MR imaging and localized proton MR spectroscopy in late infantile neuronal ceroid lipofuscinosis

PURPOSE: Late juvenile neuronal ceroid lipofuscinosis (NCL) is a lysosomal neurodegenerative disorder caused by the accumulation of lipopigment in neurons. Our purpose was to characterize the MR imaging and spectroscopic findings in three children with late infantile NCL. METHODS: Three children with late infantile NCL and three age-matched control subjects were examined by MR imaging and by localized MR spectroscopy using echo times of 135 and 5. Normalized peak integral values were calculated for N-acetylaspartate (NAA), choline, creatine, myo-inositol, and glutamate/glutamine. RESULTS: MR imaging revealed volume loss of the CNS, most prominently in the cerebellum. The T2-weighted images showed a hypointense thalamus and hyperintense periventricular white matter. Proton MR spectra revealed progressive changes, with a reduction of NAA and an increase of myo-inositol and glutamate/glutamine. In long-standing late infantile NCL, myo-inositol became the most prominent resonance. Lactate was not detectable. CONCLUSION: MR imaging in combination with proton MR spectroscopy can facilitate the diagnosis of late infantile NCL and help to differentiate NCL from other neurometabolic disorders, such as mitochondrial or peroxisomal encephalopathies.     

Neurochemical alterations in asymptomatic abstinent cocaine users: a proton magnetic resonance spectroscopy study

Cocaine can cause a variety of neuropsychiatric and neurobehavioral complications; however, it is uncertain whether cocaine causes persistent cerebral structural and neurochemical abnormalities in asymptomatic users. We studied 52 African-American men (26 human immunodeficiency virus-negative asymptomatic heavy cocaine users and 26 normal subjects). Ventricle-to-brain ratio (VBR) and white matter lesions (WML) were quantified on magnetic resonance imaging. N-acetyl-containing compounds (NA), total creatine, choline-containing compounds, myo-inositol, and glutamate + glutamine were measured with in vivo proton magnetic resonance spectroscopy, VBR and WML were not significantly different in the cocaine users compared to the normal controls. Elevated creatine (+7%; p = .05) and myo-inositol (+18%; p = .01) in the white matter were associated with cocaine use. NA, primarily a measure of N-acetyl aspartate and neuronal content, was normal. Normal NA suggest no neuronal loss or damage in the brain regions examined in these cocaine users. Therefore, we conclude that neurochemical abnormalities observed might result from alterations in nonneuronal brain tissue.     

In vivo injection of [1-13C]glucose and [1,2-13C]acetate combined with ex vivo 13C nuclear magnetic resonance spectroscopy: a novel approach to the study of middle cerebral artery occlusion in the rat

Astrocytes play a pivotal role in cerebral glutamate homeostasis. After 90 minutes of middle cerebral artery occlusion in the rat, the changes induced in neuronal and astrocytic metabolism and in the neuronal-astrocytic interactions were studied by combining in vivo injection of [1-13C]glucose and [1,2-13C]acetate with ex vivo 13C nuclear magnetic resonance spectroscopy and HPLC analysis of amino acids of the lateral caudoputamen and lower parietal cortex, representing the putative ischemic core, and the upper frontoparietal cortex, corresponding to the putative penumbra. In the putative ischemic core, evidence of compromised de novo glutamate synthesis located specifically in the glutamatergic neurons was detected, and a larger proportion of glutamate was derived from astrocytic glutamine. In the same region, pyruvate carboxylase activity, representing the anaplerotic pathway in the brain and exclusively located in astrocytes, was abolished. However, astrocytic glutamate uptake and conversion to glutamine took place, and cycling of intermediates in the astrocytic tricarboxylic acid cycle was elevated. In the putative penumbra, glutamate synthesis was improved compared with the ischemic core, the difference appeared to be brought on by better neuronal de novo glutamate synthesis, combined with normal levels of glutamate formed from astrocytic glutamine. In both ischemic regions, gamma-aminobutyric acid synthesis directly from glucose was reduced to about half, indicating impaired pyruvate dehydrogenase activity; still, gamma-aminobutyric acid reuptake and cycling was increased. The results obtained in the current study demonstrate that by combining in vivo injection of [1-13C]glucose and [1,2-13C]acetate with ex vivo 13C nuclear magnetic resonance spectroscopy, specific metabolic alterations in small regions within the rat brain suffering a focal ischemic lesion can be studied.     

