Metabolism of [3-13C]pyruvate in TCA cycle mutants of yeast.

The utilization of pyruvate and acetate by Saccharomyces cerevisiae was examined using 13C and 1H NMR methodology in intact wild-type yeast cells and mutant yeast cells lacking Krebs tricarboxylic acid (TCA) cycle enzymes. These mutant cells lacked either mitochondrial (NAD) isocitrate dehydrogenase (NAD-ICDH1),alpha-ketoglutarate dehydrogenase complex (alpha KGDC), or mitochondrial malate dehydrogenase (MDH1). These mutant strains have the common phenotype of being unable to grow on acetate. [3-13C]-Pyruvate was utilized efficiently by wild-type yeast with the major intermediates being [13C]glutamate, [13C]acetate, and [13C]alanine. Deletion of any one of these Krebs TCA cycle enzymes changed the metabolic pattern such that the major synthetic product was [13C]galactose instead of [13C]glutamate, with some formation of [13C]acetate and [13C]alanine. The fact that glutamate formation did not occur readily in these mutants despite the metabolic capacity to synthesize glutamate from pyruvate is difficult to explain. We discuss the possibility that these data support the metabolon hypothesis of Krebs TCA cycle enzyme organization.     

Tetrapyrrole biosynthesis in Anacystis nidulans; incorporation of [1-13C]-, [2-13C]-, [1,2-13C]- and [2-13C, 2-2H3]acetate

Labelling experiments with [2-¹³C]- and [1,2-¹³C]acetate showed that both photopigments of Anacystis nidulans, chlorophyll a and phycocyanobilin, share a common biosynthetic pathway from glutamate. The fate of deuterium during these biosynthetic events was studied using [2-¹³C, 2-²H₃]acetate as a precursor and determining the labelling pattern by ¹³C NMR spectroscopy with simultaneous [¹H, ²H]-broadband decoupling. The loss of ²H (ca 20%) from the precursor occurred at an early stage during the tricarboxylic acid cycle. After formation of glutamate there was no further loss of ²H in the assembly of the cyclic tetrapyrrole intermediates or during decarboxylation and modification of the side-chains. Thus the labelling data support a divergence in the pathway to cyclic and linear tetrapyrroles after protoporphyrin IX.     

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.     

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.     

Metabolism of [1,6-13C]Glucose and [U-13C]Glutamine and Depolarization Induced GABA Release in Superfused Mouse Cerebral Cortical Mini-slices

Mouse cerebral cortical mini-slices were used in a superfusion system to monitor depolarization-induced (55 mM K⁺) release of preloaded [2,3-³H]GABA and to investigate the biosynthesis of glutamate, GABA and aspartate during physiological and depolarizing (55 mM K⁺) conditions from either [1,6-¹³C]glucose or [U-¹³C]glutamine. Depolarization-induced GABA release could be reduced (50%) by the GABA transport inhibitor tiagabine (25 μM) or by replacing Ca²⁺ with Co²⁺. In the presence of both tiagabine and Co²⁺ (1 mM), release was abolished completely. The release observed in the presence of 25 μM tiagabine thus represents vesicular release. Superfusion in the presence of [1,6-¹³C]glucose led to considerable labeling in the three amino acids, the labeling in glutamate and aspartate being increased after depolarization. This condition had no effect on GABA labeling. For all three amino acids, the distribution of label in the different carbon atoms revealed on increased tricarboxylic acid (TCA) activity during depolarization. When [U-¹³C]glutamine was used as substrate, labeling in glutamate was higher than that in GABA and aspartate and the fraction of glutamate and aspartate being synthesized by participation of the TCA cycle was increased by depolarization, an effect not seen for GABA. However, GABA synthesis reflected TCA cycle involvement to a much higher extent than for glutamate and aspartate. The results show that this preparation of brain tissue with intact cellular networks is well suited to study metabolism and release of neurotransmitter amino acids under conditions mimicking neural activity. Special issue article in honor of Dr. Ricardo Tapia.     

