Double pulsed field gradient diffusion MRI to assess skeletal muscle microstructure.

PURPOSE: Preliminary study to determine whether double pulsed field gradient (PFG) diffusion MRI is sensitive to key features of muscle microstructure related to function. METHODS: The restricted diffusion profile of molecules in models of muscle microstructure derived from histology were systematically simulated using a numerical simulation approach. Diffusion tensor subspace imaging analysis of the diffusion signal was performed, and spherical anisotropy (SA) was calculated for each model. Linear regression was used to determine the predictive capacity of SA on the fiber area, fiber diameter, and surface area to volume ratio of the models. Additionally, a rat model of muscle hypertrophy was scanned using a single PFG and a double PFG pulse sequence, and the restricted diffusion measurements were compared with histological measurements of microstructure. RESULTS: Excellent agreement between SA and muscle fiber area (r2 = 0.71; p < 0.0001), fiber diameter (r2 = 0.83; p < 0.0001), and surface area to volume ratio (r2 = 0.97; p < 0.0001) in simulated models was found. In a scanned rat leg, the distribution of these microstructural features measured from histology was broad and demonstrated that there is a wide variance in the microstructural features observed, similar to the SA distributions. However, the distribution of fractional anisotropy measurements in the same tissue was narrow. CONCLUSIONS: This study demonstrates that SA-a scalar value from diffusion tensor subspace imaging analysis-is highly sensitive to muscle microstructural features predictive of function. Furthermore, these techniques and analysis tools can be translated to real experiments in skeletal muscle. The increased dynamic range of SA compared with fractional anisotropy in the same tissue suggests increased sensitivity to detecting changes in tissue microstructure.

Non-contrast MR lymphography of rare lymphatic abnormalities.

Detailed imaging of the lymphatic system often requires direct injection of contrast into lymph nodes which can be technically challenging, time consuming, and produce painful stimuli. We sought to describe the use of non-contrast MR lymphography (NCMRL) for normal controls and patients with a variety of rare disorders associated with lymphatic pathologies. Two control subjects and five affected patients (decompensated Fontan circulation, central conducting lymphatic abnormality, familial lymphedema and two with Gorham-Stout disease) were studied. NCMRL images were segmented in a semiautomated fashion and the major lymphatic channels and thoracic duct (TD) highlighted for identification. Adequate imaging was available for both controls and 4/5 affected patients; the youngest patient could not be imaged given patient motion. For the two controls, the TD was seen in the expected anatomic location. For the decompensated Fontan patient, there were numerous tortuous lymphatic channels, predominantly in the upper chest and neck. For the familial lymphedema patient, a TD was not identified; instead, peripheral lymphatic collaterals near the lateral chest walls. For the first Gorham- Stout patient, the TD was enlarged with large intrathoracic lymph collections. For the second Gorham-Stout patient, there were bilateral TD with lymph collections in vertebral bodies. Using NCMRL, we were able to image normal and abnormal lymphatic systems. An important learning point is the potential need for sedation for younger patients due to long image acquisition times and fine resolution of the structures of interest.

Estimation of visceral fat in 9- to 13-year-old girls using dual-energy X-ray absorptiometry (DXA) and anthropometry.

OBJECTIVES: Accumulation of visceral fat (VF) in children increases the risk of cardiovascular disease and type 2 diabetes, and measurement of VF in children using computed tomography and magnetic resonance imaging (MRI) is expensive. Dual-energy X-ray absorptiometry (DXA) may provide a low-cost alternative. This study aims to determine if DXA VF estimates can accurately estimate VF in young girls, determine if adding anthropometry would improve the estimate and determine if other DXA fat measures, with and without anthropometry, could be used to estimate VF in young girls. METHODS: Visceral fat was measured at lumbar intervertebral sites (L1-L2, L2-L3, L3-L4 and L4-L5) using 3.0T MRI on 32 young girls (mean age 11.3 ± 1.3 years). VF was estimated using the GE CoreScan application. Measurement of DXA android and total body fat was performed. Weight, height and waist circumference (WC) measurements were also obtained. RESULTS: Waist circumference and body mass index were both strongly correlated with MRI, although WC was the best anthropometric covariate. Per cent fat (%fat) variables had the strongest correlation and did best in regression models. DXA %VF (GE CoreScan) and DXA android %fat and total body %fat accounted for 65% to 74% of the variation in MRI VF. CONCLUSION: Waist circumference predicted MRI VF almost as well as DXA estimates in this population, and a combination of WC and DXA fat improves the predictability of VF. DXA VF estimate was improved by the addition of WC; however, DXA android %fat with WC was better at predicting MRI VF.

