Localized 13C NMR Spectroscopy in the Human Brain of Amino Acid Labeling from d-[1-13C]Glucose

Abstract: Cerebral metabolism of d [1-¹³C]glucose was studied with localized ¹³C NMR spectroscopy during intravenous infusion of enriched [1-¹³C]glucose in four healthy subjects. The use of three-dimensional localization resulted in the complete elimination of triacylglycerol resonance that originated in scalp and subcutaneous fat. The sensitivity and resolution were sufficient to allow 4 min of time-resolved observation of label incorporation into the C3 and C4 resonances of glutamate and C4 of glutamine, as well as C3 of aspartate with lower time resolution. [4-¹³C]Glutamate labeled rapidly reaching close to maximum labeling at 60 min. The label flow into [3-¹³C]glutamate clearly lagged behind that of [4-¹³C]glutamate and peaked at t = 110–140 min. Multiplets due to homonuclear ¹³C-¹³C coupling between the C3 and C4 peaks of the glutamate molecule were observed in vivo. Isotopomer analysis of spectra acquired between 120 and 180 min yielded a ¹³C isotopic fraction at C4 glutamate of 27 ± 2% (n = 4), which was slightly less than one-half the enrichment of the C1 position of plasma glucose (63 ± 1%), p < 0.05. By comparison with an external standard the total amount of [4-¹³C]glutamate was directly quantified to be 2.4 ± 0.1 µmol/ml-brain. Together with the isotopomer data this gave a calculated brain glutamate concentration of 9.1 ± 0.7 µmol/ml, which agrees with previous estimates of total brain glutamate concentrations. The agreement suggests that essentially all of the brain glutamate is derived from glucose in healthy human brain.     

Carbon-13 nuclear magnetic resonance studies of myocardial glycogen metabolism in live guinea pigs

Myocardial glycogen metabolism was studied in live guinea pigs by 13C NMR at 20.19 MHz. Open-chest surgery was used to expose the heart, which was then positioned within a solenoidal radio frequency coil for NMR measurements. The time course of myocardial glycogen synthesis during 1-h infusions of 0.5 g of D-[1-13C]glucose (and insulin) into the jugular vein was investigated. The possible turnover of the 13C-labeled glycogen was also studied in vivo by following the labeled glucose infusion with a similar infusion of unlabeled glucose. The degree of 13C enrichment of the C-1 glycogen carbons during these infusions was measured in heart extracts by 1H NMR at 360 MHz. High-quality proton-decoupled 13C NMR spectra of the labeled C-1 carbons of myocardial glycogen in vivo were obtained in 1 min of data accumulation. This time resolution allowed measurement of the time course of glycogenolysis of the 13C-labeled glycogen during anoxia by 13C NMR in vivo. With the solenoidal coil used for 13C NMR, the spin-lattice relaxation time of the labeled C-1 carbons of myocardial glycogen could be measured in vivo. For a comparison, spin-lattice relaxation times of heart glycogen were measured in vitro at 90.55 MHz. Natural abundance 13C NMR studies of the quantitative hydrolysis of extracted heart glycogen in vitro at 90.55 MHz showed that virtually all the carbons in heart glycogen contribute to the 13C NMR signals. The same result was obtained in 13C NMR studies of glycogen hydrolysis in excised guinea pig heart.     

Direct observation of glycogenesis and glucagon-stimulated glycogenolysis in the rat liver in vivo by high-field carbon-13 surface coil NMR

