Selective resonance suppression 1H‐[13C] NMR spectroscopy with asymmetric adiabatic RF pulses

Despite obvious improvements in spectral resolution at high magnetic field, the detection of ¹³C labeling by ¹H‐[¹³C] NMR spectroscopy remains hampered by spectral overlap, such as in the spectral region of ¹H resonances bound to C3 of glutamate (Glu) and glutamine (Gln), and C6 of N‐acetylaspartate (NAA). The aim of this study was to develop, implement, and apply a novel ¹H‐[¹³C] NMR spectroscopic editing scheme, dubbed “selective Resonance suppression by Adiabatic Carbon Editing and Decoupling single‐voxel STimulated Echo Acquisition Mode” (RACED‐STEAM). The sequence is based on the application of two asymmetric narrow‐transition‐band adiabatic RF inversion pulses at the resonance frequency of the ¹³C coupled to the protons that need to be suppressed during the mixing time (TM) period, alternating the inversion band downfield and upfield from the ¹³C resonance on odd and even scans, respectively, thus suppressing the detection of ¹H resonances bound to ¹³C within the transition band of the inversion pulse. The results demonstrate the efficient suppression of ¹H resonances bound to C3 of Glu and Gln, and C4 of Glu, which allows the ¹H resonances bound to C6 of NAA and C4 of Gln to be revealed. The measured time course of the resolved labeling into NAA C6 with the new scheme was consistent with the slow turnover of NAA. Magn Reson Med 61:260–266, 2009. © 2009 Wiley‐Liss, Inc.     

Methods for metabolic evaluation of prostate cancer cells using proton and 13C HR‐MAS spectroscopy and [3‐13C] pyruvate as a metabolic substrate

Prostate cancer has been shown to undergo unique metabolic changes associated with neoplastic transformation, with associated changes in citrate, alanine, and lactate concentrations. ¹³C high resolution‐magic angle spinning (HR‐MAS) spectroscopy provides an opportunity to simultaneously investigate the metabolic pathways implicated in these changes by using ¹³C‐labeled substrates as metabolic probes. In this work, a method to reproducibly interrogate metabolism in prostate cancer cells in primary culture was developed using HR‐MAS spectroscopy. Optimization of cell culture protocols, labeling parameters, harvesting, storage, and transfer was performed. Using [3‐¹³C] pyruvate as a metabolic probe, ¹H and ¹³C HR‐MAS spectroscopy was used to quantify the net amount and fractional enrichment of several labeled metabolites that evolved in multiple cell samples from each of five different prostate cancers. Average enrichment across all cancers was 32.4 ± 5.4% for [3‐¹³C] alanine, 24.5 ± 5.4% for [4‐¹³C] glutamate, 9.1 ± 2.5% for [3‐¹³C] glutamate, 25.2 ± 5.7% for [3‐¹³C] aspartate, and 4.2 ± 1.0% for [3‐¹³C] lactate. Cell samples from the same parent population demonstrated reproducible fractional enrichments of alanine, glutamate, and aspartate to within 12%, 10%, and 10%, respectively. Furthermore, the cells produced a significant amount of [4‐¹³C] glutamate, which supports the bioenergetic theory for prostate cancer. These methods will allow further characterization of metabolic properties of prostate cancer cells in the future. Magn Reson Med, 2009. © 2009 Wiley‐Liss, Inc.     

In vivo 13C magnetic resonance spectroscopy of human brain on a clinical 3 T scanner using [2‐13C]glucose infusion and low‐power stochastic decoupling

This study presents the detection of [2‐¹³C]glucose metabolism in the carboxylic/amide region in the human brain, and demonstrates that the cerebral metabolism of [2‐¹³C]glucose can be studied in human subjects in the presence of severe hardware constraints of widely available 3 T clinical scanners and with low‐power stochastic decoupling. In the carboxylic/amide region of human brain, the primary products of ¹³C label incorporation from [2‐¹³C]glucose into glutamate, glutamine, aspartate, γ‐aminobutyric acid, and N‐acetylaspartate were detected. Unlike the commonly used alkanyl region where lipid signals spread over a broad frequency range, the carboxylic carbon signal of lipids was found to be confined to a narrow range centered at 172.5 ppm and present no spectral interference in the absence of lipid suppression. Comparison using phantoms shows that stochastic decoupling is far superior to the commonly used WALTZ sequence at very low decoupling power at 3 T. It was found that glutamine C1 and C5 can be decoupled using stochastic decoupling at 2.2 W, although glutamine protons span a frequency range of ≈700 Hz. Detailed specific absorption rate analysis was also performed using finite difference time domain numerical simulation. Magn Reson Med, 2009. © 2009 Wiley‐Liss, Inc.     