Metabolism of [U-<Superscript>13</Superscript>C]Glutamine and [U-<Superscript>13</Superscript>C]Glutamate in Isolated Rat Brain Mitochondria Suggests Functional Phosphate-Activated Glutaminase Activity in Matrix

One of the forms of phosphate activated glutaminase (PAG) is associated with the inner mitochondrial membrane. It has been debated whether glutamate formed from glutamine in the reaction catalyzed by PAG has direct access to mitochondrial or cytosolic metabolism. In this study, metabolism of [U-¹³C]glutamine (3 mM) or [U-¹³C]glutamate (10 mM) was investigated in isolated rat brain mitochondria. The presence of a functional tricarboxylic (TCA) cycle in the mitochondria was tested using [U-¹³C]succinate as substrate and extensive labeling in aspartate was seen. Accumulation of glutamine into the mitochondrial matrix was inhibited by histidine (15 mM). Extracts of mitochondria were analyzed for labeling in glutamine, glutamate and aspartate using liquid chromatography-mass spectrometry. Formation of [U-¹³C]glutamate from exogenous [U-¹³C]glutamine was decreased about 50% (P < 0.001) in the presence of histidine. In addition, the ¹³C-labeled skeleton of [U-¹³C]glutamine was metabolized more vividly in the tricarboxylic acid (TCA) cycle than that from [U-¹³C]glutamate, even though glutamate was labeled to a higher extent in the latter condition. Collectively the results show that transport of glutamine into the mitochondrial matrix may be a prerequisite for deamidation by PAG. Special issue article in honor of Dr. Frode Fonnum. Lasse K. Bak and Elżbieta Ziemińska contributed equally to the experimental work described in this paper.     

Effects of pentylenetetrazole and glutamate on metabolism of [U-(13)C]glucose in cultured cerebellar granule neurons.

This study was performed to analyze the effects of glutamate and the epileptogenic agent pentylenetetrazole (PTZ) on neuronal glucose metabolism. Cerebellar granule neurons were incubated for 2 h in medium containing 3 mM [U-(13)C]glucose, with and without 0.25 mM glutamate and/or 10 mM PTZ. In the presence of PTZ, decreased glucose consumption with unchanged lactate release was observed, indicating decreased glucose oxidation. PTZ also slowed down tricarboxylic acid (TCA) cycle activity as evidenced by the decreased amounts of labeled aspartate and [1,2-(13)C]glutamate. When glutamate was present, glucose consumption was also decreased. However, the amount of glutamate, derived from [U-(13)C]glucose via the first turn of the TCA cycle, was increased. The decreased amount of [1,2-(13)C]glutamate, derived from the second turn in the TCA cycle, and increased amount of aspartate indicated the dilution of label due to the entrance of unlabeled glutamate into TCA cycle. In the presence of glutamate plus PTZ, the effect of PTZ was enhanced by glutamate. Labeled alanine was detected only in the presence of glutamate plus PTZ, which indicated that oxaloacetate was a better amino acid acceptor than pyruvate. Furthermore, there was also evidence for intracellular compartmentation of oxaloacetate metabolism. Glutamate and PTZ caused similar metabolic changes, however, via different mechanisms. Glutamate substituted for glucose as energy substrate in the TCA cycle, whereas, PTZ appeared to decrease mitochondrial activity.     

Metabolism of (1-(13)C) glucose and (2-(13)C, 2-(2)H(3)) acetate in the neuronal and glial compartments of the adult rat brain as detected by [(13)C, (2)H] NMR spectroscopy