Quantitative volumetric analyses of brain magnetic resonance imaging from rat with chronic neuroinflammation.

Brain inflammation may have a pathogenic role in many neurodegenerative diseases, including Alzheimer's disease. In the present study, we investigated the effects of chronic neuroinflammation upon anatomical changes in two regions of interest in the temporal lobe using high-resolution magnetic resonance imaging techniques. We show that chronic infusion of lipopolysaccharide into the fourth ventricle for 4 consecutive weeks enlarged the lateral ventricles and significantly decreased the size of the hippocampal formation and the temporal lobe region. These changes are comparable to those observed in humans during the early stages of Alzheimer's disease.

In vivo imaging of extracellular pH using 1H MRSI.

Tumor pH is physiologically important since it influences a number of processes relevant to tumorigenesis and therapy. Hence, knowledge of localized pH within tumors would contribute to understanding these processes. The destructiveness, poor spatial resolution, and poor signal-to-noise ratio (SNR) of current technologies (e.g., microelectrodes, 31P magnetic resonance spectroscopy) have limited such studies. An extrinsic chemical extracellular pH (pHe) probe is described that is used in combination with 1H magnetic resonance spectroscopic imaging to yield pHe maps with a spatial resolution of 1 x 1 x 4 mm3. The principle of the technique is demonstrated on a phantom. Further data are shown to demonstrate its application in vivo, and results agree with previously reported pH values. The accuracy of the reported pH measurements is <0.1 pH units, as derived from a detailed analysis of the errors associated with the technique, the description of which is included.

Early increases in breast tumor xenograft water mobility in response to paclitaxel therapy detected by non-invasive diffusion magnetic resonance imaging.

An important goal in cancer chemotherapy is to sensitively and quantitatively monitor the response of individual patients' tumors to successful, or unsuccessful, therapy so that regimens can be altered iteratively. Currently, tumor response is monitored by frank changes in tumor morphology, yet these markers take long to manifest and are not quantitative. Recent studies suggest that the apparent diffusion coefficient of water (ADCw), measured noninvasively with magnetic resonance imaging, is sensitively and reliably increased in response to successful CTx. In the present study, we investigate the combination chemotherapy response of human breast cancer tumor xenografts sensitive or resistant to Paclitaxel by monitoring changes in the ADCw. Our results indicate that there is a clear, substantial, and early increase in the ADCw after successful therapy in drug sensitive tumors and that there is no change in the ADCw in p-glycoprotein-positive tumors, which are resistant to Paclitaxel. The mechanism underlying these changes is unknown yet is consistent with apoptotic cell shrinkage and a concomitant increase in the extracellular water fraction.

Hemodialysis increases apparent diffusion coefficient of brain water in nephrectomized rats measured by isotropic diffusion-weighted magnetic resonance imaging.

The nature of brain edema in dialysis disequilibrium syndrome (DDS) was investigated by diffusion-weighted magnetic resonance imaging (DWI). DWI was performed on normal or bilaterally nephrectomized rats before, and immediately after, hemodialysis. Hemodialysis was performed with a custom-made dialyzer (surface area 150 cm2) against a bicarbonate-buffered bath for 90 min with or without 70 mM urea. Hemodialysis with non-urea bath decreased plasma urea by 21 mM, and plasma osmolality by 22 mosmol/kg H2O, and increased brain water content by 8.0% (all < 0.05), while hemodialysis with urea bath did not affect plasma urea, osmolality, or brain water content. Three sets of axial DWI images of the brain were obtained at different gradient weighing factors with an in-plane resolution of 0.39 mm2. The apparent diffusion coefficient (Dapp) of the brain water was not affected by bilateral nephrectomy, or by hemodialysis in normal rats. In nephrectomized rats, brain Dapp was significantly increased after dialysis with non-urea bath (1.15 +/- 0.08 vs 0.89 +/- 0.07 x 10(-9)m2/sec, P < 0.01). No significant changes of brain water Dapp could be observed after dialysis with urea bath. The increased Dapp associated with DDS indicates that brain extracellular water increases and/or intracellular water decreases after hemodialysis. Our results strongly suggest that the brain edema induced by hemodialysis in uremic rats is due to interstitial edema rather than cytotoxic edema. Furthermore, our results support a primary role for the "reverse urea effect" in the pathogenesis of brain edema in DDS.DWI may be a useful diagnostic tool for DDS in patients with end-stage renal disease.