13C MR spectroscopy measurements of glutaminase activity in human hepatocellular carcinoma cells using hyperpolarized 13C‐labeled glutamine

Dynamic nuclear polarization (DNP) is an emerging technique for increasing the sensitivity of ¹³C MR spectroscopy (MRS). [5‐¹³C₁]Glutamine was hyperpolarized using this technique by up to 5%, representing a 6000‐fold increase in sensitivity. The conversion of hyperpolarized glutamine to glutamate by mitochondrial glutaminase was demonstrated using ¹³C‐MRS measurements in cultured human hepatoma cells (HepG2). These results represent the first step in developing an imaging technique for detecting glutamine metabolism in vivo. Furthermore, since glutamine utilization has been correlated with cell proliferation, the study suggests a new technique for detecting changes in tumor cell proliferation. Magn Reson Med 60:253–257, 2008. © 2008 Wiley‐Liss, Inc.     

Diffusion‐weighted NMR spectroscopy allows probing of 13C labeling of glutamate inside distinct metabolic compartments in the brain

In the present work, diffusion‐weighted (DW)‐NMR spectroscopy of glutamate was performed during a ¹³C‐labeled glucose infusion in monkey brain (six experiments). It is shown that glutamate ¹³C labeling occurs significantly faster at higher diffusion weightings—slightly for glutamate in position C4, and more markedly for glutamate in position C3. This demonstrates the existence of different diffusion compartments for glutamate, associated with different metabolic rates. Metabolic modeling of ¹³C enrichment time‐courses suggests that these compartments might be gray and white matter, each having a specific oxidative metabolism rate possibly paralleled by a specific glutamate diffusion coefficient. Magn Reson Med 60:306–311, 2008. © 2008 Wiley‐Liss, Inc.     

Which prior knowledge? Quantification of in vivo brain 13C MR spectra following 13C glucose infusion using AMARES

The recent developments in high magnetic field ¹³C magnetic resonance spectroscopy with improved localization and shimming techniques have led to important gains in sensitivity and spectral resolution of ¹³C in vivo spectra in the rodent brain, enabling the separation of several ¹³C isotopomers of glutamate and glutamine. In this context, the assumptions used in spectral quantification might have a significant impact on the determination of the ¹³C concentrations and the related metabolic fluxes. In this study, the time domain spectral quantification algorithm AMARES (advanced method for accurate, robust and efficient spectral fitting) was applied to ¹³C magnetic resonance spectroscopy spectra acquired in the rat brain at 9.4 T, following infusion of [1,6‐¹³C₂] glucose. Using both Monte Carlo simulations and in vivo data, the goal of this work was: (1) to validate the quantification of in vivo ¹³C isotopomers using AMARES; (2) to assess the impact of the prior knowledge on the quantification of in vivo ¹³C isotopomers using AMARES; (3) to compare AMARES and LCModel (linear combination of model spectra) for the quantification of in vivo ¹³C spectra. AMARES led to accurate and reliable ¹³C spectral quantification similar to those obtained using LCModel, when the frequency shifts, J‐coupling constants and phase patterns of the different ¹³C isotopomers were included as prior knowledge in the analysis. Magn Reson Med, 2013. © 2012 Wiley Periodicals, Inc.     

Metabolic imaging in the anesthetized rat brain using hyperpolarized [1‐13C] pyruvate and [1‐13C] ethyl pyruvate

Formulation, polarization, and dissolution conditions were developed to obtain a stable hyperpolarized solution of [1‐¹³C]‐ethyl pyruvate. A maximum tolerated concentration and injection rate were determined, and ¹³C spectroscopic imaging was used to compare the uptake of hyperpolarized [1‐¹³C]‐ethyl pyruvate relative to hyperpolarized [1‐¹³C]‐pyruvate into anesthetized rat brain. Hyperpolarized [1‐¹³C]‐ethyl pyruvate and [1‐¹³C]‐pyruvate metabolic imaging in normal brain is demonstrated and quantified in this feasibility and range‐finding study. Magn Reson Med 63:1137–1143, 2010. © 2010 Wiley‐Liss, Inc.     

