Quantification of hepatic transaldolase exchange activity and its effects on tracer measurements of indirect pathway flux in humans.

Exchange of hepatic glucose-6-phosphate (G6P) and glyceraldehyde-3-phosphate via transaldolase modifies hepatic G6P enrichment from glucose or gluconeogenic tracers. Transaldolase exchange was quantified in five healthy, fed subjects following an oral bolus of [1,2,3-(13)C(3)]glycerol (25-30 mg/kg) and paracetamol (10-12 mg/kg). (13)C Isotopomers of hepatic G6P were quantified by (13)C NMR spectroscopy of urinary glucuronide. [1,2,3-(13)C(3)]- and [4,5,6-(13)C(3)]glucuronide isotopomers, representing the conversion of [1,2,3-(13)C(3)]glycerol to G6P via dihydroxyacetone phosphate, were resolved from [1,2-(13)C(2)]- and [5,6-(13)C(2)]glucuronide (13)C-isotopomers, derived from metabolism of [1,2,3-(13)C(3)]glycerol via pyruvate and phosphoenolpyruvate. Enrichment of [1,2,3-(13)C(3)]glucuronide was significantly less than that of [4,5,6-(13)C(3)]glucuronide (1.30 +/- 0.57% versus 1.67 +/- 0.42%, P < 0.05). Also, [1,2-(13)C(2)]glucuronide enrichment was significantly less than that of [5,6-(13)C(2)]glucuronide (0.28 +/- 0.08% versus 0.36 +/- 0.03%, P < 0.05). Transaldolase and triose phosphate isomerase exchange activities were estimated by applying the (13)C-isotopomer data to a model of hepatic sugar phosphate metabolism. Triose phosphate isomerase exchange was approximately 99% complete and did not contribute significantly to the unequal (13)C-isotopomer distributions of the glucuronide triose halves. Instead, this was attributable to 25 +/- 23% of hepatic G6P flux undergoing transaldolase exchange. This results in substantial overestimates of indirect pathway contributions to hepatic glycogen synthesis with tracers such as [5-(3)H]glucose and (2)H(2)O.     

Simultaneous separation of intracellular and extracellular lactate NMR signals of human erythrocytes.

Intracellular/extracellular lactate (Lac) distribution has been determined before in human and animal erythrocytes (red blood cells [RBCs]) with various methods. However, all previous methods determine intra- and extracellular Lac separately or indirectly. Now, (13)C-NMR spectroscopy has been used to monitor intra- and extracellular Lac simultaneously in intact RBCs. Isolated human RBCs were incubated with [3-(13)C]-Lac, [3-(13)C]-pyruvate (Pyr), and [1-(13)C]-glucose (Gluc). A distortionless enhancement by polarization transfer (DEPT) sequence was used (TR = 3.3 s, N = 128) to monitor the (13)C-NMR resonances in both compartments. The intra- and extracellular methyl group resonances of Lac and Pyr were clearly separated by 9.6 Hz and 7.0 Hz, respectively, under normoxic conditions due to the RBC chemical-shift effect. The results show that the chemical-shift effect of RBCs is convenient to monitor intra- and extracellular Lac simultaneously in intact RBCs under normoxic conditions.     

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.     

A versatile multinuclear probe designed for in vivo NMR spectroscopy: applications to subcutaneous human tumors in mice

We describe a versatile NMR probe that is designed for a variety of in vivo spectroscopic studies on small animals in vertical wide-bore magnets. Replaceable brackets enable the coils to be exchanged readily in order to observe 1H, 13C, 31P, and other nuclei, and to carry out double-resonance experiments. Two solenoidal coil designs are described and applied to observe 31P, 13C, and 1H natural abundance spectra of subcutaneously implanted human tumors in mice. For 31P and 31C observation with 1H decoupling, a concentric coil arrangement was employed with a broadband inner coil and the outer coil tuned to 1H at 400 MHz. A single coil tuned to 400 MHz was used to observe 1H resonances. A thin copper foil design was found to be superior with respect to S/N and resolution to previously described Faraday shields used to shield the NMR signals originating from nontumor tissues. 31P spectra of in vivo tumor tissue were compared to spectra of in vitro perfused tumor cells of the same origin. Tumor tissue in vivo exhibited much higher levels of inorganic phosphate and phosphocreatine. Signals from [13C2]glucose and its major metabolite, [13C2]lactate, were readily observed and monitored in an unobstructed region of the 13C spectra of tumor tissue in vivo following the injection of [13C2]glucose in adjacent tissues. A 1H spectrum of tumor tissue, characterized by five broad resonances, was observed with excellent water suppression.     

