Effect of strong homonuclear proton coupling on localized (13)C detection using PRESS.

The effect of strong homonuclear proton coupling on (13)C incorporation measurements by either indirect or direct means was investigated (and illustrated with glutamate) both numerically and experimentally at 3.0 T. In particular, two sequences were considered, each using a proton PRESS sequence for localization. The indirect (13)C detection method incorporated the POCE (proton observe carbon edited) technique onto PRESS, and for direct (13)C detection a DEPT (distortionless enhancement by polarization transfer) sequence was appended to the PRESS localization. Both analysis and experiment demonstrate that when strong homonuclear coupling of protons is additional to heteronuclear coupling with (13)C spins, the (13)C measures derived from either the indirect PRESS-POCE sequence or the direct-but-enhanced PRESS-DEPT sequence are significantly modified. Specifically, the MR lineshapes of both (13)C-bonded and nonbonded protons are changed during (13)C incorporation, giving rise, for example, to a potential cross-contamination of < or =30% between glutamate (13)C(3) and (13)C(4) measures from the PRESS-POCE indirect method. During direct-but-enhanced detection, the DEPT enhancement is reduced for glutamate (13)C(2), (13)C(3), and (13)C(4) but not equally, and the reduction is further exacerbated by proton PRESS localization, which gives rise to enhancements that are strong functions of PRESS TE(1) and TE(2).     

1H-localized broadband 13C NMR spectroscopy of the rat brain in vivo at 9.4 T.

Localized (13)C NMR spectra were obtained from the rat brain in vivo over a broad spectral range (15-100 ppm) with minimal chemical-shift displacement error (<10%) using semi-adiabatic distortionless enhancement by polarization transfer (DEPT) combined with (1)H localization. A new gradient dephasing scheme was employed to eliminate unwanted coherences generated by DEPT when using surface coils with highly inhomogeneous B(1) fields. Excellent sensitivity was evident from the simultaneous detection of natural abundance signals for N-acetylaspartate, myo-inositol, and glutamate in the rat brain in vivo at 9.4 T. After infusion of (13)C-labeled glucose, up to 18 (13)C resonances were simultaneously measured in the rat brain, including glutamate C2, C3, C4, glutamine C2, C3, C4, aspartate C2, C3, glucose C1, C6, N-acetyl-aspartate C2, C3, C6, as well as GABA C2, lactate C3, and alanine C3. (13)C-(13)C multiplets corresponding to multiply labeled compounds were clearly observed, suggesting that extensive isotopomer analysis is possible in vivo. This unprecedented amount of information will be useful for metabolic modeling studies aimed at understanding brain energy metabolism and neurotransmission in the rodent brain.     

3D localized 1H-13C heteronuclear single-quantum coherence correlation spectroscopy in vivo.

A method for spatially three-dimensional (3D) localized two-dimensional (2D) 1H-13C correlation spectroscopy, localized HSQC, is proposed. This method has the following special feature in the preparation period. The 180 degrees (13C) and 180 degrees (1H) pulses are separated in time, and the 180 degrees (13C) pulse is applied at 1/4 1JCH) before the 90 degrees (1H) polarization transfer pulse. The preparation (echo) period 2tau can then be set substantially longer than 1/(2 1JCH), so that even in a whole-body system, slice-selective 90 degrees (1H) pulses and gradient pulses can be applied in that period. The localization capabilities of this method were confirmed in a phantom experiment. The 3D localized 2D 1H-13C correlation spectra from a monkey brain in vivo were obtained after [1-13C]glucose injection, and amino acid metabolism was detected; that is, [4-13C]glutamate appeared immediately after the injection, followed by the appearance of [2-13C]glutamate, [3-13C]glutamate, and [4-13C]glutamine.     

Design of a 13C (1H) RF probe for monitoring the in vivo metabolism of [1-13C]glucose in primate brain

The design of an RF probe suitable for obtaining proton-decoupled 13C spectra from a subhuman primate brain is described. Two orthogonal saddle coils, one tuned to the resonant frequency of 13C and the other to the resonant frequency of 1H, were used to monitor the in vivo metabolism of [1-13C]glucose in rhesus monkey brain at 2.1 T. Difference spectra showed the appearance of 13C-enriched glutamate and glutamine 30 to 40 min after a bolus injection of [1-13C]glucose.     

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.     

Novel strategy for cerebral 13C MRS using very low RF power for proton decoupling.