Increase of GPC levels in cultured mammalian cells during acidosis. A 31P MR spectroscopy study using a continuous bioreactor system.

The purpose of this study was to study the metabolic events during a slow acidosis in three different cell lines by combining 31P magnetic resonance spectroscopy and hollow fiber bioreactor technology. The rate of change in intracellular pH, glycerophosphorylcholine (GPC), phophorylcholine (PCho), and nucleoside-triphosphate (NTP) levels were measured during 8 h of acidosis and 16 h of recovery in EPO, EAT, and RN1a cells, three cultured mammalians cell lines. Our results show a significant increase in GPC levels to 330 +/- 21.540 +/- 25, and 220 +/- 21% of their initial value correlated to a decrease of PCho levels to 57 +/- 14.58 +/- 17 and 45 +/- 15% of their initial value in EAT, RN1a, and EPO cells, respectively. These changes are discussed in terms of perturbation of energetic metabolism in cells undergoing a slow acidosis.

Design and application of NMR-compatible bioreactor circuits for extended perfusion of high-density mammalian cell cultures.

MR spectroscopy of cultured cells allows non-invasive analyses of the metabolism of cells with specific phenotypes under defined conditions. This technique can be used to investigate the intracellular metabolism of cells or extended to critically evaluate phenomena observed by in vivo MRS. In this paper, a cell maintenance system is described which allows MR analyses with unparalleled spectral resolution, S/N and stability. This system consists of a 25 mm diameter hollow fiber bioreactor and a supporting circuit. The hollow fiber reactor was chosen because it yields a large filling factor which can be perfused through defined volumes. The fibers were 300 microns diameter microporous (0.2 micron) cellulose acetate/cellulose nitrate membranes with high porosity, which allow bulk convective flow throughout the extracapillary space. This flow (Starling flow) is necessary to disrupt steady-state gradients in substrates and waste products. In many respects, the design of the supporting circuit is more important than the bioreactor itself, since it provides the reactor with the proper chemical and physical environment. Hence, this circuit can be applied to a variety of bioreactor configurations. The circuit consists of a hollow fiber oxygenator and a bleed-and-feed system housed in a temperature-controlled cabinet. Culture of mammalian cells in this reactor yields 31P spectra which have excellent spectral and temporal resolution. At confluence, endogenous 31P line widths were typically < 10 Hz (at 162 MHz) and well resolved spectra were obtained in < 30 s.

Energetic metabolism of glucose, mannose and galactose in glucose-starved rat insulinoma cells anchored on microcarrier beads. A phosphorus-31 NMR study.

Insulin-secreting cells (RINm5F) have successfully been grown on a large scale on poly-L-lysine coated-polystyrene microcarriers, providing a high cell number in a restricted volume under conditions that respect the metabolic integrity of these anchorage-dependent cells. The energetic metabolism of the perfused cells has been followed non-invasively by phosphorus-31 nuclear magnetic resonance spectroscopy. Glucose starvation induced a rapid decrease in nucleoside triphosphates (mainly ATP) pools, correlated with an increase in Pi level. The initial ATP level was rapidly recovered when the cells were refed with glucose or with mannose, but not with galactose, even after 2 h of perfusion. These differential effects of hexoses on energetic metabolism might be related to their various insulin-release actions on tumor islet cells.

A multinuclear magnetic resonance study of a cls11 mutant showing the Pet- phenotype of Saccharomyces cerevisiae.