Comparison of lactate and glucose metabolism in cultured neocortical neurons and astrocytes using 13C-NMR spectroscopy

In cerebral cortical neurons, synthesis of the tricarboxylic acid (TCA) cycle-derived amino acids, glutamate and aspartate as well as the neurotransmitter of these neurons, gamma-aminobutyrate (GABA), was studied incubating the cells in media containing 0.5 mM [U-13C]glucose in the absence or presence of glutamine (0.5 mM). Lyophilized cell extracts were analyzed by 13C nuclear magnetic resonance (NMR) spectroscopy and HPLC. The present findings were compared to results previously obtained using 1.0 mM [U-13C]lactate as the labeled substrate for the neurons. Regardless of the amino acids studied, incubation periods of 1 and 4 h resulted in identical amounts of 13C incorporated. Furthermore, the metabolism of lactate was studied under analogous conditions in cultured cerebral cortical astrocytes. The incorporation of 13C from lactate into glutamate was much lower in the astrocytes than in the neurons. In cerebral cortical neurons the total amount of 13C in GABA, glutamate and aspartate was independent of the labeled substrate. The enrichment in glutamate and aspartate was, however, higher in neurons incubated with lactate. Thus, lactate appears to be equivalent to glucose with regard to its access to the TCA cycle and subsequent labeling of glutamate, aspartate and GABA. It should be noted, however, that incubation with lactate in place of glucose led to lower cellular contents of glutamate and aspartate. The presence of glutamine affected the metabolism of glucose and lactate differently, suggesting that the metabolism of these substrates may be compartmentalized.     

15N-NMR spectroscopy studies of ammonia transport and glutamine synthesis in the hyperammonemic rat brain

Ammonia transport and glutamine synthesis were studied in the hyperammonaemic rat brain in vivo using 15N-NMR spectroscopy at a plasma ammonia level of approximately 0.39 mM raised via an intravenous [15N]-ammonium acetate infusion. The initial slope of the time course of the summed cerebral 15N-labelled metabolites was used to determine the rate of ammonia net transport during hyperammonemia as 0.13 +/- 0.02 micromol/min/g (mean +/- SD; n = 5). Based on the total accumulation of glutamine and the 1:2 stoichiometric relationship between fluxes of four-carbon skeletons and nitrogen atoms, the rate of de novo glutamine synthesis through anaplerosis and subsequent glutamate dehydrogenase action was calculated to be 0.065 +/- 0.01 micromol/min/g. The rate of total glutamine synthesis was estimated to be 0.20 +/- 0.06 micromol/min/g (n = 5) by fitting the [5-15N]glutamine time course to a previously described model of glutamate-glutamine cycling between astrocytes and neurones. A large dilution was also observed in [2-15N]glutamine, which supports the glutamate-glutamine cycle as being an important pathway for neuronal glutamate repletion in vivo.     

Exogenous glutamate concentration regulates the metabolic fate of glutamate in astrocytes

The metabolic fate of glutamate in astrocytes has been controversial since several studies reported > 80% of glutamate was metabolized to glutamine; however, other studies have shown that half of the glutamate was metabolized via the tricarboxylic acid (TCA) cycle and half converted to glutamine. Studies were initiated to determine the metabolic fate of increasing concentrations of [U-13C] glutamate in primary cultures of cerebral cortical astrocytes from rat brain. When astrocytes from rat brain were incubated with 0.1 mM [U-13C] glutamate 85% of the 13C metabolized was converted to glutamine. The formation of [1,2,3-13C3] glutamate demonstrated metabolism of the labeled glutamate via the TCA cycle. When astrocytes were incubated with 0.2-0.5 mM glutamate, 13C from glutamate was also incorporated into intracellular aspartate and into lactate that was released into the media. The amount of [13C] lactate was essentially unchanged within the range of 0.2-0.5 mM glutamate, whereas the amount of [13C] aspartate continued to increase in parallel with the increase in glutamate concentration. The amount of glutamate metabolized via the TCA cycle progressively increased from 15.3 to 42.7% as the extracellular glutamate concentration increased from 0.1 to 0.5 mM, suggesting that the concentration of glutamate is a major factor determining the metabolic fate of glutamate in astrocytes. Previous studies using glutamate concentrations from 0.01 to 0.5 mM and astrocytes from both rat and mouse brain are consistent with these findings.     