One of the major difficulties of in vivo 13C MRS is the need to decouple the large, one-bond, 1H-13C scalar couplings in order to obtain useful signal-to-noise ratios (SNRs) and spectral resolution at magnetic field strengths that are accessible to clinical studies. In this report a new strategy for in vivo cerebral 13C MRS is proposed. We realized that the turnover kinetics of glutamate (Glu) C5 from exogenous [2-(13)C]glucose (Glc) is identical to that of Glu C4 from exogenous [1-(13)C]Glc. The carboxylic/amide carbons are only coupled to protons via very weak long-range 1H-13C scalar couplings. As such, they can be effectively decoupled at very low RF power. Therefore, decoupling of the large 1H-13C scalar couplings can be avoided by the use of [2-(13)C]Glc. An additional advantage of this strategy is the lack of contamination from subcutaneous lipids because there are no overlapping fat signals in the vicinity of the Glu C5 and glutamine (Gln) C5 peaks. The feasibility of this strategy was demonstrated using 13C MRS on rhesus monkey brains at 4.7T.     

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.     

Multiple bond 13C-13C spin-spin coupling provides complementary information in a 13C NMR isotopomer analysis of glutamate.

Most 13C nuclear magnetic resonance (NMR) isotopomer analyses relate a metabolic index of interest to populations of 13C isotopomers as reported by one-bond 13C-13C spin-spin couplings. Metabolic conditions that produce highly enriched citric acid cycle intermediates often lead to 13C NMR spectra of metabolites such as glutamate that show extra multiplets due to long-range couplings. It can be demonstrated from 13C NMR spectra of hearts perfused with mixtures of acetate plus propionate that multiplets in glutamate C2 arising from 3J25 coupling provide a direct readout of acetyl-CoA fractional enrichment (FC1 and FC3), while multiplets in glutamate C5 arising from 2J35 and 3J25 couplings quantitatively reflect enrichment of the anaplerotic substrate.     

Alternative 1-(13)C glucose infusion protocols for clinical (13)C MRS examinations of the brain.

Clinical utility of (13)C MRS is limited by cost and long examination times. Three 1-(13)C glucose infusion protocols-a high-dose i.v., low-dose i.v., and oral administration of 1-(13)C glucose-were compared on a GE 1.5T MR scanner. Resolution and sensitivity were sufficient to identify (13)C glucose (1alpha and 1beta), glutamate (C1-C4), glutamine (C1-C4), aspartate (C2 and C3), lactate, alanine, and bicarbonate in brain spectra. The three protocols were efficacious, as measured by cerebral enrichment of 1-(13)C glucose (62%, 42%, and 38%) and its principal metabolite, 4-(13)C glutamate (13%, 11%, and 16%), respectively. Intravenous infusion of 1-(13)C glucose 0.23 g/kg body weight (low dose) provides equivalent information at one third the cost of previous regimes. Magn Reson Med 46:39-48, 2001.     

Proton-observed carbon-edited NMR spectroscopy in strongly coupled second-order spin systems.

Proton-observed carbon-edited (POCE) NMR spectroscopy is commonly used to measure 13C labeling with higher sensitivity compared to direct 13C NMR spectroscopy, at the expense of spectral resolution. For weakly coupled first-order spin systems, the multiplet signal at a specific proton chemical shift in POCE spectra directly reflects 13C enrichment of the carbon attached to this proton. The present study demonstrates that this is not necessarily the case for strongly coupled second-order spin systems. In such cases NMR signals can be detected in the POCE spectra even at chemical shifts corresponding to protons bound to 12C. This effect is demonstrated theoretically with density matrix calculations and simulations, and experimentally with measured POCE spectra of [3-13C]glutamate.     

Localized sensitivity enhanced in vivo 13C MRS to detect glucose metabolism in the mouse brain.

The application of in vivo 13C MR spectroscopy to mouse brain models is potentially valuable for improving the understanding of cerebral carbohydrate metabolism and glutamatergic neurotransmission in various neuropathologies. However, the low sensitivity of 13C nuclei and contaminating signals of lipids in the relatively small mouse brain make this application rather challenging. To meet these technical challenges, localized semi-adiabatic distortionless enhanced polarization transfer (DEPT) MR spectroscopy in combination with a continuous intravenous [1,6-13C2] glucose infusion was implemented to detect glucose metabolism in isoflurane-anesthetized mice at 7T. The signal enhancement and high spectral resolution obtained in these experiments enabled the separate determination of 13C label incorporation into as much as 13 metabolites from a 175 microL volume. Signal increases of glucose (C6), glutamine (C3, C4), and glutamate (C3, C4) were determined with a time resolution of 8.6 min. This study demonstrates an optimized MR method for the application of in vivo 13C MRS in mouse brain.     

Localized in vivo 1H NMR detection of neurotransmitter labeling in rat brain during infusion of [1-13C] D-glucose.