Ex vivo ?(13)C, (2)H? NMR spectroscopy allowed to estimate the relative sizes of neuronal and glial glutamate pools and the relative contributions of (1-(13)C) glucose and (2-(13)C, 2-(2)H(3)) acetate to the neuronal and glial tricarboxylic acid cycles of the adult rat brain. Rats were infused during 60 min in the right jugular vein with solutions containing (2-(13)C, 2-(2)H(3)) acetate and (1-(13)C) glucose or (2-(13)C, 2-(2)H(3)) acetate only. At the end of the infusion the brains were frozen in situ and perchloric acid extracts were prepared and analyzed by high resolution (13)C NMR spectroscopy (90.5 MHz). The relative sizes of the neuronal and glial glutamate pools and the contributions of acetyl-CoA molecules derived from (2-(13)C, (2)H(3)) acetate or (1-(13)C) glucose entering the tricarboxylic acid cycles of both compartments, could be determined by the analysis of (2)H-(13)C multiplets and (2)H induced isotopic shifts observed in the C4 carbon resonances of glutamate and glutamine. During the infusions with (2-(13)C, 2-(2)H(3)) acetate and (1-(13)C) glucose, the glial glutamate pool contributed 9% of total cerebral glutamate being derived from (2-(13)C, 2-(2)H(3)) acetyl-CoA (4%), (2-(13)C) acetyl-CoA (3%) and recycled (2-(13)C, 2-(2)H) acetyl-CoA (2%). The neuronal glutamate pool accounted for 91% of the total cerebral glutamate being mainly originated from (2-(13)C) acetyl-CoA (86%) and (2-(13)C, 2-(2)H) acetyl-CoA (5%). During the infusions of (2-(13)C, 2-(2)H(3)) acetate only, the glial glutamate pool contributed 73% of the cerebral glutamate, being derived from (2-(13)C, 2-(2)H(3)) acetyl-CoA (36%), (2-(13)C, 2-(2)H) acetyl-CoA (27%) and (2-(13)C) acetyl-CoA (10%). The neuronal pool contributed 27% of cerebral glutamate being formed from (2-(13)C) acetyl-CoA (11%) and recycled (2-(13)C, 2-(2)H) acetyl-CoA (16%). These results illustrate the potential of ?(13)C, (2)H? NMR spectroscopy as a novel approach to investigate substrate selection and metabolic compartmentation in the adult mammalian brain.     

A (13)C isotopomer kinetic analysis of cardiac metabolism: influence of altered cytosolic redox and [Ca(2+)](o)

Rat hearts were perfused with mixtures of [3-(13)C]pyruvate and [3-(13)C]lactate (to alter cytosolic redox) at low (0.5 mM) or high (2.5 mM) Ca(2+) concentrations to alter contractility. Hearts were frozen at various times after exposure to these substrates, were extracted, and were then analyzed by (13)C NMR spectroscopy. The time-dependent multiplets observed in the (13)C NMR resonances of glutamate in all hearts and in malate and aspartate in hearts perfused with high-pyruvate/low-lactate concentrations were analyzed using a kinetic model of the tricarboxylic acid (TCA) cycle. The analysis showed that TCA cycle flux (V(TCA)) and exchange flux (V(X)) that involved cycle intermediates were both sensitive to cell redox and altered Ca(2+) concentration, and the ratio of these fluxes (V(X)/V(TCA)) varied >10-fold.     

Recovery of (13)CO2 during rest and exercise after [1-(13)C]acetate, [2-(13)C]acetate, and NaH(13)CO3 infusions