Angular dependence of electron paramagnetic resonances of an azide-NO complex of cytochrome c oxidase: orientation of the haem-copper axis in cytochrome aa3 from ox heart

In localized proton magnetic resonance spectroscopy very short echo times (TE) are achieved to diminish signal loss due to T2 relaxation and to avoid phase distortions due to J-coupling. A sequence for single volume spectroscopy in human brain is described with a TE as low as 5 ms. Examinations were performed on a 1.5 T whole-body imager with actively shielded gradients. A self-designed stimulated echo acquisition mode (STEAM) sequence with very high amplitude spoiling gradients of 24 mT/m was used to take advantage of the whole potential of the gradient system. Optimization of TE was carried out by controlling spectral quality and localization in both phantom and volunteer measurements. Proton spectra of human brain were acquired in 21 healthy volunteers. Spectra of occipital white matter, parieto-occipital grey/white matter, and cerebellum revealed none or only small eddy current distortions at a TE of 5 ms. The volume of interest was 8-12 ml, repetition time was 1.5 s, and mixing time was 5 ms. Peak ratios of major metabolites referring to creatine were estimated and the relative standard deviations were calculated to determine interindividual reproducibility. The relative standard deviation of myo-inositol ranged from 6% to 11% within these brain regions whereas for glutamine and glutamate 7% to 16% were found.     

New insights into the compartmentation of glutamate and glutamine in cultured rat brain astrocytes

Studies from several groups have provided evidence that glutamate and glutamine are metabolized in different compartments in astrocytes. In the present study we measured the rates of 14CO2 production from U-[14C]glutamate and U-[14C]glutamine, and utilized both substrate competition experiments and the transaminase inhibitor aminooxyacetic acid (AOAA) to obtain more information about the compartmentation of these substrates in cultured rat brain astrocytes. The rates of oxidation of 1 mM glutamine and glutamate were 26.4 +/- 1.4 and 63.0 +/- 7.4 nmol/h/mg protein, respectively. The addition of 1 mM glutamate decreased the rate of oxidation of glutamine to 26.3% of the control rate, demonstrating that glutamate can effectively compete with the oxidation of glutamine by astrocytes. In contrast, the addition of 1 mM glutamine had little or no effect on the rate of oxidation of glutamate by astrocytes, demonstrating that the glutamate produced intracellularly from exogenous glutamine does not dilute the glutamate taken up from the media. The addition of 5 mM AOAA decreased the rate of 14CO2 production from glutamine to 29.2% of the control rate, consistent with earlier studies by our group. The addition of 5 mM AOAA decreased the rate of oxidation of concentrations of glutamate < or = 0.1 mM by approximately 50%, but decreased the oxidation of 0.5-1 mM glutamate by only approximately 20%, demonstrating that a substantial portion of glutamate enters the tricarboxylic acid (TCA) cycle via glutamate dehydrogenase (GDH) rather than transamination, and that as the concentration of glutamate increases the relative proportion entering the TCA cycle via GDH also increases. To determine if the presence of an amino group acceptor (i.e. a ketoacid) would increase the rate of metabolism of glutamate, pyruvate was added in some experiments. Addition of 1 mM pyruvate increased the rate of oxidation of glutamate, and the increase was inhibited by AOAA, consistent with enhanced entry of glutamate into the TCA cycle via transamination in the presence of pyruvate. Enzymatic studies showed that pyruvate increased the activity of mitochondrial aspartate aminotransferase (AAT). Overall, the data demonstrate that glutamate formed intracellularly from glutamine enters the TCA cycle primarily via transamination, but does not enter the same TCA cycle compartment as glutamate taken up from the extracellular milieu. In contrast, extracellular glutamate enters the TCA cycle in astrocytes via both transamination and GDH, and can compete with, or dilute, the oxidation of glutamate produced intracellularly from glutamine.     

