Fast (13)C-glucose metabolite mapping in rat brain using (1)H echo planar spectroscopic imaging technique at 2T

For fast (13)C metabolite mapping in rat brains, (1)H-detected (13)C NMR spectroscopy using gradient-enhanced heteronuclear multiple-quantum coherence and (1)H echo planar spectroscopic imaging were combined. (13)C glucose and 3-/4-(13)C-Glu/Gln images of rat brain were successfully constructed with 35-minute temporal resolution under a 2T magnetic field. In the ischemic region of the suture middle cerebral artery occlusion model, glucose and Glu/Gln signals decreased and lactate signals appeared. J. Magn. Reson. Imaging 2001;13:787-791.     

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.     

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.     

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.     

Cerebral activation by fasting induces lactate accumulation in the hypothalamus

Carbon‐13 (¹³C) high‐resolution magic angle spinning (HR‐MAS) spectroscopy was used to investigate the neuroglial coupling mechanisms underlying appetite regulation in the brain of C57BL/6J mice metabolizing [1‐¹³C]glucose. Control fed or overnight fasted mice received [1‐¹³C]glucose (20 μmol/g intraperitoneally [i.p.]), 15 min prior to brain fixation by focused microwaves. The hypothalamic region was dissected from the rest of the brain and ¹³C HR‐MAS spectra were obtained from both biopsies. Fasting resulted in a significant increase in hypothalamic [3‐¹³C]lactate and [2‐¹³C]γ‐aminobutyric acid (GABA) relative to the remaining brain. Administration of the orexigenic peptide ghrelin (0.3 nmol/g i.p.) did not increase hypothalamic [3‐¹³C]lactate or [2‐¹³C]GABA, suggesting that ghrelin signaling is not sufficient to elicit all the metabolic consequences of hypothalamic activation by fasting. Our results indicate that the hypothalamic regulation of appetite involves, in addition to the well‐known neuropeptide signaling, increased neuroglial lactate shuttling and augmented GABA concentrations. Magn Reson Med, 2009. © 2009 Wiley‐Liss, Inc.     

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.     

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.     

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 13CNMR spectroscopy of glucose metabolism of RIF-1 tumors

An efficient method for measuring in vivo 13C NMR spectra of tumors has been developed and employed to monitor glucose metabolism in radiation-induced fibrosarcomas (RIF-1) subcutaneously implanted in C3H/HeN mice. [1-13C]Glucose was injected directly into the tumors at a dose of 1 g/kg body wt. Spectra were obtained with a Bruker AM 360-WB spectrometer (8.4 T/8.9 cm bore) employing a homebuilt probe equipped with a four-turn solenoidal coil (1.5 cm outer diameter) for detection of 13C signals and a Helmholtz coil (two 3-cm turns separated by a 3-cm gap, oriented orthogonally to the 13C coil) for 1H decoupling. In addition to the natural abundance 13C resonances of the tumors, signals were detected from the alpha- and beta-anomers of labeled glucose. Within 15 min following injection of labeled glucose [3-13C]lactate and [3-13C]alanine were detected. Lactate labeling approached steady state levels within about 50 min after glucose injection: in contrast, alanine labeling increased continuously over the duration of the experiment (70 min). Sixty minutes after glucose injection, the ratio of the intensity of [3-13C]lactate to the principal lipid methylene resonance (30 ppm from external tetramethylsilane), which served as an internal intensity reference, was correlated with tumor size, whereas the corresponding ratio of the [3-13C]alanine resonance was not. Labeling of glutamate was below the level of detection in the in vivo spectra; however, labeling of C4-glutamate at a level approximately 50-fold lower than the level of [3-13C]lactate was detected in perchloric acid extracts. Incorporation of 13C label into C2- and C3-glutamate and C2-lactate was also observed.     