For estimating the oxidation rates (Rox) of glucose and other substrates by use of (13)C-labeled tracers, we obtained correction factors to account for label dilution in endogenous bicarbonate pools and TCA cycle exchange reactions. Fractional recoveries of (13)C label in respiratory gases were determined during 225 min of rest and 90 min of leg cycle ergometry at 45 and 65% peak oxygen uptake (VO(2 peak)) after continuous infusions of [1-(13)C]acetate, [2-(13)C]acetate, or NaH(13)CO(3). In parallel trials, [6,6-(2)H]glucose and [1-(13)C]glucose were given. Experiments were conducted after an overnight fast with exercise commencing 12 h after the last meal. During the transition from rest to exercise, CO(2) production increased (P < 0.05) in an intensity-dependent manner. Significant differences were observed in the fractional recoveries of (13)C label as (13)CO(2) at rest (NaH(13)CO(3), 77.5 +/- 2.8%; [1-(13)C]acetate, 49.8 +/- 2.4%; [2-(13)C]acetate, 26.1 +/- 1.4%). During exercise, fractional recoveries of (13)C label from [1-(13)C]acetate, [2-(13)C]acetate, and NaH(13)CO(3) were increased compared with rest. Magnitudes of label recoveries during both exercise intensities were tracer specific (NaH(13)CO(3), 93%; [1-(13)C]acetate, 80%; [2-(13)C]acetate, 65%). Use of an acetate-derived correction factor for estimating glucose oxidation resulted in Rox values in excess (P < 0.05) of glucose rate of disappearance during hard exercise. We conclude that, after an overnight fast: 1) recovery of (13)C label as (13)CO(2) from [(13)C]acetate is decreased compared with bicarbonate; 2) the position of (13)C acetate label affects carbon dilution estimations; 3) recovery of (13)C label increases in the transition from rest to exercise in an isotope-dependent manner; and 4) application of an acetate correction factor in glucose oxidation measurements results in oxidation rates in excess of glucose disappearance during exercise at 65% of VO(2 peak). Therefore, bicarbonate, not acetate, correction factors are advocated for estimating glucose oxidation from carbon tracers in exercising men.     

TCA cycle kinetics in the rat heart by analysis of (13)C isotopomers using indirect (1)H

This study was designed to test the hypothesis that indirect (1)H[(13)C] detection of tricarboxylic acid (TCA) cycle intermediates using heteronuclear multiple quantum correlation-total correlation spectroscopy (HMQC-TOCSY) nuclear magnetic resonance (NMR) spectroscopy provides additional (13)C isotopomer information that better describes the kinetic exchanges that occur between intracellular compartments than direct (13)C NMR detection. NMR data were collected on extracts of rat hearts perfused at various times with combinations of [2-(13)C]acetate, propionate, the transaminase inhibitor aminooxyacetate, and (13)C multiplet areas derived from spectra of tissue glutamate were fit to a standard kinetic model of the TCA cycle. Although the two NMR methods detect different populations of (13)C isotopomers, similar values were found for TCA cycle and exchange fluxes by analyzing the two data sets. Perfusion of hearts with unlabeled propionate in addition to [2-(13)C]acetate resulted in an increase in the pool size of all four-carbon TCA cycle intermediates. This allowed the addition of isotopomer data from aspartate and malate in addition to the more abundant glutamate. This study illustrates that metabolic inhibitors can provide new insights into metabolic transport processes in intact tissues.     

Measurements of the anaplerotic rate in the human cerebral cortex using 13C magnetic resonance spectroscopy and [1-13C] and [2-13C] glucose

Recent studies in rodent and human cerebral cortex have shown that glutamate-glutamine neurotransmitter cycling is rapid and the major pathway of neuronal glutamate repletion. The rate of the cycle remains controversial in humans, because glutamine may come either from cycling or from anaplerosis via glial pyruvate carboxylase. Most studies have determined cycling from isotopic labeling of glutamine and glutamate using a [1-(13)C]glucose tracer, which provides label through neuronal and glial pyruvate dehydrogenase or via glial pyruvate carboxylase. To measure the anaplerotic contribution, we measured (13)C incorporation into glutamate and glutamine in the occipital-parietal region of awake humans while infusing [2-(13)C]glucose, which labels the C2 and C3 positions of glutamine and glutamate exclusively via pyruvate carboxylase. Relative to [1-(13)C]glucose, [2-(13)C]glucose provided little label to C2 and C3 glutamine and glutamate. Metabolic modeling of the labeling data indicated that pyruvate carboxylase accounts for 6 +/- 4% of the rate of glutamine synthesis, or 0.02 micromol/g/min. Comparison with estimates of human brain glutamine efflux suggests that the majority of the pyruvate carboxylase flux is used for replacing glutamate lost due to glial oxidation and therefore can be considered to support neurotransmitter trafficking. These results are consistent with observations made with arterial-venous differences and radiotracer methods.     

