similar trends in metabolite changes during the course of disease, while the changes were much more pronounced in cerebellum. Striatum showed differences in metabolic response when compared to the other brain regions. Clinical relevance of these findings remain to be determined. We conclude that different brain regions are differentially susceptible to the metabolic consequences of CLD, a field which warrants further study.

From the Editor’s Desk: September 2019

From the Editor’s Desk: September 2019

Longitudinal neurometabolic changes in the hippocampus of a rat model of chronic hepatic encephalopathy

The sequence of events in hepatic encephalopathy (HE) remains unclear. Using the advantages of in vivo 1H-MRS (9.4T) we aimed to analyse the time-course of disease in an established model of type C HE by analysing the longitudinal changes in a large number of brain metabolites together with biochemical, histological and behavioural assessment. We hypothesized that neurometabolic changes are detectable very early, and that these early changes will offer insight into the primary events underpinning HE. Wistar rats underwent bile-duct ligation (BDL) and were studied before BDL and at post-operative weeks 2, 4, 6 and 8 (n = 26). In vivo short echo-time 1H-MRS (9.4T) of the hippocampus was performed in a longitudinal manner, as were biochemical (plasma), histological and behavioural tests. Plasma ammonium increased early after BDL and remained high during the study. Brain glutamine increased (+47%) as early as 2-4 weeks post-BDL while creatine (-8%) and ascorbate (-12%) decreased. Brain glutamine and ascorbate correlated closely with rising plasma ammonium, while brain creatine correlated with brain glutamine. The increases in brain glutamine and plasma ammonium were correlated, while plasma ammonium correlated negatively with distance moved. Changes in astrocyte morphology were observed at 4 weeks. These early changes were further accentuated at 6-8 weeks post-BDL, concurrently with the known decreases in brain organic osmolytes. Using a multimodal, in vivo and longitudinal approach we have shown that neurometabolic changes are already noticeable 2 weeks after BDL. These early changes are suggestive of osmotic/oxidative stress and are likely the premise of some later changes. Early decreases in cerebral creatine and ascorbate are novel findings offering new avenues to explore neuroprotective strategies for HE treatment. The sequence of events in chronic hepatic encephalopathy (HE) remains unclear, therefore using the advantages of in vivo proton magnetic resonance spectroscopy at 9.4T we aimed to test the hypothesis that neurometabolic changes are detectable very early in an established model of type C HE, offering insight into the primary events underpinning HE, before advanced liver disease confounds the findings. These early, previously unreported neurometabolic changes occurred as early as 2 to 4 weeks after bile-duct ligation, namely an increase in plasma ammonium and brain glutamine, a decrease in brain creatine and ascorbate together with behavioural and astrocyte morphology changes, and continued to progress throughout the 8-week course of the disease.

Modeling human methamphetamine use patterns in mice: chronic and binge methamphetamine exposure, reward function and neurochemistry.

Different methamphetamine use patterns in human subjects may contribute to inconsistent findings regarding the effects of methamphetamine abuse on brain and behavior. The present study investigated whether human-derived chronic and binge methamphetamine use patterns have differential effects on reward and neurochemistry in mice. Brain reward function in mice was evaluated during acute/prolonged withdrawal, and in response to methamphetamine challenge using the intracranial self-stimulation procedure. Brain dopaminergic, serotonergic and glutamatergic neurochemistry was determined with high-performance liquid chromatography. Chronic and binge regimens induced withdrawal-related decreases in reward function that were more severe during the binge regimen during cycles 1-2. Despite large differences in methamphetamine dose, both regimens induced similar reward deficits during cycles 3-4. Neither methamphetamine regimen led to persistent alterations in the sensitivity to the reward-enhancing effects of acute methamphetamine challenge. The binge regimen severely depleted striatal dopamine levels and increased brain glutamine levels. The chronic regimen had milder effects on striatal dopamine levels and altered cortical dopamine and serotonin levels. This work highlights that the magnitude of acute/prolonged withdrawal may not reflect amount or frequency of methamphetamine intake. In contrast, the array of underlying neurochemical alterations was methamphetamine regimen dependent. Thus, stratifying methamphetamine-dependent individuals based on use pattern may help to cater therapeutic interventions more appropriately by targeting use pattern-specific neurotransmitter systems.

