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.     

Enzymatic syntheses of carbamyl phosphate, L-citrulline, and N-carbamyl L-aspartate labeled with either 13N or 11C

[13N]- and [11C]carbamyl phosphate, L-[omega-13N]citrulline, L-[ureido-11C]citrulline, [carbamyl-13N]- and [carbamyl-11C]carbamyl-L-aspartate were synthesized using carbamyl phosphate synthetase co-immobilized with either aspartate transcarbamylase or ornithine transcarbamylase. Carbamyl L-[13N]aspartate was enzymatically prepared from carbamyl phosphate and L-[13N]aspartate. The tissue distribution of radioactivity in mice after injection of radiolabeled ammonia, carbamyl phosphate or citrulline was studied. The tissue distribution of isotope derived from [13N]carbamyl phosphate and [13N]ammonia were similar, with the exception of liver, brain and pancreas, in which 13NH3 uptake was higher after retroorbital injection. The distribution of label derived from L-[omega-13N]- and L-[ureido-11C]citrulline was similar. Substantial tumor (Sarcoma-180) uptake of label from L-citrulline was observed.     

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.     

Analysis of the Radiorespirometric pattern after administration of [U-14C] glucose to Ehrlich ascites tumor-bearing mice

Ehrlich ascites tumor-bearing mice were subjected to 14CO2 radiorespirometric analysis after administration of [U-14C]glucose, and the results were compared with the levels of host liver glycolytic enzyme activities and the uptake of the radioactivity into the liver. After IP administration of [U-14C]glucose, there was a progressive decline in respiratory 14CO2 after the transplantation of the Ehrlich ascites tumor cells. The peak time (PT) was about 10 min on day 1, but thereafter was increasingly delayed, and could not be determined on day 13. Peak height (PH) and yield value (YV) were both considerably decreased, and again the magnitudes of the changes increased with the time after transplantation of the tumor cells. Glycolytic enzyme activities in the host liver were at normal levels 13 days after transplantation of the tumor cells. The uptake of the radioactivity into the liver after IP administration of [U-14C]glucose began to decline from day 5 and was 50% the value in normal mice 13 days after transplantation of the tumor cells. THese results indicate that the radiorespirometric patterns with [U-14C]glucose reflects hepatic biochemical changes rather well.     

Detecting response of rat C6 glioma tumors to radiotherapy using hyperpolarized [1‐13C]pyruvate and 13C magnetic resonance spectroscopic imaging

We show here that hyperpolarized [1‐¹³C]pyruvate can be used to detect treatment response in a glioma tumor model; a tumor type where detection of response with ¹⁸fluoro‐2‐deoxyglucose, using positron emission tomography, is limited by the high background signals from normal brain tissue. ¹³C chemical shift images acquired following intravenous injection of hyperpolarized [1‐¹³C]pyruvate into rats with implanted C6 gliomas showed significant labeling of lactate within the tumors but comparatively low levels in surrounding brain.Labeled pyruvate was observed at high levels in blood vessels above the brain and from other major vessels elsewhere but was detected at only low levels in tumor and brain.The ratio of hyperpolarized ¹³C label in tumor lactate compared to the maximum pyruvate signal in the blood vessels was decreased from 0.38 ± 0.16 to 0.23 ± 0.13, (a reduction of 34%) by 72 h following whole brain irradiation with 15 Gy. Magn Reson Med, 2011. © 2010 Wiley‐Liss, Inc.     

Magnetization transfer measurements of exchange between hyperpolarized [1‐13C]pyruvate and [1‐13C]lactate in a murine lymphoma

Measurements of the conversion of hyperpolarized [1‐¹³C]pyruvate into lactate, in the reaction catalyzed by lactate dehydrogenase, have shown promise as a metabolic marker for the presence of disease and response to treatment. However, it is unclear whether this represents net flux of label from pyruvate to lactate or exchange of isotope between metabolites that are close to chemical equilibrium. Using saturation and inversion transfer experiments, we show that there is significant exchange of label between lactate and pyruvate in a murine lymphoma in vivo. The rate constants estimated from the magnetization transfer experiments, at specific points during the time course of label exchange, were similar to those obtained by fitting the changes in peak intensities during the entire exchange time course to a kinetic model for two‐site exchange. These magnetization transfer experiments may therefore provide an alternative and more rapid way of estimating flux between pyruvate and lactate to serial measurements of pyruvate and lactate ¹³C peak intensities following injection of hyperpolarized [1‐¹³C]pyruvate. Magn Reson Med 63:872–880, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Metabolic consequences of anoxia in the isolated, perfused guinea pig heart: anaerobic metabolism of endogenous amino acids

The metabolic consequences of anoxia in the isolated, perfused guinea pig heart were examined by 13C NMR spectroscopy of 13C-labeled metabolites in situ. Upon addition of [3-13C]pyruvate to the perfusate during normoxic conditions, label is detected in several metabolites, including alanine (C3), glutamate (C2, C3, and C4), and aspartate (C2 and C3), reaching steady state levels 10-15 min after the labeled precursor reaches the heart. During anoxia, the label in glutamate and aspartate decreases and label appears in C2(3) of succinate. This real-time observation demonstrates that in the isolated intact heart, anaerobic metabolism of the amino acids aspartate and glutamate to succinate occurs. These pathways, which were first noted to occur in skeletal muscle of diving mammals, may provide a mechanism supplemental to glycolysis for the production of nucleoside triphosphates during periods of anoxia.     

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.     

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.     

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.     

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.     

Localized proton NMR observation of [3-13C]lactate in stroke after [1-13C]glucose infusion.