Compartmentation of metabolism probed by [2-13C]alanine: improved 13C NMR sensitivity using a CryoProbe detects evidence of a glial metabolon

The labelling of metabolites with the NMR active nucleus 13C allows not only metabolite enrichments to be monitored, but also the relative fluxes through competing pathways to be delineated. [2-13C, 15N]alanine was used as a metabolic probe to investigate compartmentation in superfused cerebral slices. Perchloric acid extracts of the tissue were investigated using 13C NMR spectroscopy. The spectra were obtained using a CryoProbe optimised for 13C detection (dual CryoProbe [13C, 1H]) in which the receiver and transmitter coils are cooled to approximately 20K to reduce contributions to noise in the signal obtained. Compared with conventional inverse geometry probe, the signal-to-noise ratio (S/N) was increased by approximately 17-fold using this device. A large proportion of alanine was initially metabolised over the first 20 min by glial cells, as indicated by the relative importance of the glial, only enzyme pyruvate carboxylase to the labelling pattern of glutamate, with the ratio of pyruvate carboxylase to pyruvate dehydrogenase derived glutamate being 0.25, and exported [2-13C, 15N]aspartate.Using the increased sensitivity of the CryoProbe, [2-13C, 15N]aspartate was also detected in the extracts of cerebral tissue. This metabolite could only have been derived via the pyruvate carboxylase pathway, and given the large export of the metabolite into the superfusion buffer suggests the occurrence of a "metabolon" arrangement of enzymes within glial cells.     

Compartmentation of citric acid cycle metabolism in brain: effect of aminooxyacetic acid, ouabain and Ca2+ on the labelling of glutamate, glutamine, aspartate and gaba by [1-14C]acetate, [U-14C]glutamate and [U-14C]asparate

—(1) The effects of aminooxyacetic acid, ouabain and Ca²⁺ on the compartmentation of amino acid metabolism have been studied in slices of brain incubated with sodium-[1-¹⁴C]acetate, l-[U-¹⁴C]glutamate and l-[U-¹⁴C]aspartate as tracer metabolites. (2) Aminooxyacetic acid (10^-3 m) inhibited the labelling of aspartate from [¹⁴C]acetate and [¹⁴C]glutamate, as well as the incorporation of label from [¹⁴C]aspartate into glutamate and glutamine. It also inhibited the labelling of GABA from all three radioactive precursors, as would be anticipated if there was inhibition of several transaminases as well as glutamate decarboxylase. The RSA of glutamine labelled from [1-¹⁴C]acetate was increased. This finding indicated that the glutamate pool which is utilized for glutamine formation is associated with glutamate dehydrogenase, and this enzyme appears to be related to the ‘synthetic tricarboxylic acid cycle’. AOAA exerted its major inhibitory effects on the citric acid‘energy cycle’with which transaminases are associated. (3) Ouabain (10^-5 m) inhibited the labelling of glutamine to a much greater extent than the labelling of glutamate from [1-¹⁴C]acetate. It also caused leakage of amino acids from the tissue into the medium. Its effect on the glutamate–glutamine system was interpreted to be a selective inhibition of the 'synthetic’citric acid cycle. (4) The omission of Ca²⁺ from the incubation medium was associated with formation of glutamine with RSA less than 1·0 when labelled from [U-¹⁴C]glutamate, [U-¹⁴C]aspartate and lower than normal when labelled from [1-¹⁴C]acetate.     

