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.     

Sources of acetyl-CoA entering the tricarboxylic acid cycle as determined by analysis of succinate 13C isotopomers

A new 13C NMR technique for measuring substrate utilization by the citric acid cycle based on an analysis of succinate 13C isotopomers is presented. The relative contribution of up to three different labeling patterns in acetyl-CoA entering the citric acid cycle may be determined under non-steady-state conditions. We present experimental data from perfused rat hearts subjected to a brief period of ischemia, where both succinate and glutamate resonances were observed in the 13C spectrum. The contributions of labeled exogenous acetate and lactate and unlabeled sources to the acetyl-CoA pool were compared using this succinate analysis and a previously published glutamate analysis [Malloy et al. (1990) Biochemistry 29, 6756-6761], and the two methods give identical results. This indicates that the succinate and glutamate isotopomers originated from a common alpha-ketoglutarate pool, verifying that glutamate is in isotopomeric equilibrium with alpha-ketoglutarate under these conditions.     

Compartmentation of lactate and glucose metabolism in C6 glioma cells. A 13c and 1H NMR study

13C and 1H NMR spectroscopy was used to investigate the metabolism of L-lactate and D-glucose in C6 glioma cells. The changing of lactate and glucose concentration in the extracellular medium of C6 glioma cells incubated with 5.5 mM glucose and 11 mM lactate indicated a net production of lactate as the consequence of an active aerobic glycolysis. The 13C enrichments of various metabolites were determined after 4-h cell incubation in media containing both substrates, each of them being alternatively labeled in the form of either [3-13C]L-lactate or [1-13C]D-glucose. Using 11 mM [3-13C]L-lactate, the enrichment of glutamate C4, 69%, was found higher than that of alanine C3, 32%, when that of acetyl-CoA C2 was 78%. These results indicated that exogenous lactate was the major substrate for the oxidative metabolism of the cells. Nevertheless, an active glycolysis occurred, leading to a net lactate production. This lactate was, however, metabolically different from the exogenous lactate as both lactate species did not mix into a unique compartment. The results were actually consistent with the concept of the existence of two pools of both lactate and pyruvate, wherein one pool was closely connected with exogenous lactate and was the main fuel for the oxidative metabolism, and the other pool was closely related to aerobic glycolysis.     

Quantification of menstrual and diurnal periodicities in rates of cholesterol and fat synthesis in humans

The mass isotopomer distribution analysis (MIDA) technique is applied here in men and menstruating women to quantify periodicities in the biosynthesis of serum cholesterol and very low density lipoprotein (VLDL)-palmitate. The isotopic enrichment of the true biosynthetic precursor (intracellular acetyl-CoA) during oral or intravenous administration of sodium[1-13C]- or [2-13C]acetate was calculated from mass isotopomer fractional abundances in free cholesterol and VLDL-palmitate, determined by gas chromatography-mass spectrometry (GC-MS). To convert fractional into absolute cholesterol synthesis rates, decay rate constants of plasma cholesterol were determined from the die-away curves of endogenously labeled high-mass isotopomers. Oral [13C]acetate was a 3-4 times more efficient means of labeling the precursor pool for VLDL-palmitate than was intravenous [13C]acetate, consistent with a splanchnic site of VLDL-fatty acid synthesis, whereas the precursor for free cholesterol had an intermediate enrichment, suggesting a contribution from extra-splanchnic tissues as well. Endogenous synthesis of serum cholesterol was 8-11 mg/kg per day (an estimated 65-75% of input into serum cholesterol); it was 1.5- to 3-fold higher at night than during the day (37-49 mg/h at night compared to 9-23 mg/h during the day) and did not vary over the menstrual cycle (608-697 mg/day). In contrast, endogenous synthesis of fatty acids made a relatively minor contribution to body fat pools (1/10-1/20) of input into VLDL-palmitate) compared to dietary fat intake; it was greater in the day-time, and was influenced by menstrual cycle (3-fold elevated in the follicular phase compared to the luteal phase), and body composition (higher in obese men than normal weight men, r2 = 0.59 for lipogenesis vs. body mass index). Factors responsible for periodicities in endogenous lipid synthesis can be studied in humans using this approach.     