Brain magnetic resonance in experimental acute‐on‐chronic liver failure

Acute-on-chronic liver failure is the term that refers to sustained liver injury with acute decompensation, usually induced by a precipitating factor. A common link between ensuing failures of various organs is impairment of the vascular supply, which may also induce vasogenic oedema in the brain. The aim of this study was to perform magnetic resonance (MR) study of the brain in a rat model combining bile duct ligation (BDL) and lipopolysaccharide (LPS) administration to investigate brain oedema in liver failure. Bile duct-ligated rats underwent in vivo brain MR imaging at 4, 5 and 6 weeks, and after superimposed administration of LPS. The MR techniques applied enabled assessment of brain metabolites, and intra- or extracellular water distribution. Brain water content was assessed by gravimetry. MR spectroscopy showed an increase in brain glutamine and a decrease in myo-inositol and choline in relation to progression of liver disease. BDL rats showed a slight, progressive increase in the amount of cortical brain water that was significant after LPS injection. These changes did not modify the apparent diffusion coefficient, supporting a mixed origin of brain oedema (vasogenic and cytotoxic). The mechanisms leading to the development of brain oedema in an experimental liver disease model were related to the time course of liver failure and to pro-inflammatory stimuli. MR findings support the presence of cytotoxic and vasogenic mechanisms in induced brain oedema in BDL rats exposed to LPS.

Molecular and physiological responses to long-term sublethal ammonia exposure in Atlantic salmon (Salmo salar)

The objective of this study was to determine the underlying physiological and molecular responses to long-term sublethal ammonia exposure in Atlantic salmon (Salmo salar) parr. Previous studies have predominately focused on mechanisms during acute, short-term exposure. For that purpose Atlantic salmon parr were exposed to four ammonia concentrations between 4 and 1800μmoll−1 total ammonia nitrogen (TAN), and subjected to two feeding regimes for 15 weeks. Elevated environmental ammonia and full feeding strength caused an initial increase in plasma ammonia levels ([Tamm]) after 22 days of exposure, which thereafter declined and remained similar to the control animals towards the end of the study. On the other hand, a progressive decrease in plasma urea levels was evident throughout the entire exposure period and depended on the concentration of environmental ammonia, with the largest decrease in urea levels observed at the highest ammonia concentrations (1700 and 1800μmoll−1 TAN). We hypothesized that the successful adaptation to long-term elevated ammonia levels would involve an increased capacity for carrier-facilitated branchial excretion. This hypothesis was strengthened by the first evidence of an up-regulation of branchial transcription of the genes encoding the Rhesus (Rh) glycoproteins, Rhcg1 and Rhcg2, urea transporter (UT) and aquaporin 3a (Aqp3a), during long-term exposure. Of the Rhesus glycoprotein (Rh) mRNAs, Rhcg1 was up-regulated at all tested ammonia levels, while Rhcg2 showed a concentration-sensitive increase. Increased transcription levels of V-type H+-ATPase (H+-ATPase) were observed at the highest ammonia concentrations (1700 and 1800μmoll−1 TAN) and coincided with an up-regulation of Rhcg2 at these concentrations. Transcription of UT and Aqp3a was increased after 15 weeks of exposure to low ammonia levels (470 and 480μmoll−1 TAN). A significant increase in brain glutamine (Gln) concentration was observed for full fed Atlantic salmon after 22 days and in fish with restricted feeding after 105 days of exposure to 1800 and 1700μmoll−1 TAN, respectively, without any concomitant decrease in brain glutamate (Glu) concentrations. These results suggest that Gln synthesis is an ammonia detoxifying strategy employed in the brain of Atlantic salmon parr during long-term sublethal ammonia exposure. Full feed strength had an additive effect on plasma [Tamm], while the restricted feeding regime postponed the majority of the observed physiological and molecular responses. In conclusion, Atlantic salmon parr adapts to the long-term sublethal ammonia concentrations with increased branchial transcription levels of ammonia and urea transporting proteins and ammonia detoxification in the brain.

Topiramate raises anterior cingulate cortex glutamine levels in healthy men; a 4.0 T magnetic resonance spectroscopy study

Potential mechanisms of action of topiramate include alterations of glutamatergic and GABAergic systems. In particular, topiramate has been shown to increase occipital cortex GABA levels, as measured using proton magnetic resonance spectroscopy (MRS). The purpose of this study was to measure the effect of acute oral topiramate on the GABA precursors glutamate and glutamine in the anterior cingulate cortex (ACC) and occipital lobe (OL) using high-field (4.0 T) proton MRS (1H MRS). Proton MR spectra were acquired from healthy men at three times: at baseline and 2 and 6 h after ingesting 50 (N=5) or 100 mg (N=5) of topiramate. Blood samples were acquired prior to each scan for the purpose of obtaining serum topiramate levels. A 100-mg dose of topiramate significantly increased ACC glutamine levels within 2 h of ingestion and OL glutamine levels within 6 h of ingestion. There were no measured significant effects of topiramate on ACC or OL glutamate levels. A 100-mg dose of oral topiramate increased serum topiramate and ACC glutamine levels within 2 h. OL glutamine levels increased within 6 h. Increased brain glutamine levels may be a consequence of topiramate positively modulating GABAA receptors. This result is of interest given the possible role for topiramate in the treatment of epilepsy, migraine headache, bipolar disorder, eating disorders, and alcohol dependence.