High-field 13C surface coil nuclear magnetic resonance has been employed to investigate glucose and glycogen metabolism in rat liver in vivo. Natural abundance and isotopically enriched proton-decoupled 13C NMR experiments were conducted at 90.56 MHz on a standard commercial spectrometer utilizing a laboratory-built high-sensitivity double-resonance coaxial coil probe. At variance with a previous preliminary report, natural abundance spectra of the liver in vivo from a rat fed ad libitum reveal resonances of substantial intensity from hepatic glycogen with approximately 10 min of signal averaging. The response of hepatic glycogen levels to an intravenous injection of the hormone glucagon was continuously monitored through the glycogen C-1 carbon resonance intensity; this revealed an average 60% depletion of hepatic glycogen stores in vivo within approximately 1 h. In a complementary study utilizing fasted rats, 100 mg of D-[1-13C]glucose (90% enriched) was administered via a peripheral vein injection and continuously monitored by 13C NMR with 3-min time resolution as it was incorporated into hepatic glycogen. The C-1 carbon resonances of hepatic glucose and glycogen are well-resolved in vivo enabling the time course for the relative change in concentration for both metabolites to be established simultaneously. The 13C label incorporated into the glycogen pool reaches a steady-state level in approximately 40 min.     

Direct,noninvasive measurement of brain glycogen metabolism in humans

The concentration and metabolism of the primary carbohydrate store in the brain, glycogen, is unknown in the conscious human brain. This study reports the first direct detection and measurement of glycogen metabolism in the human brain, which was achieved using localized 13C NMR spectroscopy. To enhance the NMR signal, the isotopic enrichment of the glucosyl moieties was increased by administration of 80 g of 99% enriched [1-13C]glucose in four subjects. 3 h after the start of the label administration, the 13C NMR signal of brain glycogen C1 was detected (0.36+/-0.07 micromol/g, mean+/-S.D., n=4). Based on the rate of 13C label incorporation into glycogen and the isotopic enrichment of plasma glucose, the flux through glycogen synthase was estimated at 0.17+/-0.05 micromol/(gh). This study establishes that brain glycogen can be measured in humans and indicates that its metabolism is very slow in the conscious human. The noninvasive detection of human brain glycogen opens the prospect of understanding the role and function of this important energy reserve under various physiological and pathophysiological conditions.     

The metabolism of [3-(13)C]lactate in the rat brain is specific of a pyruvate carboxylase-deprived compartment

Lactate metabolism in the adult rat brain was investigated in relation with the concept of lactate trafficking between astrocytes and neurons. Wistar rats were infused intravenously with a solution containing either [3-(13)C]lactate (534 mM) or both glucose (750 mM) and [3-(13)C]lactate (534 mM). The time courses of both the concentration and (13)C enrichment of blood glucose and lactate were determined. The data indicated the occurrence of [3-(13)C]lactate recycling through liver gluconeogenesis. The yield of glucose labeling was, however, reduced when using the glucose-containing infusate. After a 20-min or 1-h infusion, perchloric acid extracts of the brain tissue were prepared and subsequently analyzed by (13)C- and (1)H-observed/(13)C-edited NMR spectroscopy. The (13)C labeling of amino acids indicated that [3-(13)C]lactate was metabolized in the brain. Based on the alanine C3 enrichment, lactate contribution to brain metabolism amounted to 35% under the most favorable conditions used. By contrast with what happens with [1-(13)C]glucose metabolism, no difference in glutamine C2 and C3 labeling was evidenced, indicating that lactate was metabolized in a compartment deprived of pyruvate carboxylase activity. This result confirms, for the first time from an in vivo study, that lactate is more specifically a neuronal substrate.     

Measurement of amino acid metabolism derived from [1-13C]glucose in the rat brain using13C magnetic resonance spectroscopy