Cerebral metabolic compartmentation as revealed by nuclear magnetic resonance analysis of D-[1-13C]glucose metabolism

Nuclear magnetic resonance (NMR) was used to study the metabolic pathways involved in the conversion of glucose to glutamate, gamma-aminobutyrate (GABA), glutamine, and aspartate. D-[1-13C]Glucose was administered to rats intraperitoneally, and 6, 15, 30, or 45 min later the rats were killed and extracts from the forebrain were prepared for 13C-NMR analysis and amino acid analysis. The absolute amount of 13C present within each carbonatom pool was determined for C-2, C-3, and C-4 of glutamate, glutamine, and GABA, for C-2 and C-3 of aspartate, and for C-3 of lactate. The natural abundance 13C present in extracts from control rats was also determined for each of these compounds and for N-acetylaspartate and taurine. The pattern of labeling within glutamate and GABA indicates that these amino acids were synthesized primarily within compartments in which glucose was metabolized to pyruvate, followed by decarboxylation to acetyl-CoA for entry into the tricarboxylic acid cycle. In contrast, the labeling pattern for glutamine and aspartate indicates that appreciable amounts of these amino acids were synthesized within a compartment in which glucose was metabolized to pyruvate, followed by carboxylation to oxaloacetate. These results are consistent with the concept that pyruvate carboxylase and glutamine synthetase are glia-specific enzymes, and that this partially accounts for the unusual metabolic compartmentation in CNS tissues. The results of our study also support the concept that there are several pools of glutamate, with different metabolic turnover rates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lactate turnover in rat glioma measured by in vivo nuclear magnetic resonance spectroscopy

Elevated tissue lactate concentrations typically found in tumors can be measured by in vivo nuclear magnetic resonance (NMR) spectroscopy. In this study, lactate turnover in rat C6 glioma was determined from in vivo 1H NMR measurements of [3-13C]lactate buildup during steady-state hyperglycemia with [1-13C]glucose. With this tumor model, a narrow range of values was observed for the first-order rate constant that describes lactate efflux, k2 = 0.043 +/- 0.007 (n = 12) SD min-1. For individual animals, the standard error in k2 was small (< 18%), which indicated that the NMR data fit the kinetic model well. Lactate measurements before and after infusing [1-13C]glucose showed that the majority of the tumor lactate pool was metabolically active. Signals from 13C-labeled glutamate in tumors were at least 10-fold smaller than the [3-13C]lactate signal, whereas spectra of the contralateral hemispheres revealed the expected labeling of [4-13C]glutamate, as well as [2-13C] and [3-13C]glutamate, which indicates that label cycled through the tricarboxylic acid cycle in the brain tissue. Lack of significant 13C labeling of glutamate was consistent with low respiratory metabolism in this glioma. It is concluded that lactate in rat C6 glioma is actively turning over and that the kinetics of lactate efflux can be quantified noninvasively by 1H NMR detection of 13C label. This noninvasive NMR approach may offer a valuable tool to help evaluate tumor growth and metabolic responsiveness to therapies.     

Use of [6,6-2H2]glucose and of low-enrichment [U-13C6]-glucose for sequential or simultaneous measurements of glucose turnover by gas chromatography-mass spectrometry

We developed gas chromatography-mass spectrometric methods for assaying the enrichment of 99 at.% [6,6-2H2]glucose and 30 at.% [U-13C6]glucose, although both tracers are mostly M + 2. 13C enrichment is determined either by the C-1 to C-5 fragment of glucose aldonitrile pentaacetate or by oxidation of glucose to glucarate. 2H enrichment is assayed as the difference between the 13C enrichment of glucarate and the 2H + 13C enrichment of glucose. The techniques, which were validated in in vivo experiments, are applicable to the determination of simultaneous or sequential measurements of the rate of glucose appearance before and after an intervention. They could also be applied to the simultaneous determination of (i) gluconeogenesis by incorporation of a 13C-labeled precursor into glucose and (ii) the rate of glucose appearance by [6,6-2H2]glucose infusion.     

Quantification of the GABA shunt and the importance of the GABA shunt versus the 2-oxoglutarate dehydrogenase pathway in GABAergic neurons