To assess whether elevated lactate in stable stroke is being actively produced from blood glucose localized 1H NMR stimulated echo spectra were obtained from a patient in the region of a 32-day-old cortical infarct before and 60-100 min after infusion of [1-13C]glucose. Prior to the infusion the spectrum from the region of the infarct contained an elevated resonance from C3 lactate and a greatly reduced resonance from N-acetyl groups relative to an unaffected contralateral region. After the infusion two additional resonances were observed at 62 and -64 Hz relative to the unlabeled resonance of C3 lactate which were assigned on the basis of chemical shift and relative intensity to [3-13C]lactate. The [3-13C]lactate fractional enrichment in the infarct region was measured to be 32% which is within error one-half the average [1-13C]plasma glucose enrichment during the postinfusion NMR measurement. The result suggests that the stroke lactate pool was completely derived from infused glucose.     

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.     

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.     

NMR measurement of brain oxidative metabolism in monkeys using 13C-labeled glucose without a 13C radiofrequency channel.

We detected glutamate C4 and C3 labeling in the monkey brain during an infusion of [U-13C6]glucose, using a simple 1H PRESS sequence without 13C editing or decoupling. Point-resolved spectroscopy (PRESS) spectra revealed decreases in 12C-bonded protons, and increases in 13C-bonded protons of glutamate. To take full advantage of the simultaneous detection of 12C- and 13C-bonded protons, we implemented a quantitation procedure to properly measure both glutamate C4 and C3 enrichments. This procedure relies on LCModel analysis with a basis set to account for simultaneous signal changes of protons bound to 12C and 13C. Signal changes were mainly attributed to 12C- and 13C-bonded protons of glutamate. As a result, we were able to measure the tricarboxylic acid (TCA) cycle flux in a 3.9 cm3 voxel centered in the monkey brain on a whole-body 3 Tesla system (VTCA = 0.55 +/- 0.04 micromol x g(-1) x min(-1), N = 4). This work demonstrates that oxidative metabolism can be quantified in deep structures of the brain on clinical MRI systems, without the need for a 13C radiofrequency (RF) channel.     

Noninvaive observation of hepatic glycogen formation in man by13C MRS after Orl and intravenous glucose administration

The formation of glycogen in the liver of normal volunteers was followed noninvasively with ¹³C manetic resonance spectroscopy (MRS) under tow different conditions; a) intravenous infusion of [₁-¹³C] glucose under hyperglycemic and hyperinsullinemic clamp conditions, and b) oral Intake of glucose in the form of a bolus. For the intravenous infusion, [₁-¹³C]glucose with an enrichment level of 99% was employed. The C₁ signals of α- and β-glucose could be detected in the human liver already after an infusion period of 8 min. However, an increase in the glycogen signal was observed only after a prolonged infusion of about 60 min. Changes in the glycogen signal correlated well with the time course of insulin and glucagon during the measurement. Experiments showed also that liver glycogen formation in man can be followed noninvasively by¹³C-MRS using nonlabeled glucose or [₁-¹³C]glucose with a low level of enrichment (6.6%). The use of nonlabeled glucose may therefore simplify the quantitation of net liver glycogen synthesis since it can be based directly on changes in the natural abundance ¹³C MRS glycogen signal, avoiding label dilution through the various metabolic pathways of glucose. The glucose uptake, estimated from the increase in the glycogen signal, was consistent with findings from more complex and invasive studies of glucose uptake in the liver. The average liver glycogen concentration in 12 h overnight fasted volunteers (n = 18) without any special dietary preparation was assessed to be 229 ± 34 mM (minimum = 160 mM; maximum = 274 mM).     

Glucose production pathways by 2H and 13C NMR in patients with HIV-associated lipoatrophy.

Patients with HIV taking protease inhibitors were selected for the presence (five subjects) or absence (five subjects) of lipoatrophy. Following an overnight fast, subjects were given oral (2)H(2)O in divided doses (5 mL/kg body water), [U-(13)C(3)] propionate (10 mg/kg), and acetaminophen (1000 mg). Glucose (from plasma) or acetaminophen glucuronide (from urine) were converted to monoacetone glucose for (2)H NMR and (13)C NMR analysis. The fraction of plasma glucose derived from gluconeogenesis was not significantly different between groups. However, flux from glycerol into gluconeogenesis relative to glucose production was increased from 0.20 +/- 0.13 among subjects without lipoatrophy to 0.42 +/- 0.12 (P < 0.05) among subjects with lipoatrophy, and the TCA cycle contribution was reduced. Lipoatrophy was associated with an abnormal profile of glucose production as assessed by (13)C and (2)H NMR of plasma and urine.     

In vivo 13C nuclear magnetic resonance investigations of choline metabolism in rabbit brain

The metabolism of choline in rabbit brain was studied by the noninvasive approach of in vivo 13C NMR spectroscopy. 13C-Enriched precursors were introduced into the brain. Surgery of the head skin was avoided through controlled localization of the surface coil. For long-term accumulation studies in brain, repeated subcutaneous injections proved to be advantageous over other forms of application. The resorption kinetics was calculated to be zero order which suggests slow delivery from the subcutaneous depots. Choline metabolism was studied by two approaches: [N-13CH3]choline and S-[13CH3]methionine were administered separately to adult and myelinating rabbits (Days 5 to 32), respectively, over 4 weeks. [N-13CH3]Choline and the 13CH3 group of methionine were incorporated into lecithin and sphingomyelin of brain myelin. In vivo kinetic studies of the turnover of these labeled structures were carried out. Choline and methionine are readily transported through the blood-brain barrier and utilized by the myelinating brain for the biosynthesis of its phospholipids.     

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.