To clarify the unique characteristics of amino acid metabolism derived from glucose in the central nervous system (CNS), we injected [1-¹³C]glucose intraperitoneally to the rat, and extracted the free amino acids from several kinds of tissues and measured the amount of incorporation of¹³C derived from [1-¹³C]glucose into each amino acid using¹³C-magnetic resonance spectroscopy (NMR). In the adult rat brain, the intensities of resonances from¹³C-amino acids were observed in the following order: glutamate, glutamine, aspartate, -aminobutyrate (GABA) and alanine. There seemed no regional difference on this labeling pattern in the brain. However, only in the striatum and thalamus, the intensities of resonances from [2-¹³C]GABA were larger than that from [2,3-¹³C]aspartate. In the other tissues, such as heart, kidney, liver, spleen, muscle, lung and small intestine, the resonances from GABA were not detected and every intensity of resonances from¹³C-amino acids, except¹³C-alanine, was much smaller than those in the brain and spinal cord. In the serum,¹³C-amino acid was not detected at all. When the rats were decapitated, in the brain, the resonances from [1-¹³C]glucose greatly reduced and the intensities of resonances from [3-¹³C]lactate, [3-¹³C]alanine, [2, 3, 4-¹³C]GABA and [2-¹³C]glutamine became larger as compared with those in the case that the rats were sacrificed with microwave. In other tissues, the resonances from [1-¹³C]glucose were clearly detected even after the decapitation. In the glioma induced by nitrosoethylurea in the spinal cord, the large resonances from glutamine and alanine were observed; however, the intensities of resonances from glutamate were considerably reduced and the resonances from GABA and aspartate were not detected. These results show that the pattern of¹³C label incorporation into amino acids is unique in the central nervous tissues and also suggest that the metabolic compartmentalization could exist in the CNS through the metabolic trafficking between neurons and astroglia.Abbreviations NMR nuclear magnetic resonance - GABA -aminobutyrate - GFAP glial fibrillary acidic protein Special issue dedicated to Dr. Bernard W. Agranoff.     

Metabolism of [1-(13)C)glucose and [2-(13)C]acetate in the hypoxic rat brain.

The effects of hypoxia on the metabolism of the central nervous system were investigated in rats submitted to a low oxygen atmosphere (8% O(2); 92% N(2)). [1-(13)C]glucose and [2-(13)C]acetate were used as substrates, this latter being preferentially metabolized by glial cells. After 1-h substrate infusion, the incorporation of 13C in brain metabolites was determined by NMR spectroscopy. Under hypoxia, an important hyperglycemia was noted. As a consequence, when using labeled glucose, the specific enrichment of brain glucose C1 was lower (48.2+/-5.1%) than under normoxia (66.9+/-2.5%). However, relative to this specific enrichment, the (13)C incorporation in amino acids was increased under hypoxia. This suggested primarily a decreased exchange between blood and brain lactate. The glutamate C2/C4 enrichment ratio was higher under hypoxia (0.62+/-0.01) than normoxia (0.51+/-0.06), indicating a lower glutamate turnover relative to the neuronal TCA cycle activity. The glutamine C2/C4 enrichment ratio was also higher under hypoxia (0.87+/-0.07 instead of 0.65+/-0.11), indicating a new balance in the contributions of different carbon sources at the acetyl-CoA level. When using [2-(13)C]acetate as substrate, no difference in glutamine enrichment appeared under hypoxia, whereas a significant decrease in glutamate, aspartate, alanine and lactate enrichments was noted. This indicated a lower trafficking between astrocytes and neurons and a reduced tricarboxylic acid cycle intermediate recycling of pyruvate.     

Brain pyruvate recycling and peripheral metabolism: an NMR analysis ex vivo of acetate and glucose metabolism in the rat

The occurrence of pyruvate recycling in the rat brain was studied in either pentobarbital anesthetized animals or awake animals receiving a light analgesic dose of morphine, which were infused with either [1-13C]glucose + acetate or glucose + [2-13C]acetate for various periods of time. Metabolite enrichments in the brain, blood and the liver were determined from NMR analyses of tissue extracts. They indicated that: (i) Pyruvate recycling was revealed in the brain of both the anesthetized and awake animals, as well as from lactate and alanine enrichments as from glutamate isotopomer composition, but only after infusion of glucose + [2-13C]acetate. (ii) Brain glucose was labelled from [2-13C]acetate at the same level in anaesthetized and awake rats (approximately 4%). Comparing its enrichment with that of blood and liver glucose indicated that brain glucose labelling resulted from hepatic gluconeogenesis. (iii) Analysing glucose 13C-13C coupling in the brain, blood and the liver confirmed that brain glucose could be labelled in the liver through the activities of both pyruvate recycling and gluconeogenesis. (iv) The rate of appearance and the amount of brain glutamate C4-C5 coupling, a marker of pyruvate recycling when starting from [2-13C]acetate, were lower than those of brain glucose labelling from hepatic metabolism. (v) The evaluation of the contributions of glucose and acetate to glutamate metabolism revealed that more than 60% of brain glutamate was synthesized from glucose whereas only 7% was from acetate and that glutamate C4-C5 coupling was mainly due to the metabolism of glucose labelled through hepatic gluconeogenesis. All these results indicate that, under the present conditions, the pyruvate recycling observed through the labelling of brain metabolites mainly originates from peripheral metabolism.     