We investigated the activity of the cerebral GABA shunt relative to the overall cerebral tricarboxylic acid (TCA) cycle and the importance of the GABA shunt versus 2-oxoglutarate dehydrogenase for the conversion of 2-oxoglutarate into succinate in GABAergic neurons. Awake mice were dosed with [1-(13)C]glucose, and brain extracts were analyzed by 13C NMR spectroscopy. The percent enrichments of GABA C-2 and glutamate C-4 were the same: 5.0 +/- 1.6 and 5.1 +/- 0.2%, respectively (mean +/- SD). This, together with previous data, indicates that the flux through the GABA shunt relative to the overall cerebral TCA cycle flux equals the GABA/glutamate pool size ratio, which in the mouse is 17%. It has previously been shown that under the experimental conditions used in this study, the 13C labeling of aspartate from [1-(13)C]-glucose specifically reflects the metabolic activity of GABAergic neurons. In the present study, the reduction in the formation of [13C]aspartate during inhibition of the GABA shunt by gamma-vinyl-GABA indicated that not more than half the flux from 2-oxoglutarate to succinate in GABAergic neurons goes via the GABA shunt. Therefore, because fluxes through the GABA shunt and 2-oxoglutarate dehydrogenase in GABAergic neurons are approximately the same, the TCA cycle activity of GABAergic neurons could account for one-third of the overall cerebral TCA cycle activity in the mouse. Treatment with gamma-vinyl-GABA, which increased GABA levels dramatically, caused changes in the 13C labeling of glutamate and glutamine, which indicated a reduction in the transfer of glutamate from neurons to glia, implying reduced glutamatergic neurotransmission. In the most severely affected animals these alterations were associated with convulsions.     

PET-characterization of [carbonyl-11C]WAY-100635 binding to 5-HT1A receptors in the primate brain

A method for the accurate determination of 13C enrichments in nonprotonated carbon atoms of organic compounds that makes use of unresolved 13C satellites of proton(s) bonded to the vicinal carbon atom was developed. Using glutamate as a model molecule, this 1H nuclear magnetic resonance (NMR) inverse spin-echo difference spectroscopy method was calibrated for inversion efficiency and relaxation effects which were then shown to cause only a minor loss of the measured 13C satellite amplitude (2% for glutamate C-1 and 7% for glutamate C-5). The determination of 13C enrichments in nonprotonated glutamate carbon atoms by this method was shown to be more precise than 13C NMR. As a first application, a [5-13C]glucose labeling experiment with Corynebacterium glutamicum ASK1 was performed. The labeling patterns of glutamate and arginine extracted from cellular protein were determined using the newly developed method and standard 1H NMR with and without broadband 13C decoupling. Determination of the 13C enrichment in C-5 of glutamate and arginine, respectively, by the two methods showed good agreement. From the deduced labeling pattern of 2-oxoglutarate, an in vivo carbon flux distribution within the central metabolism of C. glutamicum ASK1 was calculated. Thus, the relative flux toward oxaloacetate via the tricarboxylic acid cycle enzyme malate dehydrogenase was determined as 45%, whereas that via anaplerotic C3 carboxylation was determined as 55%.     

Kinetic analysis of dynamic 13C NMR spectra: metabolic flux, regulation, and compartmentation in hearts

Control of oxidative metabolism was studied using 13C NMR spectroscopy to detect rate-limiting steps in 13C labeling of glutamate. 13C NMR spectra were acquired every 1 or 2 min from isolated rabbit hearts perfused with either 2.5 mM [2-13C]acetate or 2.5 mM [2-13C]butyrate with or without KCl arrest. Tricarboxylic acid cycle flux (VTCA) and the exchange rate between alpha-ketoglutarate and glutamate (F1) were determined by least-square fitting of a kinetic model to NMR data. Rates were compared to measured kinetics of the cardiac glutamate-oxaloacetate transaminase (GOT). Despite similar oxygen use, hearts oxidizing butyrate instead of acetate showed delayed incorporation of 13C label into glutamate and lower VTCA, because of the influence of beta-oxidation: butyrate = 7.1 +/- 0.2 mumol/min/g dry wt; acetate = 10.1 +/- 0.2; butyrate + KCl = 1.8 +/- 0.1; acetate + KCl = 3.1 +/- 0.1 (mean +/- SD). F1 ranged from a low of 4.4 +/- 1.0 mumol/min/g (butyrate + KCl) to 9.3 +/- 0.6 (acetate), at least 20-fold slower than GOT flux, and proved to be rate limiting for isotope turnover in the glutamate pool. Therefore, dynamic 13C NMR observations were sensitive not only to TCA cycle flux but also to the interconversion between TCA cycle intermediates and glutamate.     