Unambiguous assignment of the H3S and H3R deuterations of cerebral (2‐13C) glutamate by 13C NMR at 18.8 tesla

We used high‐field ¹³C NMR (18.8 T) to assign unambiguously the isotopic shifts induced by the deuterium substitutions of the H3_proR and H3_proS hydrogens of (2‐¹³C) glutamate in extracts of the brain from deuterated animals. Monodeuterated H3R or H3S glutamate diastereoisomers were produced stereospecifically either by chemical synthesis or by coupling the reactions of isocitrate dehydrogenase and aspartate aminotransferase in deuterated medium, respectively. We show that the (3S‐²H) or (3R‐²H) deuterations induce characteristic small (Δ₂ = ?0.058 parts per million (ppm)) or large (Δ₂ = ?0.071 ppm) vicinal isotopic shifts upfield of the perprotonated (2‐¹³C) glutamate resonance (at 55.5 ppm). Isotopically shifted (2‐¹³C, 3S‐²H) or (2‐¹³C, 3R‐²H) glutamate singlets are conveniently observed by high‐field ¹³C NMR in brain extracts from deuterated rats. Since the (3S‐²H) or (3R‐²H) glutamate diastereoisomers are produced stereospecifically by the cytosolic or mitochondrial isoforms of aconitase and isocitrate dehydrogenase, our results will facilitate the ¹³C NMR investigation of these enzymatic activities and their role in subcellular glutamate trafficking. Magn Reson Med 63:1088–1091, 2010. © 2010 Wiley‐Liss, Inc.     

Detection of [1,6-13C2]-glucose metabolism in rat brain by in vivo 1H-[13C]-NMR spectroscopy.

Localized, water-suppressed (1)H-[(13)C]-NMR spectroscopy was used to detect (13)C-label accumulation in cerebral metabolites following the intravenous infusion of [1,6-(13)C(2)]-glucose (Glc). The (1)H-[(13)C]-NMR method, based on adiabatic RF pulses, 3D image-selected in vivo spectroscopy (ISIS) localization, and optimal shimming, yielded high-quality (1)H-[(13)C]-NMR spectra with optimal NMR sensitivity. As a result, the (13)C labeling of [4-(13)C]-glutamate (Glu) and [4-(13)C]-glutamine (Gln) could be detected from relatively small volumes (100 microL) with a high temporal resolution. The formation of [n-(13)C]-Glu, [n-(13)C]-Gln (n = 2 or 3), [2-(13)C]-aspartate (Asp), [3-(13)C]-Asp, [3-(13)C]-alanine (Ala), and [3-(13)C]-lactate (Lac) was also observed to be reproducible. The (13)C-label incorporation curves of [4-(13)C]-Glu and [4-(13)C]-Gln provided direct information on metabolic pathways. Using a two-compartment metabolic model, the tricarboxylic acid (TCA) cycle flux was determined as 0.52 +/- 0.04 micromol/min/g, while the glutamatergic neurotransmitter flux equaled 0.25 +/- 0.05 micromol/min/g, in good correspondence with previously determined values.     

Comparison of [3,4‐13C2]glucose to [6,6‐2H2]glucose as a tracer for glucose turnover by nuclear magnetic resonance

A recently introduced tracer, [3,4‐¹³C₂]glucose, was compared to the widely used tracer, [6,6‐²H₂]glucose, for measurement of whole‐body glucose turnover. The rate of glucose production (GP) was measured in rats after primed infusions of [3,4‐¹³C₂]glucose, [6,6‐²H₂]glucose, or both tracers simultaneously followed by a constant infusion of tracer(s) over 90 min. Blood glucose was purified and converted into monoacetone glucose for analysis by ¹³C NMR (for [3,4‐¹³C₂]glucose) or ¹H and ²H NMR (for [6,6‐²H₂]glucose). The values of GP measured during infusion of each single tracer were not significantly different. In rats infused with both tracers simultaneously, GP was identical as reported by each tracer, 42 ± 4 μmol/kg/min. Since ²H and ¹³C enrichment in glucose is typically much less than 2% for in vivo studies, [3,4‐¹³C₂]glucose does not interfere with measurements of ¹³C or ²H enrichment patterns and therefore is valuable when multiple metabolic pathways are being evaluated simultaneously. Magn Reson Med 53:1479–1483, 2005. © 2005 Wiley‐Liss, Inc.     