Hepatic glycogen synthesis from duodenal glucose and alanine. An in situ 13C NMR study

An in situ and in vivo surface coil 13C NMR study was performed to study hepatic glycogen synthesis from [3-13C]alanine and [1-13C]glucose administered by intraduodenal infusion in 18-h fasted male Sprague-Dawley rats. Combined, equimolar amounts of alanine and glucose were given. Hepatic appearance and disappearance of substrate and concurrent glycogen synthesis was followed over 150 min, with 5-min time resolution. Active glycogen synthesis from glucose via the direct (glucose----glycogen) and indirect (glucose----lactate----glycogen) pathways and from alanine via gluconeogenesis was observed. The indirect pathway of glycogen synthesis from [1-13C]glucose accounted for 30% (+/- 6 S.E.) of total glycogen formed from labeled glucose. This estimate does not take into account dilution of label in the hepatic oxaloacetate pool and is, therefore, somewhat uncertain. Hepatic levels of [3-13C]alanine achieved were significantly lower than levels of [1-13C]glucose in the liver, and the period of active glycogen synthesis from [3-13C]alanine was longer than from glucose. However, the overall pseudo-first-order rate constant during the period of active glycogen synthesis from [3-13C]alanine (0.075 min-1 +/- 0.026 S.E.) was almost 3 times that from [1-13C]glucose via the direct pathway (0.025 min-1 +/- 0.005 S.E.). The most likely reason for the small rate constant governing direct glycogen formation from duodenally administered glucose compared to that from duodenally administered alanine is a low level of glucose phosphorylating capacity in the liver.     

Glycogen metabolism as detected by in vivo and in vitro 13C-NMR spectroscopy using [1,2-13C2]glucose as substrate

The metabolism of glucose to glycogen in the liver of fasted and well-fed rats was investigated with 13C nuclear magnetic resonance spectroscopy using [1,2-(13)C2]glucose as the main substrate. The unique spectroscopic feature of this molecule is the 13C-13C homonuclear coupling leading to characteristic doublets for the C-1 and C-2 resonances of glucose and its breakdown products as long as the two 13C nuclei remain bonded together. The doublet resonances of [1,2-(13)C2]glucose thus provide an ideal marker to follow the fate of this exogenous substrate through the metabolic pathways. [1,2-(13)C2]Glucose was injected intraperitoneally into anesthetized rats and the in vivo 13C-NMR measurements of the intact animals revealed the transformation of the injected glucose into liver glycogen. Glycogen was extracted from the liver and high resolution 13C-NMR spectra were obtained before and after hydrolysis of glycogen. Intact [1,2-13C2]glucose molecules give rise to doublet resonances, natural abundance [13C]glucose molecules produce singlet resonances. From an analysis of the doublet-to-singlet intensities the following conclusions were derived. (i) In fasted rats virtually 100% of the glycosyl units in glycogen were 13C-NMR visible. In contrast, the 13C-NMR visibility of glycogen decreased to 30-40% in well-fed rats. (ii) In fed rats a minimum of 67 +/- 7% of the exogenous [1,2-(13)C2]glucose was incorporated into the liver glycogen via the direct pathway. No contribution of the indirect pathway could be detected. (iii) In fasted rats externally supplied glucose appeared to be consumed in different metabolic processes and less [1,2-(13)C2]glucose was found to be incorporated into glycogen (13 +/- 1%). However, the observation of [5,6-(13)C2]glucose in liver glycogen provided evidence for the operation of the so-called indirect pathway of glycogen synthesis. The activity of the indirect pathway was at least 9% but not more than 30% of the direct pathway. (vi) The pentose phosphate pathway was of little significance for glucose but became detectable upon injection of [1-(13)C]ribose.     