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.     

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 detection of glioma glycolysis using edited 1H MRS

In vivo ¹H MRS can be used to detect and quantify the lactate resonance at 1.3 ppm provided that overlapping lipid resonances are eliminated. A homonuclear spectral editing method was developed to acquire uncontaminated ¹H spectra of lactate with adiabatic pulses. An advantage of the adiabatic pulse sequence is the ability to induce uniform flip angles and to maximize sensitivity in applications employing surface coil transmitters which produce highly inhomogeneous B₁. Glycolytic activity in an intracerebral C6 glioma in rats was monitored by using adiabatic editing sequences to observe [3-¹³C]lactate produced from infused [1-¹³C]glucose. Acute hyperglycemia (serum glucose >22 mM, n = 10) had no significant effect (P = 0.08) on the total ([¹²C]+ [¹³C]) tumor lactate signal intensity.     

In vivo monitoring of hepatic glutathione in anesthetized rats by 13C NMR.

A method for in vivo (13)C NMR monitoring of hepatic glutathione (GSH) in intact, anesthetized rats has been developed. Studies were conducted using a triple-tuned, surgically implanted surface coil designed for this animal model. The coil permitted complete decoupling and sufficient resolution in the (13)C NMR spectrum to monitor the time course of hepatic (13)C-metabolites of intravenously administered 2-(13)C-glycine, particularly GSH at 44.2 ppm and serine signals at 61.1 and 57.2 ppm, respectively. It further allowed concomitant monitoring of high-energy phosphagens and intracellular pH by (31)P NMR. To confirm in vivo NMR peak assignments, we compared high-resolution 2D (1)H[(13)C] heteronuclear multiple quantum coherence and 1D (13)C spectra of hepatic perchloric acid extracts to those of authentic standards. The fractional isotopic enrichment of hepatic (13)C-glycine increased exponentially at a rate of 1.68 h(-1) and reached its plateau level of 81% in 2 h. The (13)C fractional isotopic enrichment of GSH increased exponentially at a rate of 0.316 h(-1) and reached 55% after 4 h of 2-(13)C-glycine infusion, but without achieving a plateau. To confirm that the resonance at 44.2 ppm resulted from GSH, a rat was given an intravenous dose of 2-oxothiazolidine-4-carboxylic acid (OTC), a cysteine precursor that increases intracellular GSH. As expected, with OTC administration the hepatic (13)C GSH-to-glycine peak area increased more than sevenfold.     

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.     

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.     

Semiselective POCE NMR spectroscopy.

A new scheme is proposed to edit separately glutamate C(3) and C(4) resonances of (1)H bound to (13)C, in order to resolve these two signals which overlap at intermediate magnetic fields (1.5 T-3 T), commonly available for human brain studies. The two edited spectra are obtained by combining the individual acquisitions from a four-scan measurement in two different ways. The four acquisitions correspond to the two steps of the classical POCE scheme combined with another two-scan module, where the relative phases of the C(3) and C(4) (1)H resonances are manipulated using zero quantum and double quantum coherence pathways. This new technique exhibits the same sensitivity as POCE and allows the (13)C labeling of C(3) and C(4) glutamate from [1-(13)C]glucose to be monitored separately in the rat brain at 3 T.     

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.     

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.     

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.     

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.     

Hepatic gluconeogenesis from alanine: 13C nuclear magnetic resonance methodology for in vivo studies

This paper describes an experimental protocol designed to optimize 13C NMR spectra from the liver of the living anesthetized rat at 1.9 T. The protocol involves the use of a Helmholtz NMR coil which is positioned around the liver after surgical exposure. 1H decoupling is facilitated by double tuning this coil to both the 1H and the 13C frequencies. The protocol was shown to be suitable for studying the hepatic metabolism of 13C-labeled substrates in vivo by investigating the metabolism of [3-13C]alanine. Labeled glucose, glutamate, glutamine, and aspartate were formed and detected by 13C NMR in vivo in this experiment. The labeling patterns in these metabolites provided evidence that the major flow of alanine carbon into the Krebs cycle is via the pyruvate carboxylase reaction rather than through pyruvate dehydrogenase.     

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.     

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.     

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.