In vivo investigation of glutamate-glutamine metabolism in hyperammonemic monkey brain using 13C-magnetic resonance spectroscopy

To investigate the metabolism of glutamate and glutamine in living monkey brain, a system of in vivo 13C magnetic resonance spectroscopy (MRS) using 1H-decoupled 13C spectroscopy combined with monitoring temperature changes in the brain by MR phase mapping was developed. Serial 13C-NMR spectra of the amino acids glutamate and glutamine were acquired non-invasively over 4 h from anesthetized monkey brain after the intravenous administration of [1-13C]glucose (0.5-1.0 g/kg). In the acute hyperammonemic state induced by the administration of ammonium acetate (77 mg/kg bolus), it was observed that 13C incorporation into glutamine-4 was clearly accelerated, without changes of 13C incorporation into glutamate-4. During hyperammonemia, it was shown directly by [2-13C]glucose administration that the anaplerotic pathway for the TCA cycle was also augmented, contributing to the formation of glutamine in the astroglia.     

Organic osmolytes in the kidney of domesticated red deer, Cervus elaphus

The cortex, inner and outer medulla, and papilla of kidneys of domestic red deer were analysed. In hydrated animals the urine concentration was found to be 672 +/- 45 mOsm.l-1. The medullary and papillary regions of the kidney were rich in the osmolytes betaine, glycerophosphorylcholine (GPC), inositol and sorbitol, all of which showed a steep rise in concentration from cortex to papilla. The kidney was rich in free amino acids, in particular taurine, glutamate (+glutamine), glycine and alanine, which were present at concentrations sufficient to suggest a possible role as osmolytes.     

Measurement of glutamate and glutamine in the medial prefrontal cortex of never-treated schizophrenic patients and healthy controls by proton magnetic resonance spectroscopy

BACKGROUND: Positron emission tomographic and postmortem studies comparing schizophrenic patients with healthy control subjects have found medial prefrontal cortical and anterior cingulate abnormalities that suggest dysfunction in glutamatergic neurons. The glutamate used for nerve signal transduction is predominantly derived from glutamine. After signal transduction, glutamate released into the synapse is converted to glutamine in glial cells, transported back to the presynaptic neuron, and reconverted to glutamate for reuse. In this study, levels of glutamate and glutamine were examined by means of in vivo proton (1H) magnetic resonance spectroscopy. METHODS: Localized in vivo 1H spectra were acquired from a 4.5-cm3 volume in the left medial prefrontal cortex encompassing portions of Brodmann areas 24, 32, and 9 in 10 never-treated schizophrenic subjects and 10 healthy controls of comparable age, sex, handedness, education, and parental education. From each spectrum, metabolite levels were estimated for glutamate and glutamine, as well as 10 other metabolites and 3 macromolecules, by means of a noninteractive computer program that combined modeled in vitro spectra of every metabolite to reconstruct each in vivo spectrum. RESULTS: A significant increase in glutamine level was found in the medial prefrontal cortex of the schizophrenic patients compared with controls. N-acetylaspartate and other measured metabolites and macromolecules were not significantly changed in schizophrenics. CONCLUSION: Increased glutamine levels in the medial prefrontal region most likely reflect decreased glutamatergic activity in this region in never-treated schizophrenic patients compared with healthy controls.     