Beta-adrenergic stimulation of C6 glioma cells: effects of cAMP overproduction on cellular metabolites. A multinuclear NMR study.

We used 31P-NMR spectroscopy to investigate the response of living C6 glioma cells to stimulation by a beta-adrenergic agonist, isoproterenol. In the presence of 3-isobutyl-1-methylxanthine, stimulation induced an accumulation of cAMP, making possible the NMR detection of the second messenger in living cells grown on microcarrier beads and perfused in the NMR tube. The cAMP signal rose to a maximum level within 20-25 min of stimulation; thereafter it decreased to the detection threshold within 60 min. At the same time, 40% increases of phosphomonoester and diphosphodiester signals were observed, whereas no significant change in phosphocreatine and nucleotide signals was detected. The kinetics of changes of the cellular content in phosphorylated metabolites were analyzed after recording 31P-NMR spectra of cell perchloric acid extracts as a function of time of stimulation. cAMP accumulation in stimulated cells was evidenced by a near linear increase of its NMR signal as a function of incubation time (from 0 to 60 min). Concomitantly with the production of cAMP, the data showed 30% decreases of phosphocreatine and ATP levels within 60 min of stimulation, and an unexpected redistribution of pyrimidine and purine nucleoside triphosphates. At the same time, levels of phosphomonoesters (phosphorylcholine and phosphorylethanolamine) and phosphodiesters (glycerophosphorylcholine and glycerophosphorylethanolamine) rose (50% increase). 13C-NMR spectra of cell perchloric acid extracts prepared after isoproterenol stimulation of cells incubated in the presence of [1-13C]glucose indicated a higher glucose content in stimulated cells, whereas the resonance of ribose C1 was diminished. Moreover, the resonances of C1 of ethanolamine and choline (and their derivatives) were increased in spectra of stimulated cells, whereas that of C3 of serine was decreased. In addition, the 13C-NMR data indicated that neither the pattern of glutamate carbon enrichment nor the glutamate/glutamine ratio was modified in stimulated cells. On the other hand, the heteronuclear coupling pattern of the lactate (methyl group) resonance in 1H-NMR spectra of cell incubation media indicated that no change occurred in the carbon flux through the pentose-phosphate shunt under stimulation. The results of this multinuclear NMR approach are discussed in terms of metabolic responses of C6 cells to beta-adrenergic stimulation and cAMP overproduction.     

13C NMR evidence of the failure of human erythrocytes to metabolize ascorbate and dehydroascorbate to lactate

13C-NMR spectroscopy was used to record time courses of the metabolism of [1-(13)C]-L-ascorbic acid (AA) and [2-(13)C]-L-ascorbic acid and their dehydro-counterparts (DHAA) by human erythrocytes. Under a range of experimental conditions, but most notably in the absence of glucose in the incubation medium, no (13)C-NMR signal for lactate emerged during any of the 5 h time courses. The NMR resonances that did emerge over time were assigned to diketogulonic (DKG) acid and CO(2). Only very minor resonances from degradation products of DKG appeared from samples that contained physiologically high concentrations of DHAA. These results are in contrast with those in a recent report that lactate is derived from AA in human erythrocytes. However, an explanation for this possible artifact is given.     