Energetic and intermediary metabolism was studied in a Pet- mutant of Saccharomyces cerevisiae with a calcium-sensitive phenotype that shows an inability to grow when cultured in a medium containing non-fermentable substrates. The perchloric acid extracts were prepared from suspensions of cls11 mutant and wild-type cells incubated with [1,3-13C]glycerol or [2-13]acetate, and analyzed by 31P, 13C and 1H NMR. 31P- and 1H-NMR spectra showed significant differences between cls11 and wild-type cells at the level of amino acids, the storage carbohydrate trehalose (higher in mutant cells), and sugar phosphates (higher in wild-type cells). 13C-NMR spectra revealed major differences in the steady-state labelling of glutamate carbons. For incubations with [1,3-13C]glycerol, we estimated from the relative 13C enrichment of glutamate carbons that acetyl-CoA C2 is 43% C13 labelled in wild-type and 10% 13C labelled in mutant cells, respectively. For incubations with [2-13C]acetate, we calculated that the ratio of the relative flux through the glyoxylate shunt versus oxidative reactions is 58% in wild-type cells and 44% in the cls11 mutant cells. Again, a dilution of the relative enrichment of C2 of acetyl-CoA was observed in the mutant cells (89%) compared to the wild-type cells (97%). These results are discussed in terms of pleiotropic defects in non-fermentable carbon metabolism in mutant cells.

Effect of VIP on the glycogen metabolism of human colon adenocarcinoma cells studied by 13C nuclear magnetic resonance spectroscopy.

Metabolic pathways of glucose utilization have been investigated in a human colon adenocarcinoma cell line (HT29) using carbon-13 Nuclear Magnetic Resonance spectroscopy. HT29 cells were adapted to grow on a polystyrene beaded microcarrier and were perfused when attached to the beads in a specially designed NMR cell. Abnormalities in carbohydrate metabolism already observed in several cancer cells were studied in HT29 cells fed with (1-13C)-enriched glucose. The cells were first perfused with a glucose-free medium for 2 h in order to deplete the intracellular store of glycogen, and they were subsequently perfused with a medium containing enriched glucose at an initial concentration of 5.5 mM. Sequential 13C-NMR spectra, recorded at 100.5 MHz (5 min accumulation), show that HT29 cells were able to utilize glucose through the glycolytic pathway while storing glucose as glycogen (glucose was utilized at a rate of 3.9 mumol/mg protein/hr). The glycolytic activity determined by the amount of lactic acid produced was 4.6 microns/mg protein/hr, corresponding to the formation of 1.2 lactic acid per glucose molecule. Glycogen accumulation corresponded to 16 micrograms/mg of protein. Treatment of HT29 with 10 nM vasoactive intestinal peptide (VIP) induced a transient decrease in the level of labelled glycogen to 50% of the initial value. Control level was recovered 12 min after VIP loading.

Metabolic changes in undifferentiated and differentiated human colon adenocarcinoma cells studied by multinuclear magnetic resonance spectroscopy.

Aspects of energetic and intermediary metabolism were studied in a colon adenocarcinoma cell line (HT29) by multinuclear magnetic resonance spectroscopy. Experiments were carried out on the HT29-D4 clone, which was isolated by limit dilution techniques. This clone, usually undifferentiated (D4-UD), can be maintained in a differentiated state (D4-D) in a glucose-free medium. Metabolic data were obtained by NMR analysis of perchloric acid extracts from D4-UD and D4-D cells. Phosphorus-31 and proton NMR spectra showed the presence of a large amount of choline and phosphorylcholine in the differentiated state (400% and 200%, respectively, of the levels found in D4-UD cells). Other differences appeared in the content of phosphocreatine (absent in D4-D cells) and myoinositol (absent in D4-UD cells). Carbon-13 spectra were recorded from perchloric acid extracts of cells incubated with [1-13C]-labeled glucose or [2-13C]-labeled acetate. The data indicated that both types of cells metabolize glucose through the glycolytic pathway to give lactate, but only D4-D cells were able to store glucose as glycogen at a very high level. A mathematical analysis of fluxes through the tricarboxylic acid (TCA) cycle was developed on the basis of models derived from previous 14C tracer studies. The model was based on the steady-state labeling of glutamate carbons by the 13C isotope and gave the fraction of labeled acetyl-Coa entering the TCA cycle, and the activity y of anaplerotic reactions relative to the flux through the citrate synthetase reaction. The data indicated that y greater than 0.3 in all cases. Only 15% and 30% of labeled acetyl CoA entered the TCA cycle in D4-UD and D4-D cells, respectively, under labeled glucose incubation: these values were significantly different upon labeled acetate feeding, reaching 55% for D4-UD cells and 85% for D4-D cells. The main result of this study is that the process of differentiation of HT29 cells is correlated with a large increase in the activity of oxidative metabolism.