In vitro and ex vivo 13C-NMR spectroscopy studies of pyruvate recycling in brain

Pyruvate recycling is a well established pathway in the liver, but in the brain, the cellular localization of pyruvate recycling remains controversial and its physiological significance is unknown. In cultured cortical astrocytes, pyruvate formed from [U-13C]glutamate was shown to re-enter the TCA cycle after conversion to acetyl-CoA, as demonstrated by the labelling patterns in aspartate C-2 and C-3, lactate C-2, and glutamate C-4, which provides evidence for pyruvate recycling in astrocytes. This finding is in agreement with previous studies of astrocytic cultures, in which pyruvate recycling has been described from [U-13C]glutamine, in the presence of glutamate, and from [U-13C]aspartate. Pyruvate recycling in brain was studied in fasted rats receiving either an intraperitoneal or a subcutaneous injection of [1,2-13C]acetate followed by decapitation 30 min later. Extracts of cortical tissue were analysed with 13C-NMR spectroscopy and total amounts of amino acids quantified by HPLC. Plasma extracts were analysed with 1H- and 13C-NMR spectroscopy, and showed a significantly larger amount of [1, 2-13C]acetate in the intraperitoneal group compared to the subcutaneous group. Furthermore, a small amount of label was detected in glucose in both groups. In the subcutaneously injected rats, [4-13C]glutamate and [2-13C]GABA were less enriched than plasma glucose, which might have been the precursor. In the intraperitoneally injected rats, however, pyruvate formation from [1, 2-13C]acetate, and re-entry of this pyruvate into the TCA cycle was demonstrated by the presence of greater 13C enrichment in [4-13C]glutamate and [4-13C]glutamine compared to the subcutaneous group, probably resulting from the significantly higher [1, 2-13C]acetate concentration in brain and plasma.     

Glutamate, glutamine, and GABA as substrates for the neuronal and glial compartments after focal cerebral ischemia in rats

BACKGROUND AND PURPOSE: Even though the utilization of substrates alternative to glucose may play an important role in the survival of brain cells under ischemic conditions, evidence on changes in substrate selection by the adult brain in vivo during ischemic episodes remains very limited. This study investigates the utilization of glutamate, glutamine, and GABA as fuel by the neuronal and glial tricarboxylic acid cycles of both cerebral hemispheres after partially reversible focal cerebral ischemia (FCI). METHODS: Right hemisphere infarct was induced in adult Long-Evans rats by permanent occlusion of the right middle cerebral artery and transitory occlusion of both common carotid arteries. (1,2-13C2) acetate was infused for 60 minutes in the right carotid artery immediately after carotid recirculation had been re-established (1-hour group) or 23 hours later (24-hour group). Extracts from both cerebral hemispheres were prepared and analyzed separately by 13C nuclear magnetic resonance and computer-assisted metabolic modeling. RESULTS: FCI decreased the oxidative metabolism of glucose in the brain in a time-dependent manner. Reduced glucose oxidation was compensated for by increased oxidations of (13C) glutamate and (13C) GABA in the astrocytes of the ipsilateral hemispheres of both groups. Increased oxidative metabolism of (13C) glutamine in the neurons was favored by increased activity of the neuronal pyruvate recycling system in the 24-hour group. CONCLUSIONS: Data were obtained consistent with time-dependent changes in the utilization of glutamate and GABA or glutamine as metabolic substrates for the glial or neuronal compartments of rat brain after FCI.     

Proton magnetic resonance spectroscopy of the primary motor cortex in patients with motor neuron disease: subgroup analysis and follow-up measurements