Brain uptake and organ distribution of 11C from 11C-labeled glucose

The time course of the distribution of carbon-11 (11C, t1/2 = 20.4 min) in brain after the i.v. administration of 11C-labeled glucose [( 11C]glucose) was studied in an effort to understand and explore its behavior in relation to the known factors concerning the catabolic fate of glucose carbon in the brain. The biodistribution of 11C from [11C]glucose was studied in rats using organ dissection. Human radiation doses were estimated from rat biodistribution data. All the rat organs except the brain cleared with a half time of 30-60 min. The brain showed delayed uptake that plateaued from 20 to 60 min. The 11C distribution in normal, non-ischemic, brain 30 min after intravenously administered [11C]glucose is due to labeled carbon incorporation into amino acids associated with tricarboxylic acid cycle intermediates. External imaging with the Massachusetts General Hospital positron camera, PC I, was performed in dogs and humans and the time course of 11C incorporation was similar to the rat brain results. Regional uptake paralleled known metabolic differences between grey and white matter in normal human volunteers. A patient with progressive dementia had less uptake in an area of decreased perfusion as demonstrated angiographically, suggesting that the image obtained 20 min after tracer administration could be used to detect abnormalities in cerebral metabolism due to pathology.     

A method for measuring cerebral glucose metabolism in vivo by 13C-NMR spectroscopy.

Current methods for estimating the rate of cerebral glucose utilization (CMR(glc)) typically measure metabolic activity for 40 min or longer subsequent to administration of [(13)C]glucose, 2-[(14)C]deoxyglucose, or 2-[(18)F]deoxyglucose. We report preliminary findings on estimating CMR(glc) for a period of 15 min by use of 2-[6-(13)C]deoxyglucose. After a 24-hr fast, rats were anesthetized, infused with [1-(13)C]glucose for 50 min, and injected with 2-[6-(13)C]deoxyglucose (500 mg/kg). During the subsequent 12.95 min the estimated value of CMR(glc) was 0.6 +/- 0.4 micromol/min/g (mean +/- SD, N = 7), in agreement with values reported for anesthetized rats studied with the 2-[(14)C]deoxyglucose method and other (13)C-NMR methods that measure CMR(glc). In rats injected with bicuculline methiodide (a known stimulant of CMR(glc)), CMR(glc) increased by more than 75% during 12.95 min following injection of bicuculline (Wilcoxon signed rank test, P = 0.042, N = 8).     

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.     

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.     

Measurement of brain glutamate using TE-averaged PRESS at 3T.

A method is introduced that provides improved in vivo spectroscopic measurements of glutamate (Glu), glutamine (Gln), choline (Cho), creatine (Cre), N-acetyl compounds (NAtot, NAA + NAAG), and the inositols (mI and sI). It was found that at 3T, TE averaging, the f1 = 0 slice of a 2D J-resolved spectrum, yielded unobstructed signals for Glu, Glu + Gln (Glx), mI, NA(tot), Cre, and Cho. The C4 protons of Glu at 2.35 ppm, and the C2 protons of Glx at 3.75 ppm were well resolved and yielded reliable measures of Glu/Gln stasis. Apparent T1/T2 values were obtained from the raw data, and metabolite tissue levels were determined relative to a readily available standard. A repeatibility error of <5%, and a coefficient of variation (CV) of <10% were observed for brain Glu levels in a study of six normal volunteers.     

Pathways for the synthesis of sorbitol from 13C-labeled hexoses, pentose, and glycerol in renal papillary tissue

Suspensions of rabbit renal papillary tissue were incubated with D-[6-13C]glucose, D-[1-13C]fructose, D-[1-13C]ribose, and [2-13C]glycerol. The perchloric acid extracts of the above incubations were investigated with 13C NMR spectroscopy. All 13C-labeled substrates give rise to 13C-labeled D-sorbitol. D-[6-13C]Glucose and D-[1-13C]fructose are converted directly into D-sorbitol via the aldose reductase and sorbitol dehydrogenase pathway, respectively, whereas D-[1-13C]ribose and [2-13C]glycerol give rise to labeling of the D-glyceraldehyde pool which on its turn causes a labeling of D-sorbitol. Label exchanges observed from incubations with glycerol and D-ribose indicate that the pentose shunt plays a role in this synthesis of D-sorbitol.     