Ammonia affects brain nitrogen metabolism but not hydration status in the Gulf toadfish ( Opsanus beta)

Laboratory rodents made hyperammonemic by infusing ammonia into the blood show symptoms of brain cell swelling and increased intracranial pressure. These symptoms could be caused in part by an increase in brain glutamine formed when brain glutamine synthetase (GS) naturally detoxifies ammonia to glutamine. Previous studies on the Gulf toadfish ( Opsanus beta) demonstrated that it is resistant to high ammonia exposure (HAE) (96 h LC 50 = 10 mM) despite an increase in brain glutamine. This study attempts to resolve whether the resistance of O. beta is mediated by special handling of brain water in the face of changing glutamine concentrations. Methionine sulfoximine (MSO), an inhibitor of GS, was used to pharmacologically manipulate glutamine concentrations, and magnetic resonance imaging (MRI) was used to assess the status of brain water. Ammonia or MSO treatment did not substantially affect blood acid–base parameters. Exposure to 3.5 mM ammonium chloride in seawater for 16 or 40 h resulted in a parallel increase in brain ammonia (3-fold) and glutamine (2-fold) and a decrease in brain glutamate (1.3-fold). Pre-treatment with MSO prevented ammonia-induced changes in glutamine and glutamate concentrations. HAE also induced an increase in plasma osmolality (by 7%) which was probably due to a disturbance of osmoregulatory processes but which did not result in broader whole body dehydration as indicated by muscle water analysis. The increase in brain glutamine was not associated with any changes in brain water in toadfish exposed to 3.5 mM ammonia for up to 40 h or even at 10, 20 and 30 mM ammonia consecutively and for one hour in each concentration. The lack of brain water accumulation implies that ammonia toxicity in toadfish appears to be via pathways other than cerebral swelling. Furthermore, toadfish pre-treated with MSO did not survive a normally sub-lethal exposure to 3.5 mM ammonia for 40 h. The enhancement of ammonia toxicity by MSO suggests that GS function is critical to ammonia tolerance in this species.

Glutamine synthetase activity and glutamine content in brain: modulation by NMDA receptors and nitric oxide

Acute intoxication with large doses of ammonia leads to rapid death. The main mechanism for ammonia elimination in brain is its reaction with glutamate to form glutamine. This reaction is catalyzed by glutamine synthetase and consumes ATP. In the course of studies on the molecular mechanism of acute ammonia toxicity, we have found that glutamine synthetase activity and glutamine content in brain are modulated by NMDA receptors and nitric oxide. The main findings can be summarized as follows.Blocking NMDA receptors prevents ammonia-induced depletion of brain ATP and death of rats but not the increase in brain glutamine, indicating that ammonia toxicity is not due to increased activity of glutamine synthetase or formation of glutamine but to excessive activation of NMDA receptors.Blocking NMDA receptors in vivo increases glutamine synthetase activity and glutamine content in brain, indicating that tonic activation of NMDA receptors maintains a tonic inhibition of glutamine synthetase.Blocking NMDA receptors in vivo increases the activity of glutamine synthetase assayed in vitro, indicating that increased activity is due to a covalent modification of the enzyme. Nitric oxide inhibits glutamine synthetase, indicating that the covalent modification that inhibits glutamine synthetase is a nitrosylation or a nitration.Inhibition of nitric oxide synthase increases the activity of glutamine synthetase, indicating that the covalent modification is reversible and it must be an enzyme that denitrosylate or denitrate glutamine synthetase.NMDA mediated activation of nitric oxide synthase is responsible only for part of the tonic inhibition of glutamine synthetase. Other sources of nitric oxide are also contributing to this tonic inhibition.Glutamine synthetase is not working at maximum rate in brain and its activity may be increased pharmacologically by manipulating NMDA receptors or nitric oxide content. This may be useful, for example, to increase ammonia detoxification in brain in hyperammonemic situations.

Role of manganese accumulation in increased brain glutamine of the cirrhotic rat.

Cirrhosis promotes increases of both manganese and glutamine in brain. Manganese is a modulator and glutamine is the product of glutamine synthetase. This work studies the relationship between manganese and glutamine synthetase in a model of cirrhosis in the rat. We administered manganese (1 g/L) in the drinking water of sham-operated and bile-duct obstructed rats. We evaluated the manganese and glutamine accumulation and the glutamine synthetase activity in frontal cortex, striatum, and pallidum after 2, 4, and 6 weeks of biliary obstruction or sham surgery. Cirrhotic rats receiving manganese increased their brain content of metal about 400%-600% after 4 weeks of treatment (P < .05) and also remarkably accumulated glutamine through time in the three regions studied (P < .05 at week 6). Interestingly, bile-duct obstructed rats treated with manganese showed no effect on glutamine synthetase activity. Results from this study suggest that manganese induces increases of brain glutamine independently of its synthesis.