OBJECTIVES: To determine the motor cortex degeneration in patients with amyotrophic lateral sclerosis (ALS) using proton magnetic resonance spectroscopy, and to prove that proton magnetic resonance spectroscopy is suited to monitor the course of disease with follow-up examinations. MATERIALS AND METHODS: We studied 33 patients with ALS whose conditions were diagnosed according to the El Escorial World Federation of Neurology criteria. Nine patients with ALS were followed up for up to 2 years. The control group included 20 healthy volunteers and 4 patients with multifocal motor neuropathy. Proton magnetic resonance spectroscopy determined levels of the brain metabolites N-acetylaspartate (NAA), choline, inositol-containing compounds, glutamate/glutamine, and phosphocreatine. RESULTS: Patients with ALS showed a significant reduction in the NAA-choline (P <.001) and NAA-phosphocreatine (P <.005) metabolite ratios and significantly elevated choline-phosphocreatine (P <.005) ratios compared with controls. Inositol-phosphocreatine ratios were also elevated in case patients, but the increase was less pronounced (P <.05). No differences in glutamate/glutamine-phosphocreatine ratios were detected between case patients and controls. An analysis of subgroups demonstrated less significant differences in NAA-choline metabolite ratios (P<.05), even in patients with pure lower motor neuron syndrome (suspected ALS). No changes in metabolite T1 and T2 relaxation times were observed. Patients with multifocal motor neuropathy showed normal metabolic ratios. Progressive alterations in affected metabolite ratios could be documented in the follow-up examinations. CONCLUSIONS: Spectroscopic changes in the motor cortices of patients with ALS correspond with a reduction in levels of NAA and an elevation in levels of choline and inositol compounds. Since NAA is exclusively expressed in neurons, the observed decrease of NAA reflects neuronal loss or dysfunction. Inositol and choline are associated with plasma membrane metabolism, so the release of these compounds may be related to membrane disorders.     

A study of the precision of confocal, ratiometric, Fura-2-based [Ca2+] measurements

Increased ammonia has been considered a key factor in the pathogenesis of hepatic encephalopathy. The high concentration of ammonia interferes with oxidative metabolism in the brain through an inhibitory effect on the tricarboxylic acid cycle (TCA). Inhibition of the TCA cycle may result in depletion of ATP. Due to the involvement of astrocytes in brain detoxification of ammonia, these cells are good candidates for studying ammonia's effect on energy stores in the brain. C6-glioma cells, which have altered glycolytic rates, may show greater sensitivity to the toxicity of ammonium chloride than astrocytes. To study the effect of ammonium chloride on energy storage of both astrocytes and C6-glioma, we observed the acute and chronic effects of NH4Cl (7.5 or 15 mM) on the metabolism of isolated astrocytes and C6-glioma cells. Primary astrocytes were isolated from the cerebral hemispheres of 1-2 day old Sprague-Dawley rats, and C6-glioma cells were purchased from the American Type Culture Collection (ATCC). Following treatment of the cells with ammonia, glucose, lactate, glutamate, ATP, and PCr were assayed. Our data showed that at 15 min following treatment with NH4Cl, there were no significant differences in the concentration of metabolites measured in astrocytes. However, following 15 min of treatment with NH4Cl, the concentration of some metabolites, for example, ATP and lactate, changed significantly in C6-glioma cells. We have shown that 24 h of treatment was sufficient time to see significant biochemical changes but not morphological changes in either cell type. Simultaneous biochemical and morphological changes were observed 48 h following treatment in C6-glioma cells and at 9-10 days following treatment in primary astrocytes. In primary astrocytes at 24 h following treatment, glucose utilization increased. This high utilization of glucose was in accordance with the increase in lactate and glutamate production and the decrease in ATP and PCr formation. In C6-glioma cells the utilization of glucose increased but this high utilization of glucose was consistent with a significant decrease in the concentration of lactate, glutamate and ATP.     

Immediate adaptation of the dog kidney to acute hypercapnia

Studies were performed to determine whether ammoniagenesis could adapt instantaneously to acidosis in the dog kidney. Following acute respiratory acidosis, renal glutamine extraction rose acutely in dogs with stable renal blood flow but did not change when the renal blood flow fell by more than 25%. Acute hypercapnia immediately increased renal ammonia production in both groups of dogs. The rate of both glutamine extraction and ammonia production in acutely hypercapnic dogs without hemodynamic changes was comparable to the rates observed in dogs with chronic metabolic acidosis. Furthermore, the renal metabolite profile observed in acute hypercapnia was similar to the pattern described in chronic metabolic acidosis, i.e., a marked fall in renal glutamate and alpha-ketoglutarate concentrations and a fivefold increase in malate and oxaloacetate concentrations. In the liver and muscle, acute hypercapnia induced no significant change in glutamine concentration but glutamate and alpha-ketoglutarate concentrations decreased. Our findings demonstrate that the dog kidney can adapt immediately to acidosis but that hemodynamic change may mask this adaptation.     