13C-NMR study of hypoglycemia-induced glycolytic changes in embryonic mouse heart

BACKGROUND: Glucose metabolites can be detected in embryonic mouse tissues using 13C-NMR spectroscopy. The advantage of this method is in its chemical specificity and the ability to follow metabolic changes. METHODS: In this study, CD-1 mice were mated and embryos excised on gestational day (GD) 10.5 (plug = GD 0.5). Hearts were isolated and cultured in 150 mg/dl glucose (normoglycemic medium) or 40 mg/dl glucose (hypoglycemic medium) for 6 hr. 13C-labeled glucose comprised 62%-64% of total glucose in the culture medium. Pre- and postculture media were treated with deuterated water (D2O), and 13C spectra were obtained using a Bruker Avance 500 MHz spectrometer operating at 11.744 tesla (125.7 MHz for 13C). NMR spectra demonstrated resonances for 13C-glucose in preculture normoglycemic and hypoglycemic media. Postculture spectra for normoglycemic and hypoglycemic media demonstrated 13C-glucose signals as well as a signal for 13C-lactate. Area under the curve (AUC) was measured for the [1-(13)C-glucose] resonance from preculture media and the [3-(13)C-lactate] resonance from postculture media. The ratios of AUC for postculture [3-(13)C-lactate] to preculture [1-(13)C-glucose] were calculated and found to be higher in hypoglycemic than in normoglycemic media. RESULTS: Our results confirm earlier findings using radiolabeled substrates and suggest that 13C-NMR spectroscopy can be used to study glucose metabolism in isolated embryonic hearts exposed to hypoglycemia. CONCLUSIONS: NMR effectively measures glucose and its metabolite, lactate, in the same spectrum and thus determines metabolic flux in the isolated embryonic heart after exposure to hypoglycemia and normoglycemia. This method could evaluate glucose metabolism in embryonic tissues following other teratogenic exposures.     

Metabolism of D-glucose in a wall-less mutant of Neurospora crassa examined by 13C and 31P nuclear magnetic resonances: effects of insulin

13C NMR and 31P NMR have been used to investigate the metabolism of glucose by a wall-less strain of Neurospora crassa (slime), grown in a supplemented nutritionally defined medium and harvested in the early stationary stage of growth. With D-[1-13C]- or D-[6-13C]glucose as substrates, the major metabolic products identified from 13C NMR spectra were [2-13C]ethanol, [3-13C]alanine, and C1- and C6-labeled trehalose. Several observations suggested the existence of a substantial hexose monophosphate (HMP) shunt: (i) a 70% greater yield of ethanol from C6- than from C1-labeled glucose; (ii) C1-labeled glucose yielded 19% C6-labeled trehalose, while C6-labeled glucose yielded only 4% C1-labeled trehalose; (iii) a substantial transfer of 13C from C2-labeled glucose to the C2-position of ethanol. 31P NMR spectra showed millimolar levels of intracellular inorganic phosphate (Pi), phosphodiesters, and diphosphates including sugar diphosphates and polyphosphate. Addition of glucose resulted in a decrease in cytoplasmic Pi and an increase in sugar monophosphates, which continued for at least 30 min. Phosphate resonances corresponding to metabolic intermediates of both the glycolytic and HMP pathways were identified in cell extracts. Addition of insulin (100 nM) with the glucose had the following effects relative to glucose alone: (i) a 24% increase (P less than 0.01) in the rate of ethanol production; (ii) a 38% increase (P less than 0.05) in the rate of alanine production; (iii) a 27% increase (P less than 0.05) in the rate of glucose disappearance. Insulin thus increases the rates of production of ethanol and alanine in these cells, in addition to increasing production of CO2 and glycogen, as previously shown.     

Cerebral metabolism of [1,2-13C2]acetate as detected by in vivo and in vitro 13C NMR