Simplified 13C metabolic modeling for simplified measurements of cerebral TCA cycle rate in vivo

¹³C NMR spectroscopy is a unique tool to measure the cerebral tricarboxylic acid (TCA) cycle rate in vivo. The measurement relies on metabolic modeling of glutamate C3 and C4 enrichment time courses during a ¹³C‐glucose intravenous infusion. Usual metabolic models require the plasma glucose and ¹³C‐glucose time courses as input functions, as well as the knowledge of Michaelis‐Menten kinetics parameters governing passage through the blood‐brain barrier. It is shown in the present work that, when using an infusion protocol yielding a rapidly stable plasma glucose fractional enrichment, metabolic modeling can be simplified in such a manner that this additional information on input function and glucose transport is no longer required, significantly simplifying the measurement of cerebral TCA cycle rate in vivo. Magn Reson Med, 2009. © 2009 Wiley‐Liss, Inc.     

13C NMR study of the generation of C2- and C3,-deuterated lactic acid by tumoral pancreatic islet cells exposed to D-[1-13C]-, D-[2-13C]- and D-[6-13C]-Glucose in 2H2O

Tumoral pancreatic islet cells of the RIN5mF line were incubated for 120 min in media prepared in ²H₂O and containing D -[1-¹³C]glucose, and D -[2-¹³C]glucose, and D -[6-¹³C]glucose. The generation of C₂- and C₃- deuterated lactic acid was assessed by ¹³C NMR. The interpretation of experimental results suggests that a) the efficiency of deuteration on the C₁ of D-fructose 6-phosphate does not exceed about 47% and 4% in the phosphoglucoisomerase and phosphomannoisomerase reactions, respectively; b) approximately 38% of the molecules of D -glyceraldehyde 3-phosphate generated from D -glucose escape deuteration in the sequence of reactions catalyzed by triose phosphate isomerase and aldolase; and c) about 41% of the molecules of pyruvate generated by glycolysis are immediately converted to lactate, the remaining 59% of pyruvate molecules undergoing first a single or double back-and-forth interconversion with L -alanine. It is proposed that this methodological approach, based on high resolution ¹³C NMR spectroscopy, may provide novel information on the regulation of back-and-forth interconversion of glycolytic intermediates in intact cells as modulated, for instance, by enzyme-to-enzyme tunneling.     

Multitissue assessment of in vivo postprandial intracellular lipid partitioning in rats using localized (1) H-[(13) C] magnetic resonance spectroscopy

Excess accumulation of lipids in nonadipose tissues such as skeletal muscle and liver has been implicated in the development of obesity‐related disorders, but the cause of this ectopic lipid overload remains unknown. The aim of this study was to determine in vivo postprandial lipid partitioning in rat skeletal muscle and liver, using localized ¹H‐[¹³C] magnetic resonance spectroscopy in combination with the oral administration of ¹³C‐labeled lipids. Six rats were measured at baseline and 5 and 24 h after administration of 400 mg [U‐¹³C]‐labeled algal lipids. Five hours after administration, fractional ¹³C enrichments of the lipid pools in muscle and liver were increased 3.9‐fold and 4.6‐fold (P < 0.05), respectively, indicating that part of the ingested lipids had been taken up by muscle and liver tissue. At 24 h, fractional ¹³C enrichments of muscle and liver lipids were decreased 1.6‐fold and 2.2‐fold (P < 0.05), respectively, compared with the 5 h values. This can be interpreted as a depletion of ¹³C‐labeled lipids from the intracellular lipid pools as a consequence of lipid turnover. In conclusion, the novel application of ¹H‐[¹³C] magnetic resonance spectroscopy in combination with the oral administration of ¹³C‐labeled lipids is applicable for the longitudinal assessment of in vivo lipid partitioning between multiple tissues. Magn Reson Med, 2012. © 2011 Wiley Periodicals, Inc.     

Broadband decoupled, 1H-localized 13C MRS of the human brain at 4 tesla

Broadband proton decoupling of the entire ¹³C spectrum was possible within power absorption guidelines and resulted in the detection of narrow (as low as 2–3 Hz), natural abundance signals from metabolites such as myo-inositol, glutamate, N-acetyl-aspartate, and glutamine from 72 cm³ volumes in the human brain. To overcome the chemical shift displacement error, three-dimensional localization on the ¹H z magnetization was combined with polarization transfer. Efficiency of the heteronuclear localization method was demonstrated by the elimination of all scalp lipid resonances. A signal-to-noise ratio of 5:1 for 0.07 mM [¹³C] was achieved in 12 min, which is approximately a fivefold improvement over the sensitivity reported at 2.1 Tesla.     