Resolved localized nuclear magnetic resonance (NMR) signals of 1H bound to 13C label in the carbon positions of glutamate C4, C3 and glutamine C4, C3, as well as in aspartate C3, lactate C3, alanine C3, gamma-aminobutyric acid C3, and glucose C1 were simultaneously observed in spectra obtained from rat brain in vivo. Time-resolved label incorporation was measured with a new adiabatic carbon editing and decoupling (ACED) single-voxel stimulated echo acquisition mode (STEAM) sequence. Adiabatic carbon broadband decoupling of 12 kHz bandwidth was achieved in vivo, which decoupled the entire 13C spectrum at 9.4 T. Resonances from N-acetyl-aspartate and creatine were also detected, consistent with natural-abundance 13C levels. These results emphasize the potential of 1H NMR for following complex biochemical pathways in localized areas of resting rat brain as well as during focal activation using infusions of 13C-labeled glucose.     

Detection of metabolites in rabbit brain by 13C NMR spectroscopy following administration of [1-13C]glucose

1H-decoupled 13C NMR spectra (20.2 MHz) of the living rabbit brain were collected with a surface coil following the intravenous infusion of [1-13C]glucose. Within 15 min of infusion, the alpha and beta anomers of glucose were detected and, shortly thereafter, the carbon atoms at positions C4, C3, and C2 of glutamate and(or) glutamine. After reductions of inspired oxygen from 30 to 5%, lactate C3 was detected. The intensity of the lactate resonance rose progressively during hypoxia and later fell during recovery with oxygen. The 13C fractional isotopic enrichment of arterial blood glucose was measured by 1H NMR providing information on the rate and extent of blood glucose labeling.     

Direct validation of in vivo localized 13C MRS measurements of brain glycogen.

With the use of localized 13C MRS in conjunction with [1-(13)C]-D-glucose infusion, it is possible to study brain glycogen metabolism in vivo. The purpose of this study was to validate in vivo 13C MRS measurements by comparing them with results from a standard biochemical assay. To increase the [1-(13)C] glycogen concentration, 11 rats were subjected to an episode of acute hypoglycemia followed by a mild hyperglycemic recovery period during which [1-(13)C]-D-glucose was infused. The total brain [1-(13)C] glycogen of the same animal was determined from the enzymatically determined total brain glycogen content, which was fixed by focused microwave irradiation (4 kW in 1.4 s) immediately after the end of the in vivo NMR measurements. The corresponding isotopic enrichment (IE) of glycogen was measured by in vitro 1H MRS of protons bound to glucose C1-alpha. The in vivo [1-(13)C] glycogen concentration was strongly correlated to the in vitro [1-(13)C] glycogen content determined by biochemical measurement in a linear manner (R=0.79). The results are consistent with the notion that localized 13C MRS measurements closely reflect 13C glycogen content in the brain.     

Measurement of brain glutamate and glutamine by spectrally-selective refocusing at 3 Tesla.

A new single-voxel proton NMR spectrally-selective refocusing method for measuring glutamate (Glu) and glutamine (Gln) in the human brain in vivo at 3T is reported. Triple-resonance selective 180 degrees RF pulses with a bandwidth of 12 Hz were implemented within point-resolved spectroscopy (PRESS) for selective detection of Glu or Gln, and simultaneous acquisition of creatine singlets for use as a reference in phase correction. The carriers of the spectrally-selective 180 degrees pulses and the echo times (TEs) were optimized with both numerical and experimental analyses of the filtering performance, which enabled measurements of the target metabolites with negligible contamination from N-acetylaspartate and glutathione. The concentrations of Glu and Gln in the prefrontal cortex were estimated to be 9.7+/-0.5 and 3.0+/-0.7 mM (mean+/-SD, N=7), with reference to Cr at 8 mM.     

Comparative reliability of proton spectroscopy techniques designed to improve detection of J‐coupled metabolites

Improved detection of J‐coupled neurometabolites through the use of modified proton magnetic resonance spectroscopy (1H‐MRS) techniques has recently been reported. TE‐averaged point‐resolved spectroscopy (PRESS) uses the J modulation effects by averaging FIDs with differing echo times to improve detection of glutamate, while standard PRESS detection of glutamate can be improved by using an appropriate single echo determined from J‐modulation simulations. In the present study, the reliabilities of TE‐averaged PRESS, standard PRESS with TE = 40 ms, and standard PRESS with TE = 30 ms in detecting metabolite levels in the cingulate gyrus of the human brain at 3T were compared in six subjects. TE‐averaged PRESS measures showed a mean variability of 9% for N‐acetyl aspartate, choline, and creatine, compared with < 4% for the 30‐ and 40‐ms PRESS techniques. The coefficients of variation for glutamate were 10%, 7%, and 5% for TE‐averaged, 30‐ms, and 40‐ms PRESS, respectively. PRESS with a TE of 40 ms also demonstrated improved reliability for GABA and glutamine concentrations. These results show that with the appropriate selection of echo time standard PRESS can be a reliable ¹H‐MRS technique for the measurement of J‐coupled neurometabolites in the human brain and, moreover, compares favorably with at least one J‐edited technique. Magn Reson Med 60:964–969, 2008. © 2008 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.     