A 13C-NMR study on the influxes into the tricarboxylic acid cycle of a renal epithelial cell line, LLC-PK1/Cl4: the metabolism of [2-13C]glycine, L-[3-13C]alanine and L-[3-13C]aspartic acid in renal epithelial cells

Perchloric acid extracts of LLC-PK1/Cl4 cells, a renal epithelial cell line, incubated with either [2-13C]glycine L-[3-13C]alanine, or D,L-[3-13C]aspartic acid were investigated by 13C-NMR spectroscopy. All amino acids, except labelled glycine, gave rise to glycolytic products and tricarboxylic acid cycle (TCA) intermediates. For the first time we also observed activity of gamma-glutamyltransferase activity and glutathione synthetase activity in LLC-PK1 cells, as is evident from enrichment of reduced glutathione. Time courses showed that only 6% of the labelled glycine was utilized in 30 min, whereas 31% of L-alanine and 60% of L-aspartic acid was utilized during the same period. 13C-NMR was also shown to be a useful tool for the determination of amino acid uptake in LLC-PK1 cells. These uptake experiments indicated that glycine, alanine and aspartic acid are transported into Cl4 cells via a sodium-dependent process. From the relative enrichment of the glutamate carbons, we calculated the activity of pyruvate dehydrogenase to be about 61% when labelled L-alanine was the only carbon source for LLC-PK1/Cl4 cells. Experiments with labelled D,L-aspartic, however, showed that about 40% of C-3-enriched oxaloacetate (arising from a de-amination of aspartic acid) reached the pyruvate pool.     

Isotopic discrimination between D-[1-(13)C]fructose and D-[2-(13)C]fructose in rat liver cells

When liver cells from either normal or hereditarily diabetic rats are exposed to (13)C-enriched D-fructose (10 mM) and unlabelled D-glucose (also 10 mM) in the presence of D(2)O, the output of (13)C-enriched D-glucose generated from D-[1-(13)C]fructose is significantly lower than that from D-[2-(13)C]fructose. This coincides with a higher generation of (13)C-enriched L-lactate and L-alanine from D-[1-(13)C]fructose, as compared to D-[2-(13)C]fructose. In absolute terms, the mean paired difference in the output of (13)C-enriched D-glucose generated from D-[1-(13)C]fructose versus D-[2-(13)C]fructose is not significantly different from the mean paired difference in the production of (13)C-enriched L-lactate and L-alanine from the same precursors, with an overall mean value of 7.01 +/- 1.59 micromol (n = 8; P < 0.005). It is proposed that these findings indicate isotopic discrimination at the phosphoglucoisomerase level between (12)C and (13)C for the carbon atom in position 1 (as compared to that in position 2) of D-fructose 6-phosphate.     

Synthesis and structure determination of [8(-13)C]guanosine 5'-diphosphate

A combined chemical and enzymatic synthesis of [8(-13)C]guanosine 5'-diphosphate (GDP) from H13COOH is described. About 35 mg nucleotide was obtained from 500 mg H13COOH. Analysis of the [8(-13)C]GDP by negative ion fast atom bombardment mass spectrometry and by 13C NMR confirmed that one atom of 13C was incorporated at the 8-position of the guanine ring at 90 +/- 10% enrichment. The chemical shift of the C(8) was 140.2 ppm downfield from internal trimethylsilylpropionate at neutral pH and room temperature, with a spin-spin coupling 1J(13C(8)-H(8] of 217 Hz and a 3J(13C(8)-H(1'] of 3.9 Hz.     

Breath [13CO2] recovery from an oral glucose load during exercise: comparison between [U-13C] and [1,2-13C]glucose.