13C-NMR spectroscopic evaluation of the citric acid cycle flux in conditions of high aspartate transaminase activity in glucose-perfused rat hearts

A new mathematical model, based on the observation of 13C-NMR spectra of two principal metabolites (glutamate and aspartate), was constructed to determine the citric acid cycle flux in the case of high aspartate transaminase activity leading to the formation of large amounts of labeled aspartate and glutamate. In this model, the labeling of glutamate and aspartate carbons by chemical and isotopic exchange with the citric acid cycle are considered to be interdependent. With [U-13C]Glc or [1,2-(13)C]acetate as a substrate, all glutamate and aspartate carbons can be labeled. The isotopic transformations of 32 glutamate isotopomers into 16 aspartate isotopomers or vice versa were studied using matrix operations; the results were compiled in two matrices. We showed how the flux constants of the citric acid cycle and the 13C-enrichment of acetyl-CoA can be deduced from 13C-NMR spectra of glutamate and/or aspartate. The citric acid cycle flux in beating Wistar rat hearts, aerobically perfused with [U-13C]glucose in the absence of insulin, was investigated by 13C-NMR spectroscopy. Surprisingly, aspartate instead of glutamate was found to be the most abundantly-labeled metabolite, indicating that aspartate transaminase (which catalyses the reversible reaction: (glutamate + oxaloacetate <--> 2-oxoglutarate + aspartate) is highly active in the absence of insulin. The amount of aspartate was about two times larger than glutamate. The quantities of glutamate (G0) or aspartate (A0) were approximately the same for all hearts and remained constant during perfusion: G0 = (0.74 +/- 0.03) micromol/g; A0 = (1.49 +/- 0.05) micromol/g. The flux constants, i.e., the fraction of glutamate and aspartate in exchange with the citric acid cycle, were about 1.45 min(-1) and 0.72 min(-1), respectively; the flux of this cycle is about (1.07 +/- 0.02) micromol min(-1) g(-1). Excellent agreement between the computed and experimental data was obtained, showing that: i) in the absence of insulin, only 41% of acetyl-CoA is formed from glucose while the rest is derived from endogenous substrates; and ii) the exchange between aspartate and oxaloacetate or between glutamate and 2-oxoglutarate is fast in comparison with the biological transformation of intermediate compounds by the citric acid cycle.     

A prospective, multicenter study of a pneumonia practice guideline

Two-dimensional 1H detected 13C NMR spectroscopy has been used to study the intracellular metabolism of [3-(13)C]pyruvate in Halobacterium salinarium. The method, resulting in considerable improvement in spectral resolution and signal-to-noise ratio, is well suited for studying transient metabolic intermediates. Pyruvate utilization by the bacterium is a double exponential function with rate constants of 49.13 and 4.67x10(-3) per min. The relative 13C enrichment is the fastest for C-3 glutamate. Glutamate C-4 labeling decreases initially and increases later on during incubation, while glutamine C-3 is high to begin with and exhibits a declining trend. The glutamate labeling indicates a high initial flux through pyruvate carboxylase and extensive randomizing of the label in the tricarboxylic acid cycle.     

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.     

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.     

Involvement of Fas/Fas ligand system-mediated apoptosis in the development of concanavalin A-induced hepatitis

A series of 2H- and 13C-labeled glutamates were used as substrates for coenzyme B12-dependent glutamate mutase, which equilibrates (S)-glutamate with (2S,3S)-3-methylaspartate. These compounds contained the isotopes at C-2, C-3, or C-4 of the carbon chain: [2-2H], [3,3-2H2], [4,4-2H2], [2,3,3,4,4-2H5], [2-13C], [3-13C], and [4-13C]glutamate. Each reaction was monitored by electron paramagnetic resonance (EPR) spectroscopy and revealed a similar signal characterized by g'xy = 2.1, g'z = 1.985, and A' = 5.0 mT. The interpretation of the spectral data was aided by simulations which gave close agreement with experiment. This approach underpinned the idea of the formation of a radical pair, consisting of cob(II)alamin interacting with an organic radical at a distance of 6.6 +/- 0.9 A. Comparison of the hyperfine couplings observed with unlabeled glutamate with those from the labeled glutamates enabled a principal contributor to the radical pair to be identified as the 4-glutamyl radical. These findings support the currently accepted mechanism for the glutamate mutase reaction, i.e., the process is initiated through hydrogen atom abstraction from C-4 of glutamate by the 5'-deoxyadenosyl radical, which is derived by homolysis of the Co-C sigma-bond of coenzyme B12.     

Biosynthesis of bikaverin in Fusarium oxysporum. Use of 13C nuclear magnetic resonance with homonuclear 13C decoupling to locate adjacent 13C labels

Bikaverin obtained by supplementing cultures of Fusarium oxysporum with singly and doubly 13C labeled acetate was enriched by approximately 0.5 atom percent with the 13C isotope. At this low enrichment 13C NMR spectra of samples labeled from (1-13C)- and (2-13C) acetate did not show, unequivocally, the pattern of isotopic incorporation. Small sample size, poor solubility and difficulties in the assignment of resonances also restricted the amount of information thacetate. The difficulty was overcome by using 13C homonuclear single-frequency decoupling in conjunction with 1H heteronuclear decoupling to locate bonded 13C-13C pairs. The carbon skeleton of bikaverin was shown to be biosynthesized entirely by condensation of acetate units and the pattern of assembly was established.     