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.     

Simultaneous in vivo monitoring of hepatic glucose and glucose-6-phosphate by (13)C-NMR spectroscopy.

Hepatic glucose-6-phosphate (G6P) was monitored non-invasively in rat liver by in vivo (13)C NMR spectroscopy after infusion of [1-(13)C] glucose. The phosphorylation of glucose to G6P yields small but characteristic displacements for all of its (13)C-NMR resonances relative to those of glucose. It is demonstrated that in vivo (13)C-NMR spectroscopy at 7 Tesla provides the spectral sensitivity and resolution to detect hepatic G6P present at sub-millimolar concentration as partially resolved low-field shoulders of the glucose C1 resonances at 96.86 ppm (C1beta) and 93. 02 ppm (C1alpha). Upon (13)C-labeling, the intracellular conversion of [1-(13)C] glucose to [1-(13)C] G6P could be monitored, which allowed the hepatic glucose-G6P substrate cycle to be assessed in situ. The close correlation found for the (13)C labeling patterns of glucose and G6P supports the concept of an active substrate cycle whose rate exceeds that of net hepatic glucose metabolism. High-resolution (13)C-NMR spectroscopy and biochemical analyses of tissue biopsies collected at the end of the experiments confirmed qualitatively the findings obtained in vivo.     

In vivo 13C saturation transfer effect of the lactate dehydrogenase reaction.

Lactate dehydrogenase (LDH, EC 1.1.1.27) catalyzes an exchange reaction between pyruvate and lactate. It is demonstrated here that this reaction is sufficiently fast to cause a significant magnetization (saturation) transfer effect when the 13C resonance of pyruvate is saturated by a continuous-wave (CW) RF pulse. Infusion of [2-(13)C]glucose was used to allow labeling of pyruvate C2 at 207.9 ppm to determine the pseudo first-order rate constant of the unidirectional lactate-->pyruvate flux in vivo. During systemic administration of GABAA receptor antagonist bicuculline, this pseudo first-order rate constant was determined to be 0.08+/-0.01 s-1 (mean+/-SD, N=4) in halothane-anesthetized adult rat brains. In 9L and C6 rat glioma models, the 13C saturation transfer effect of the LDH reaction was also detected in vivo. Our results demonstrate that the 13C magnetization transfer effect of the LDH reaction may be useful as a novel marker for utilizing noninvasive in vivo MRS to study many physiological and pathological conditions, such as cancer.     

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.     

Characterization of acetate metabolism in tumor cells in relation to cell proliferation: acetate metabolism in tumor cells

To reveal the metabolic fate of acetate in neoplasms that may characterize the accumulation patterns of [1-(11)C]acetate in tumors depicted by positron emission tomography. Four tumor cell lines (LS174T, RPMI2650, A2780, and A375) and fibroblasts in growing and resting states were used. In uptake experiments, cells were incubated with[1-(14)C]acetate for 40 min. [(14)C]CO(2) was measured in the tight-air chamber, and the metabolites in cells were identified by thin layer chromatography and paper chromatography. The glucose metabolic rate of each cell line was measured with [2,6-(3)H]2-deoxy-glucose (DG), and the growth activity of each cell line was estimated by measuring the incorporation of [(3)H]methyl thymidine into DNA. Compared with resting fibroblasts, all four tumor cell lines showed higher accumulation of (14)C activity from [1-(14)C]acetate. These tumor-to-normal ratios of [1-(14)C]acetate were larger than those of DG. Tumor cells incorporated (14)C activity into the lipid-soluble fraction, mostly of phosphatidylcholine and neutral lipids, more prominently than did fibroblasts. The lipid-soluble fraction of (14)C accumulation in cells showed a positive correlation with growth activity, whereas the water-soluble and CO(2) fractions did not. These findings suggest that the high tumor-to-normal ratio of [1-(14)C]acetate is mainly due to the enhanced lipid synthesis, which reflects the high growth activity of neoplasms. This in vitro study suggests that [1-(11)C]acetate is appropriate for estimating the growth activity of tumor cells.