Portacaval anastomosis results in altered neuron--astrocytic metabolic trafficking of amino acids: evidence from 13C-NMR studies

13C-NMR spectroscopy was used to evaluate the dynamic consequences of portacaval anastomosis on neuronal and astrocytic metabolism and metabolic trafficking between neurons and astrocytes. Glutamate is predominantly labeled from [1-13C]glucose, whereas [2-13C]acetate is more efficient in labeling glutamine, in accordance with its primary metabolism in astrocytes. Alanine and succinate labeling was only observed with [1-13C]glucose as precursor. Brain [1-13C]glucose metabolism in portacaval-shunted rats was similar to that in sham-operated controls with the exception of labeled glutamine and succinate formation, which was increased in shunted rats. The 13C enrichment was, however, decreased owing to an increase in total glutamine and succinate. Using [2-13C]acetate, on the other hand, flux of astrocytic label to neurons was severely decreased because label incorporation into glutamate, aspartate, and GABA was decreased following portacaval shunting. The latter amino acids are predominantly localized in neurons. These findings demonstrate that metabolic trafficking of amino acids from astrocytes to neurons is impaired in portacaval-shunted rats.     

Evidence that 3-aminopicolinate stimulates glutamine metabolism by rat renal cortical tissue in vitro

The hyperglycemic agent 3-aminopicolinate used at concentrations of 0.05 and 0.1 mM stimulates the removal of 5 mM glutamine. It also stimulates the accumulation of glutamate and the formation of ammonia, glucose and aspartate by isolated rat kidney-cortex tubules. These effects are consistent with a stimulation of glutaminase and an inhibition of phosphoenolpyruvate carboxy-kinase by this compound. Higher concentrations (0.5 and 1 mM) of 3-aminopicolinate fail to affect the removal of 5 mM glutamine, but greatly alter the fate of both glutamine carbon and amino nitrogen. Similar effects of 3-aminopicolinate are observed when glutamine is used at a near-physiological concentration.     

In vivo 13C NMR measurements of cerebral glutamine synthesis as evidence for glutamate-glutamine cycling

The cerebral tricarboxylic acid (TCA) cycle rate and the rate of glutamine synthesis were measured in rats in vivo under normal physiological and hyperammonemic conditions using 13C NMR spectroscopy. In the hyperammonemic animals, blood ammonia levels were raised from control values of approximately 0.05 mM to approximately 0.35 mM by an intravenous ammonium acetate infusion. Once a steady-state of cerebral metabolites was established, a [1-13C]glucose infusion was initiated, and 13C NMR spectra acquired continuously on a 7-tesla spectrometer to monitor 13C labeling of cerebral metabolites. The time courses of glutamate and glutamine C-4 labeling were fitted to a mathematical model to yield TCA cycle rate (V(TCA)) and the flux from glutamate to glutamine through the glutamine synthetase pathway (V(gln)). Under hyperammonemia the value of V(TCA) was 0.57 +/- 0.16 micromol/min per g (mean +/- SD, n = 6) and was not significantly different (unpaired t test; P > 0.10) from that measured in the control animals (0.46 +/- 0.12 micromol/min per g, n = 5). Therefore, the TCA cycle rate was not significantly altered by hyperammonemia. The measured rate of glutamine synthesis under hyperammonemia was 0.43 +/- 0.14 micromol/min per g (mean +/- SD, n = 6), which was significantly higher (unpaired t test; P < 0.01) than that measured in the control group (0.21 +/- 0.04 micromol/ min per g, n = 5). We propose that the majority of the glutamine synthetase flux under normal physiological conditions results from neurotransmitter substrate cycling between neurons and glia. Under hyperammonemia the observed increase in glutamine synthesis is comparable to the expected increase in ammonia transport into the brain and reported measurements of glutamine efflux under such conditions. Thus, under conditions of elevated plasma ammonia an increase in the rate of glutamine synthesis occurs as a means of ammonia detoxification, and this is superimposed on the constant rate of neurotransmitter cycling through glutamine synthetase.