13C isotopomer analyses in intact tissue using {13C}homonuclear decoupling

Entry of ¹³C-enriched acetyl-CoA into the citric acid cycle results in scrambling of ¹³C into the various carbon positions of all intermediate pools. The eventual result is that the ¹³C resonances of all detectable intermediates or molecules exchanging with those intermediates appear as multiplets due to nearest neighbor spin-spin couplings. We have previously shown that an isotopomer analysis of the glutamate ¹³C multiplets provides a history of ¹³C flow through the cycle pools and that relative substrate utilization and relative anaplerotic flux can be quantitated (C.R. Malloy, A.D. Sherry, and F.M.H. Jeffrey, Am. J. Physiol. 259, H987–H995 (1990)). A major limitation of the method for in vivo applications is spectral resolution of multiline resonances required for a complete isotopomer analysis. We now show that (¹³C)homonuclear decoupling of the glutamate C3 resonance collapses nine-line C4 and C2 resonances into three-line multiplets. We demonstrate that these three line ¹³C multiplets are well resolved in isolated, perfused rat hearts and present steady-state equations that allow an isotopomer analysis from data obtained in intact tissue. This advancement offers for the first time the possibility of extending ¹³C isotopomer methods to complex metabolic conditions in vivo.     

Oxidation of acetate in rabbit skeletal muscle: Detection by 13C NMR spectroscopy in vivo

The results of a proton-decoupled and Overhauser-enhanced ¹³C NMR study of acetate metabolism in skeletal muscle are reported. [2-¹³C]Acetate was infused intravenously over 2 h into anesthetized rabbits, and skeletal muscle in the lateral thigh was monitored by ¹³C NMR spectroscopy at 4.7 T. Stable ¹³C enrichment in carbons 2, 3, and 4 of glutamate was observed at the end of the infusion, and the half-time for enrichment was 17 min for glutamate C4 and 50 min for glutamate C2 and C3. The contribution of exogenous acetate to acetyl-coenzyme A was nearly equal in skeletal muscle and heart in vivo (83–87%, measured in tissue extracts), comparable with earlier perfused heart studies in which acetate was the sole available substrate. Although relative flux through the combined anaplerotic pathways (relative to citric acid cycle flux) was higher in quiescent skeletal muscle (26%) compared with hearts (3%) from the same animals, actual anaplerotic flux was estimated to be substantially higher in heart than in skeletal muscle after correcting for differences in citric acid cycle flux in the two tissues.     

1-13C]glucose MRS in chronic hepatic encephalopathy in man.

[1-13C]-labeled glucose was infused intravenously in a single dose of 0.2 g/kg body weight over 15 min in six patients with chronic hepatic encephalopathy, and three controls. Serial 13C MR spectra of the brain were acquired. Patients exhibited the following characteristics relative to normal controls: 1) Cerebral glutamine concentration was increased (12.6 +/- 3.8 vs. 6.5 +/- 1.9 mmol/kg, P < 0.006) and glutamate was reduced (8.2 +/- 1.0 vs. 9.9 +/- 0.6 mmol/kg, P < 0.02). 2) 13C incorporation into glutamate C4 and C2 positions was reduced in patients (80 min after start of infusion C4: 0.43 +/- 0.09 vs. 0.84 +/- 0.15 mmol/kg, P < 0.001; C2: 0.20 +/- 0.03 vs. 0.45 +/- 0.07 mmol/kg, P < 0.0001). 3) 13C incorporation into bicarbonate was delayed (90 +/- 21 vs. 40 +/- 10 min, P < 0.003), and the time interval between detection of glutamate C4 and C2 labeling was longer in patients (22 +/- 8 vs. 12 +/- 3 min, P < 0.03). 4) Glutamate C2 turnover time was reduced in chronic hepatic encephalopathy (17.1 +/- 6.8 vs. 49.6 +/- 8.7 min, P < 0.0002). 5) 13C accumulation into glutamine C2 relative to its substrate glutamate C2 increased progressively with the severity of clinical symptoms (r = 0.96, P < 0.01). These data indicate disturbed neurotransmitter glutamate/glutamine cycling and reduced glucose oxidation in chronic hepatic encephalopathy. [1-13C] glucose MRS provides novel insights into disease progression and the pathophysiology of chronic hepatic encephalopathy.     