Carbon-13 chemical shift imaging of [1-13C] glucose under metabolism in the rat head in vivo.

Carbon-13 chemical shift images (metabolic maps) of [1-13C] glucose in the heads of rats were obtained and compared with proton images of the same rats in terms of signal allocation. Wistar rats were kept awake or anesthetized. [1-13C] glucose was injected intravenously in a dose of 1 g per kg of body weight. The head of the Wistar rat was placed on or into circular coils. Carbon-13 images were obtained using a 7.05 Tesla system. A simple spin echo sequence was used with a chemical shift selective (CHESS) pulse. The frequency band width was set to cover the spectral breadth of the carbon-13 signal of [1-13C] glucose. The slice thickness of the image was 4 mm or 6 mm, and the field of view (FOV) was 60 mm x 60 mm, with a matrix size of 64 x 64. The total acquisition time was 36 minutes. Strong signals were observed from the scalp muscles and tissues outside the brain, but signal strength from the brain itself was minimal. This was presumably due to the metabolism of [1-13C] glucose in the brain. Little difference was recognized between [1-13C] glucose images of the heads of rats with and without anesthesia. Chemical shift imaging of carbon-13 could be useful methods for the in vivo study of physiochemical structures and metabolic pathways of living organs.     

In vivo distribution of carbon-11 phenytoin and its major metabolite, and their use in scintigraphic imaging.

Curie quantities (0.3--1.5 Ci) of H11CN were used in the synthesis of C-11-tagged phenytoin (C-11.DPH) and 5-(p-hydroxyphenyl)-5-phenylhydantoin (C-11.HPPH), using a modified Bücherer-Bergs reaction. The H11CN was produced from a mixture of 95% nitrogen and 5% hydrogen by a 45-min bombardment with 10-MeV protons at 10 muA. Following i.v. infusions of C-11 DPH (13.7 mg/kg at a rate of 29 mg/min) into the left femoral vein of Rhesus monkeys, DPH shows persistent concentration in the brain and liver fields. Extravascular administration shows significant retention at the site of administration. Intravenous bolus injection of [11C]-HPPH into a Rhesus monkey, at a dose of 6.4 mg/kg, resulted in localization of this compound in the liver, gallbladder, urinary bladder, and intestinal fields. Loss of activity from the liver region, with appearance of this activity in the intestinal field, suggests that [11C]-HPPH is secreted into the intestine via the bile. Further investigation is needed to study the potential of [11C]-DPH as a brain-scanning agent and [11C]-HPPH as a possible cholescintigraphic agent.     

Hepatic UDP-glucose 13C isotopomers from [U-13C]glucose: a simple analysis by 13C NMR of urinary menthol glucuronide.

Menthol glucuronide was isolated from the urine of a healthy 70-kg female subject following ingestion of 400 mg of peppermint oil and 6 g of 99% [U-(13)C]glucose. Glucuronide (13)C-excess enrichment levels were 4-6% and thus provided high signal-to-noise ratios (SNRs) for confident assignment of (13)C-(13)C spin-coupled multiplet components within each (13)C resonance by (13)C NMR. The [U-(13)C]glucuronide isotopomer derived via direct pathway conversion of [U-(13)C]glucose to [U-(13)C]UDP-glucose was resolved from [1,2,3-(13)C(3)]- and [1,2-(13)C(2)]glucuronide isotopomers derived via Cori cycle or indirect pathway metabolism of [U-(13)C]glucose. In a second study, a group of four overnight-fasted patients (63 +/- 10 kg) with severe heart failure were given peppermint oil and infused with [U-(13)C]glucose for 4 hr (14 mg/kg prime, 0.12 mg/kg/min constant infusion) resulting in a steady-state plasma [U-(13)C]glucose enrichment of 4.6% +/- 0.6%. Menthol glucuronide was harvested and glucuronide (13)C-isotopomers were analyzed by (13)C NMR. [U-(13)C]glucuronide enrichment was 0.6% +/- 0.1%, and the sum of [1,2,3-(13)C(3)] and [1,2-(13)C(2)]glucuronide enrichments was 0.9% +/- 0.2%. From these data, flux of plasma glucose to hepatic UDPG was estimated to be 15% +/- 4% that of endogenous glucose production (EGP), and the Cori cycle accounted for at least 32% +/- 10% of GP.