The purpose of the present experiment was to compare 13CO2 recovery at the mouth, and the corresponding exogenous glucose oxidation computed, during a 100-min exercise at 63 +/- 3% maximal O2 uptake with ingestion of glucose (1.75 g/kg) in six active male subjects, by use of [U-13C] and [1,2-13C]glucose. We hypothesized that 13C recovery and exogenous glucose oxidation could be lower with [1,2-13C] than [U-13C]glucose because both tracers provide [13C]acetate, with possible loss of 13C in the tricarboxylic acid (TCA) cycle, but decarboxylation of pyruvate from [U-13C]glucose also provides 13CO2, which is entirely recovered at the mouth during exercise. The recovery of 13C (25.8 +/- 2.3 and 27.4 +/- 1.2% over the exercise period) and the amounts of exogenous glucose oxidized computed were not significantly different with [1,2-13C] and [U-13C]glucose (28.9 +/- 2.6 and 30.7 +/- 1.3 g, between minutes 40 and 100), suggesting that no significant loss of 13C occurred in the TCA cycle. This stems from the fact that, during exercise, the rate of exogenous glucose oxidation is probably much larger than the flux of the metabolic pathways fueled from TCA cycle intermediates. It is thus unlikely that a significant portion of the 13C entering the TCA cycle could be diverted to these pathways. From a methodological standpoint, this result indicates that when a large amount of [13C]glucose is ingested and oxidized during exercise, 13CO2 production at the mouth accurately reflects the rate of glucose entry in the TCA cycle and that no correction factor is needed to compute the oxidative flux of exogenous glucose.     

Backbone dynamics of membrane proteins in lipid bilayers: the effect of two-dimensional array formation as revealed by site-directed solid-state 13C NMR studies on [3-13C]Ala- and [1-13C]Val-labeled bacteriorhodopsin

We have recorded site-directed solid-state 13C NMR spectra of [3-13C]Ala- and [1-13C]Val-labeled bacteriorhodopsin (bR) as a typical membrane protein in lipid bilayers, to examine the effect of formation of two-dimensional (2D) lattice or array of the proteins toward backbone dynamics, to search the optimum condition to be able to record full 13C NMR signals from whole area of proteins. Well-resolved 13C NMR signals were recorded for monomeric [3-13C]Ala-bR in egg phosphatidylcholine (PC) bilayer at ambient temperature, although several 13C NMR signals from the loops and transmembrane alpha-helices were still suppressed. This is because monomeric bR reconstituted into egg PC, dimyristoylphosphatidylcholine (DMPC) or dipalmytoylphosphatidylcholine (DPPC) bilayers undergoes conformational fluctuations with frequency in the order of 10(4)-10(5) Hz at ambient temperature, which is interfered with frequency of magic angle spinning or proton decoupling. It turned out, however, that the 13C NMR signals of purple membrane (PM) were almost fully recovered in gel phase lipids of DMPC or DPPC bilayers at around 0 degrees C. This finding is interpreted in terms of aggregation of bR in DMPC or DPPC bilayers to 2D hexagonal array in the presence of endogenous lipids at low temperature, resulting in favorable backbone dynamics for 13C NMR observation. It is therefore concluded that [3-13C]Ala-bR reconstituted in egg PC, DMPC or DPPC bilayers at ambient temperature, or [3-13C]Ala- and [1-13C]Val-bR at low temperature gave rise to well-resolved 13C NMR signals, although they are not always completely the same as those of 2D hexagonal lattice from PM.     

Metabolism of [1-13C]glucose in a synaptosomally enriched fraction of rat cerebrum studied by1H/13C magnetic resonance spectroscopy

This study explored the utility of¹H and¹³C magnetic resonance spectroscopy to study a standard synaptosomally enriched fraction (P2 pellet) made from rat cerebrum. The preparations contained high concentrations of N-acetylaspartate and -aminobutyric acid and low concentrations of glutamine, indicating that they were in fact rich in neuronal cytosol. The metabolic competence of the preparation was assessed by quantitative measurements of its ability to convert [1-¹³C]glucose into lactate, glutamate, aspartate, and other metabolites under well oxygenated conditions in 30 minutes. The minimum mean glycolytic rate was 0.8 mM glucose/min and the flow through the tricarboxylic acid cycle was equivalent to 0.2 mM glucose/min.Abbreviations ppm parts per million (chemical shift scale) - NMR nuclear magnetic resonance - GABA -aminobutyric acid - PBS phosphate-buffered normal saline solution - TSP 3-trimethylsilylpropionate During the performance of these studies Dr. A.P. Burlina was on leave from Instituto di Clinica delle Malattie Nervose e Mentali, University of Padua, Padua, Italy.