Hyperpolarized 13C spectroscopy and an NMR‐compatible bioreactor system for the investigation of real‐time cellular metabolism

The purpose of this study was to combine a three‐dimensional NMR‐compatible bioreactor with hyperpolarized ¹³C NMR spectroscopy in order to probe cellular metabolism in real time. JM1 (immortalized rat hepatoma) cells were cultured in a three‐dimensional NMR‐compatible fluidized bioreactor. ³¹P spectra were acquired before and after each injection of hyperpolarized [1‐¹³C] pyruvate and subsequent ¹³C spectroscopy at 11.7 T. ¹H and two‐dimensional ¹H‐¹H‐total correlation spectroscopy spectra were acquired from extracts of cells grown in uniformly labeled ¹³C‐glucose, on a 16.4 T, to determine ¹³C fractional enrichment and distribution of ¹³C label. JM1 cells were found to have a high rate of aerobic glycolysis in both two‐dimensional culture and in the bioreactor, with 85% of the ¹³C label from uniformly labeled ¹³C‐glucose being present as either lactate or alanine after 23 h. Flux measurements of pyruvate through lactate dehydrogenase and alanine aminotransferase in the bioreactor system were 12.18 ± 0.49 nmols/sec/10⁸ cells and 2.39 ± 0.30 nmols/sec/10⁸ cells, respectively, were reproducible in the same bioreactor, and were not significantly different over the course of 2 days. Although this preliminary study involved immortalized cells, this combination of technologies can be extended to the real‐time metabolic exploration of primary benign and cancerous cells and tissues prior to and after therapy. Magn Reson Med, 2010. © 2010 Wiley‐Liss, Inc.     

Investigation of tumor hyperpolarized [1‐13C]‐pyruvate dynamics using time‐resolved multiband RF excitation echo‐planar MRSI

Hyperpolarized [1‐¹³C]‐pyruvate is an exciting new agent for the in vivo study of cellular metabolism and a potential cancer biomarker. The nature of the hyperpolarized signal poses unique challenges because of its short duration and the loss of magnetization with every excitation. In this study, we applied a novel and efficient time‐resolved MR spectroscopic imaging (MRSI) method to investigate in a prostate cancer model the localized temporal dynamics of the uptake of [1‐¹³C]‐pyruvate and its conversion to metabolic products, specifically [1‐¹³C]‐lactate. This hyperpolarized ¹³C method used multiband excitation pulses for efficient use of the magnetization. This study demonstrated that regions of tumor were differentially characterized from normal tissue by the lactate dynamics, where tumors showed later lactate detection and longer lactate duration that was statistically significant (P < 0.001). Compared to late‐pathologic‐stage tumors, early‐ to intermediate‐stage tumors demonstrated significantly (P < 0.01) lower lactate total signal‐to‐noise ratio (SNR), with similar temporal dynamic parameters. Hyperpolarized pyruvate dynamics provided uptake, perfusion, and vascularization information on tumors and normal tissue. Large, heterogeneous tumors demonstrated spatially variable uptake of pyruvate and metabolic conversion that was consistent with cellularity and necrosis identified by histology. The results of this study demonstrated the potential of this new hyperpolarized MR dynamic method for improved cancer detection and characterization. Magn Reson Med 63:582–591, 2010. © 2010 Wiley‐Liss, Inc.     

In vivo detection of cortical GABA turnover from intravenously infused [1-13C]D-glucose.

In this study [2-(13)C] gamma-aminobutyric acid (GABA) was spectrally resolved in vivo and detected simultaneously with [4-(13)C]glutamate (Glu) and [4-(13)C]glutamine (Gln) in the proton spectra obtained from a localized 40 microL voxel in rat neocortex with the use of an adiabatic (1)H-observed, (13)C-edited (POCE) spectroscopy method and an 89-mm-bore vertical 11.7 Tesla microimager. The time-resolved kinetics of (13)C label incorporation from intravenously infused [1-(13)C]glucose into [4-(13)C]Glu, [4-(13)C]Gln, and [2-(13)C]GABA were measured after acute administration of gabaculine, a potent and specific inhibitor of GABA-transaminase. In contrast to previous observations of a rapid turnover of [2-(13)C]GABA from [1-(13)C]glucose in intact rat brain, the rate of (13)C incorporation from [1-(13)C]glucose into [2-(13)C]GABA in the gabaculine-treated rats was found to be significantly reduced as a result of the blockade of the GABA shunt.