Tricarboxylic acid cycle in rat brain synaptosomes. Fluxes and interactions with aspartate aminotransferase and malate/aspartate shuttle

The flux through different segments of the tricarboxylic acid cycle was measured in rat brain synaptosomes with gas chromatography-mass spectrometry using either deuterated glutamine or [13C]aspartate. The flux between 2-oxoglutarate and oxaloacetate was estimated to be 3.14 and 4.97 nmol/min/mg protein with and without glucose, respectively. These values were 3-5-fold faster than the flux between oxaloacetate and 2-oxoglutarate (0.92 nmol/min per mg protein) measured in the presence of glucose. The pattern of intermediates labeling suggests that the overall rate-controlling reaction involves either citrate synthase or pyruvate dehydrogenase but not 2-oxoglutarate or isocitrate dehydrogenase. The enrichment in [3,3,4,4-2H4]glutamate from [2,3,3,4,4-2H5]glutamine was as rapid as in [2,3,3,4,4-2H5]glutamate, which indicates that the aspartate aminotransferase reaction is severalfold faster than the flux through the tricarboxylic acid cycle. [13C]Aspartate was rapidly converted to [13C]malate, suggesting that in intact synaptosomes aspartate entry into the mitochondrion is very slow. The finding that aspartate is taken up by mitochondria as malate, along with the observed high enrichment in [3-2H]malate (from [2,3,3,4,4-2H5]glutamine), is consistent with the substantial synaptosomal activity of the malate/aspartate shuttle.     

Glucose and lactate metabolism in C6 glioma cells: evidence for the preferential utilization of lactate for cell oxidative metabolism

13C and 1H nuclear magnetic resonance spectroscopy (NMR) was used to investigate the metabolism of L-lactate and D-glucose in C6 glioma cells. The 13C enrichment of cell metabolites was examined after a 4-h incubation in media containing 5.5 mM glucose and 11 mM lactate, each metabolite being alternatively labelled with either [1-13C]D-glucose or [3-13C]L-lactate. The results indicated that exogenous lactate was the major substrate for oxidative metabolism. They were consistent with the concept of the existence of 2 pools of both lactate and pyruvate, of which 1 pool was closely connected with exogenous lactate and oxidative metabolism, and the other pool was closely related to glycolysis and disconnected from oxidative metabolism. The molecular basis of this behaviour could be related to different locations for the lactate dehydrogenase isoenzymes, as suggested by their immunohistochemical labelling.     

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.     

NMR spectroscopy of cultured astrocytes: effects of glutamine and the gliotoxin fluorocitrate

Glial synthesis of glutamine, citrate, and other carbon skeletons, as well as metabolic effects of the gliotoxin fluorocitrate, were studied in cultured astrocytes with 13C and 31P NMR spectroscopy. [2-13C]Acetate and [1-13C]glucose were used as labeled precursors. In some experiments glutamine (2.5 mM) was added to the culture medium. Fluorocitrate (20 microM) inhibited the tricarboxylic acid (TCA) cycle without affecting the level of ATP. The net export of glutamine was reduced significantly, and that of citrate increased similarly, consistent with an inhibition of aconitase. Fluorocitrate (100 microM) inhibited TCA cycle activity even more and (without addition of glutamine) caused a 40% reduction in the level of ATP. In the presence of 2.5 mM glutamine, 100 microM fluorocitrate did not affect ATP levels, although glutamine synthesis was nearly fully blocked. The consumption of the added glutamine increased with increasing concentrations of fluorocitrate, whereas the consumption of glucose decreased. This shows that glutamine fed into the TCA cycle, substituting for glucose as an energy substrate. These findings may explain how fluorocitrate selectively lowers the level of glutamine and inhibits glutamine formation in the brain in vivo, viz., not by depleting glial cells of ATP, but by causing a rerouting of 2-oxoglutarate from glutamine synthesis into the TCA cycle during inhibition of aconitase. Analysis of the 13C labeling of the C-2 versus the C-4 positions in glutamine obtained with [2-13C]acetate revealed that 57% of the TCA cycle intermediates were lost per turn of the cycle.(ABSTRACT TRUNCATED AT 250 WORDS)