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.     

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.     

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.     

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).     

Simple measurement of gluconeogenesis by direct 2H NMR analysis of menthol glucuronide enrichment from 2H2O.

The contribution of gluconeogenesis to fasting glucose production was determined by a simple measurement of urinary menthol glucuronide (MG) 2H enrichment from 2H2O. Following ingestion of 2H2O (0.5% body water) during an overnight fast and a pharmacological dose (400 mg) of a commercial peppermint oil preparation the next morning, 364 micromol MG was quantitatively recovered from a 2-h urine collection by ether extraction and a 125 micromol portion was directly analyzed by 2H NMR. The glucuronide 2H-signals were fully resolved and their relative intensities matched those of the monoacetone glucose derivative. The pharmacokinetics and yields of urinary MG after ingestion of 400 mg peppermint oil as either gelatin or enteric-coated capsules 1 h before breakfast were quantified in five healthy subjects. Gelatin capsules yielded 197 +/- 81 micromol of MG from the initial 2-h urine collection while enteric-coated capsules gave 238 +/- 84 micromol MG from the 2- to 4-h urine collection.     

A 13C NMR study of the application of [U-13C]succinate for metabolic investigations in rabbit renal proximal convoluted tubular cells

The fate of the 13C label arising from [U-13C]succinate in rabbit renal proximal convoluted tubular (PCT) cells, incubated with [U-13C]succinate, was investigated by 13C NMR spectroscopy of the perchloric acid extracts of the cell pellets. The 13C label of [U-13C]succinate was predominantly metabolized into glutamate, glutamine, and into glucose, lactate, and alanine, which is the consequence of a carbon efflux of the label from the Krebs cycle at the stage of alpha-ketoglutarate and oxaloacetate, respectively.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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).     

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.     

Sources of hepatic glycogen synthesis during an oral glucose tolerance test: Effect of transaldolase exchange on flux estimates

Sources of hepatic glycogen synthesis during an oral glucose tolerance test were evaluated in six healthy subjects by enrichment of a 75‐g glucose load with 6.67% [U‐¹³C]glucose and 3.33% [U‐²H₇]glucose and analysis of plasma glucose and hepatic uridine diphosphate–glucose enrichments (sampled as urinary menthol glucuronide) by ²H and ¹³C nuclear magnetic resonance. The direct pathway contribution, as estimated from the dilution of [U‐¹³C]glucose between plasma glucose and glucuronide, was unexpectedly low (36 ± 5%). With [U‐²H₇]glucose, direct pathway estimates based on the dilution of position 3 ²H‐enrichment between plasma glucose and glucuronide were significantly higher (51 ± 6%, P = 0.05). These differences reflect the exchange of the carbon 4, 5, and 6 moiety of fructose‐6‐phosphate and glyceraldehyde‐3‐phosphate catalyzed by transaldolase. As further evidence of this exchange, ²H‐enrichments in glucuronide positions 4 and 5 were inferior to those of position 3. From the difference in glucuronide positions 5 and 3 enrichments, the fraction of direct pathway carbons that experienced transaldolase exchange was estimated at 21 ± 4%. In conclusion, the direct pathway contributes only half of hepatic glycogen synthesis during an oral glucose tolerance test. Glucose tracers labeled in positions 4, 5, or 6 will give significant underestimates of direct pathway activity because of transaldolase exchange. Magn Reson Med, 2009. © 2009 Wiley‐Liss, Inc.     

Oxidation of lactate in rats after short-term strenuous exercise

Oxidation of lactate and glucose was investigated in rats after short-term strenuous running to exhaustion at a speed of 80-110 m.min-1, lasting about 100 sec. Immediately after the exercise, 4 microCi of [U-14C]lactate (LA and AR) or 9.4 microCi of [U-14C]glucose (GL) was injected into the aorta through an indwelling catheter. In AR, the rats ran at a speed of 25 m.min-1 for 20 min after injection of [U-14C]lactate. Expired gas was collected by a bottomless metabolism chamber while the rats were on the treadmill for 120 min. Blood lactate concentration tended to decrease faster in AR than in LA. Peak evolution of 14CO2 expiration occurred at 12.5 min recovery in LA, 7.5 min of recovery in AR, and 35 min of recovery in GL. Cumulative percent recovery of 14C as 14CO2 was 48.5% +/- 2.8% in LA, 74.0% +/- 2.9% in AR, and 18.6% +/- 1.6% (mean +/- SE) in GL. Significant differences were found in these rates between groups (P less than 0.01). It was suggested that a great deal of lactate was oxidized directly, not after conversion to glucose in rats after short-term strenuous exercise to exhaustion and mild exercise following strenuous exercise (active recovery) enhanced lactate oxidation.     

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

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

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

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