The development of low-grade cerebral edema in cirrhosis is supported by the evolution of 1H-magnetic resonance abnormalities after liver transplantation

Background/Aims : Liver failure may cause brain edema through an increase in brain glutamine. However, usually standard neuroimaging techniques do not detect brain edema in cirrhosis. We assessed magnetization transfer ratio and 1H-magnetic resonance (MR) spectroscopy before and after liver transplantation to investigate changes in brain water content in cirrhosis. Methods : Non-alcoholic cirrhotics without overt hepatic encephalopathy ( n =24) underwent 1H-MR of the brain and neuropsychological tests. 1H-MR results were compared with those of healthy controls ( n =10). In a subgroup of patients ( n =11), the study was repeated after liver transplantation. Results : Cirrhotic patients showed a decrease in magnetization transfer ratio (31.5±3.1 vs. 37.1±1.1, P <0.01) and an increase in glutamine/glutamate signal (2.22±0.47 vs. 1.46±0.26, P <0.01). The increase in glutamine/glutamate signal was correlated to the decrease in magnetization transfer ratio and to neuropsychological function. Following liver transplantation, there was a progressive normalization of magnetization transfer ratio, glutamine/glutamate signal and neuropsychological function. Accordingly, correlations between these variables were lost after liver transplantation. Conclusions : Cirrhotic patients show reversible changes in magnetization transfer ratio that are compatible with the development of low-grade cerebral edema. Minimal hepatic encephalopathy and low-grade cerebral edema appear to be the consequences of the metabolism of ammonia in the brain.

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.

Effects of valproate and other antiepileptic drugs on brain glutamate, glutamine, and GABA in patients with refractory complex partial seizures

Preclinical studies suggested valproate increased brain γ-aminobutyric acid (GABA) with no major effects on brain glutamate or glutamine. Valproate increased human cerebrospinal fluid GABA and glutamine in some studies; others reported no effect.In vivomeasurements of glutamate, glutamine, and GABA were made of a 14 cm3volume in the occipital cortex using a1H spectroscopy with a 2.1 Tesla magnetic resonance spectrometer and an 8 cm surface coil. Ten control subjects and 14 patients with refractory complex partial seizures were examined. Brain glutamine concentrations were above normal in three of five patients taking valproate and two of nine taking carbamazepine or phenytoin. Mean glutamine levels of patients taking valproate were higher than control subjects and patients taking carbamazepine or phenytoin. Brain glutamate concentrations were above normal in four of nine patients taking phenytoin or carbamazepine and two of five taking valproate. Brain GABA levels were below normal in four of nine patients taking carbamazepine or phenytoin and one of five taking valproate. Above normal glutamate or below normal GABA was present in nine of 14 patients and may contribute to their refractory epilepsy. Increased brain glutamine associated with valproate therapy may reflect mild hyperammonemia.

Chronic hyponatremia exacerbates ammonia-induced brain edema in rats after portacaval anastomosis

Background/Aim: Abnormalities in brain organic osmolytes are associated with hepatic encephalopathy and with chronic hyponatremia. In spite of the high frequency of hyponatremia in acute and chronic hepatic failure, its role in the development of neurological complications in liver disease is poorly understood. We aimed to study the effect of prior hyponatremia on the development of ammonia-induced brain edema in rats after portacaval anastomosis. In this model, brain swelling is mediated in part through an increase in brain glutamine, an organic osmolyte. Methods: Hyponatremia was induced in rats with 1-desamino—8-D-arginine vasopressin (DDAVP) administered through an osmotic minipump for 1 week. This was followed by performance of a portacaval anastomosis and ammonia infusion. At the end of the infusion, brain water (density gradient) and key brain organic osmolytes (HPLC) were measured. Results: Rats with hyponatremia showed a decrease in all three brain organic osmolytes measured: glutamine, myo-inositol and taurine. Hyperammonemia resulted in the expected rise in glutamine, with a reduction to myo-inositol and taurine. In the combined group (hyponatremia plus hyperammonemia), the rise in brain glutamine induced by ammonia infusion was attenuated (10.6±0.9 mM/kg vs. 15.5±0.8 mM/kg hyperammonemia alone; p<0.05). In spite of this limited rise in brain glutamine, ammonia infusion to hyponatremic rats exacerbated brain swelling (82.3±0.3 % vs. 80.6±0.1%; p<0.05). Conclusion: Hyponatremia worsens brain swelling in a model of ammonia-induced brain edema. The decrease in the concentration of brain organic osmolytes induced by hyponatremia does not protect the brain from the development of ammonia-induced brain edema.