The metabolism of [1,2-13C2]acetate in rat brain was studied by in vivo and in vitro 13C NMR spectroscopy, in particular by taking advantage of the homonuclear 13C-13C spin coupling patterns. Well nourished rats were infused with [1,2-13C2]acetate or [1-13C]acetate in the jugular vein, and the in situ kinetics of 13C labeling during the infusion period was followed by 13C NMR techniques. The in vivo 13C NMR spectra showed signals from (i) the C-1 carbon of [1,2-13C2] acetate or [1-13C]acetate, (ii) 13CO3H-, and (iii) the natural abundance 13C carbons of sufficiently mobile fatty acids. Methanol/HCl/perchloric acid extracts of the brains were prepared and were further analyzed by high resolution 13C NMR. The homonuclear 13C-13C spin coupling patterns after infusion of [1,2-13C2]acetate showed very different isotopomer populations in glutamate, glutamine, and gamma-aminobutyric acid. Analyzing the relative proportions of these isotopomers revealed (i) two different glutamate compartments in the rat brain characterized by the presence and absence, respectively, of glutamine synthase activity, (ii) two different tricarboxylic acid cycles, one preferentially metabolizing [(1,2-13C2]acetate, the other mainly using unlabeled acetyl-coenzyme A, (iii) a hitherto unknown cerebral pyruvate recycling system associated with the tricarboxylic acid cycle, metabolizing primarily unlabeled acetyl-coenzyme A, and (iv) a predominant production of gamma-aminobutyric acid in the glutamate compartment lacking glutamine synthase.     

Detergent delipidation and solubilization strategies for high-resolution NMR of the membrane protein bacteriorhodopsin

High-resolution NMR studies of bacteriorhodopsin require the availability of the detergent-solubilized protein with both high concentration and small rotational correlation time. A procedure is described for the optimized preparation of such samples. Bacteriorhodopsin was first delipidated by detergent treatment of purple membrane under nonsolubilizing conditions for the protein. The delipidated aggregated protein could then be solubilized into monomers at concentration close to millimolar by selected detergents. The solubilizing detergent had an important effect on the rotational correlation time of the protein as shown by measuring in each case the temperature-dependent stability of the protein, the size of the detergent-protein complex, and the detergent viscosity. Consistently, a strong influence of the detergent was also found on spectral resolution in 13C NMR spectra of solubilized bacteriorhodopsin labeled with [1-13C]phenylalanine. Best resolution was obtained using n-dodecylmaltoside as detergent, with which relatively narrow well resolved 13C NMR resonances were observed at 50 degrees C. It is suggested that high-resolution NMR studies performed with this detergent may contribute to the structural resolution of bacteriorhodopsin.     

Uniform 13C isotope labeling of proteins with sodium acetate for NMR studies: application to human carbonic anhydrase II

Uniform double labeling of proteins for NMR studies can be prohibitively expensive, even with an efficient expression and purification scheme, due largely to the high cost of [13C6, 99%]glucose. We demonstrate here that uniformly (greater than 95%) 13C and 15N double-labeled proteins can be prepared for NMR structure/function studies by growing cells in defined media containing sodium [1,2-13C2, 99%]acetate as the sole carbon source and [15N, 99%]ammonium chloride as the sole nitrogen source. In addition, we demonstrate that this labeling scheme can be extended to include uniform carbon isotope labeling to any desired level (below 50%) by utilizing media containing equal amounts of sodium [1-13C, 99%]acetate and sodium [2-13C, 99%]acetate in conjunction with unlabeled sodium acetate. This technique is less labor intensive and more straightforward than labeling using isotope-enriched algal hydrolysates. These labeling schemes have been used to successfully prepare NMR quantities of isotopically enriched human carbonic anhydrase II. The activity and the 1H NMR spectra of the protein labeled by this technique are the same as those obtained from the protein produced from media containing labeled glucose; however, the cost of the sodium [1,2-13C2, 99%]acetate growth media is considerably less than the cost of the [13C6, 99%]glucose growth media. We report here the first published 13C and 15N NMR spectra of human carbonic anhydrase II as an important step leading to the assignment of this 29-kDa zinc metalloenzyme.     

Real-time detection of 13C NMR labeling kinetics in perfused EMT6 mouse mammary tumor cells and betaHC9 mouse insulinomas

A method was developed for obtaining high signal-to-noise 13C NMR spectra of intracellular compounds in metabolically active cultured cells. The method allows TCA cycle labeling kinetics to be determined in real time without significant oxygen transport limitations. Cells were immobilized on the surface of nonporous microcarriers that were either uncoated or coated with polypeptides and used in a 12-cm3 packed bed. The methods were tested with two EMT6 mouse mammary tumor cell lines, one strongly adherent and the other moderately adherent, and a weakly adherent mouse insulinoma line (betaHC9). For both EMT6 lines, NTP and oxygen consumption measurements indicated that the number of cells in the spectrometer ranged from 6 x 10(8) to 1 x 10(9). During infusion of [1-13C]glucose, labeling in C-4 glutamate (indicative of flux into the first half of the TCA cycle) could be detected with 15-min resolution. However, labeling for C-3 and C-2 glutamate (indicative of complete TCA cycle activity) was fivefold lower and difficult to quantify. To increase TCA cycle labeling, cells were infused with medium containing [1,6-13C2]glucose. A 2.5-fold increase was observed in C-4 glutamate labeling and C-3 and C-2 glutamate labeling could be monitored with 30-min resolution. Citrate synthase activity was indirectly detected in real time, as [3,4-13C2]glutamate was formed from [2-13C]oxaloacetate and [2-13C]acetate (of acetyl-CoA). Cell mass levels observed with betaHC9 cells were somewhat lower. However, the 13C S/N was sufficient to allow real-time monitoring of the response of intracellular metabolite labeling to a step change in glucose and a combined glutamine/serum pulse.     

Delayed labelling of brain glutamate after an intra-arterial [13C]glucose bolus: evidence for aerobic metabolism of guinea pig brain glycogen store.

Glycogen in glial cells is the largest store of glucose equivalents in the brain. Here we describe evidence that brain glycogen contributes to aerobic energy metabolism of the guinea pig brain in vivo. Five min after an intra-arterial bolus injection of d-[U-14C]glucose, 28+/-11% of the radioactivity in brain tissue was associated with the glycogen fraction, indicating that a significant proportion of labelled glucose taken up by the brain is converted to glycogen shortly after bolus infusion. Incorporation of 13C-label into lactate generated by brains made ischaemic after d-[1-13C]glucose injection confirms that these glucose equivalents can be mobilised for anaerobic glucose metabolism. Aerobic metabolism was monitored by following the time course of 13C-incorporation into glutamate in guinea pig cortex and cerebellum in vivo. After an intra-arterial bolus injection of d-[1-13C]glucose, glutamate labelling reached a maximum 40-60 min after injection, suggesting that a slowly metabolised pool of labelled glucose equivalents was present. As the concentration of 13C-labelled glucose in blood was shown to decrease below detectable levels within 5 min of bolus injection, this late phase of glutamate labelling must occur with mobilisation of a brain storage pool of labelled glucose equivalents. We interpret this as evidence that glucose equivalents in glycogen may contribute to energy metabolism in the aerobic guinea pig brain.     

Magnetic resonance spectroscopy and metabolism. Applications of proton and 13C NMR to the study of glutamate metabolism in cultured glial cells and human brain in vivo.

Nuclear magnetic resonance (NMR) spectroscopy was used to study the metabolism of cells from the central nervous system both in vitro on perchloric acid extracts obtained either from cultured tumoral cells (C6 rat glioma) or rat astrocytes in primary culture, and in vivo within the human brain. Analysis of carbon 13 NMR spectra of perchloric acid extracts prepared from cultured cells in the presence of NMR [1-13C] glucose as substrate allowed determination of the glutamate and glutamine enrichments in both normal and tumoral cells. Preliminary results indicated large changes in the metabolism of these amino acids (and also of aspartate and alanine) in the C6 cell as compared to its normal counterpart. Localized proton NMR spectra of the human brain in vivo were obtained at 1.5 T, in order to evaluate the content of various metabolites, including glutamate, in peritumoral edema from a selected volume of 2 x 2 x 2 cm3. N-acetyl aspartate, glutamate, phosphocreatine, creatine, choline and inositol derivative resonances were observed in 15 min spectra. N-acetyl-aspartate was found to be at a lower level in contrast to glutamate which was detected at a higher level in the injured area as compared to the contralateral unaffected side.