Phosphorus-31 nuclear magnetic resonance study of the C6 glioma cell line cultured on microcarrier beads.

The energetic metabolism of perfused C6 glioma cells anchored and cultured on polystyrene microcarrier beads has been studied by phosphorus-31 nuclear magnetic spectroscopy (NMR). The observation of intracellular phosphorylated compounds demonstrates the metabolic long-lasting viability of the perfused cells. The effect of glucose deprivation on energetic metabolism and intracellular pH illustrates the existence of an active aerobic glycolysis. The non-invasive study of anchored C6 cells by NMR provides a direct means to investigate the metabolism of glioma cells.

Entrapment of gadolinium-DTPA in liposomes. Characterization of vesicles by P-31 NMR spectroscopy.

The use of paramagnetic ion chelates to enhance contrast between pathologic and surrounding parenchyma is extensively documented in the magnetic resonance imaging (MRI) literature. Liposomes can be used to increase chelate concentration in the pathologic area, thereby enhancing the efficiency of paramagnetic compounds as contrast agents. Liposomes (50 +/- 20 nm) were prepared by sonicating a solution of dipalmitoylphosphatidylcholine and cholesterol containing 16.5 mM Gadolinium-DTPA (Gd-DTPA) in pharmaceutical formulation (Schering Laboratories, France) and 25 mM inorganic phosphate (Pi). The solutions were dialyzed against 0.9% NaCl before analysis by phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy. Spectra of liposomes displayed a sharp resonance ascribed to Pi and a broad signal arising from the phosphate groups of the phospholipid bilayer. The content of Gd-DTPA in liposomes was directly estimated, based on specific modifications of the longitudinal relaxation rate of intraliposomal Pi. Entrapment ratio was estimated by P-31 NMR spectroscopy and atomic absorption spectroscopy to represent 2.5% to 5% of the initial Gd-DTPA content in the solution. This work illustrates the usefulness of NMR spectroscopy in the characterization of liposomes to be used for MRI applications.

Growth in serum-free medium of human colonic adenocarcinoma cell lines on microcarriers: a two-step method allowing optimal cell spreading and growth.

Human colonic adenocarcinoma cells have been successfully grown on polystyrene microcarriers by modifying the culture conditions used in monolayer culture. The method can be divided into two culture phases: a) a phase of spreading, wherein cells were seeded in presence of serum-supplemented medium; b) a phase of active growth wherein spread cells on the beads were allowed to grow in a serum-free medium. Under these conditions, optimal spreading and growth of HT 29 and HRT 18 cells on the microcarriers were obtained. A differential propagation was observed between HT 29-D4 and HT 29-D9 cells (both clonal populations derived from HT 29 cells) on the microcarriers that is tentatively related to the discrepancy observed in the spreading efficiency of these clonal cells on serum-coated culture flasks. An index of spreading efficiency (IS index) has been defined to quantify the efficiency of spreading of each cell line on microcarriers. These data gave the opportunity to develop serum-free, scale-up methods to culture cells like HT 29 which release potentially useful products.

Growth of a human colonic adenocarcinoma cell line (HT 29) on microcarrier beads: metabolic studies by 31phosphorus nuclear magnetic resonance spectroscopy.

A method allowing the growth of a human colon adenocarcinoma cell line (HT 29) on beaded polystyrene microcarriers has been developed by modifying the culture conditions used in monolayer cultures. Under optimized conditions, the cells became confluent 7 days after seeding and reached a density of 2.8 X 10(5) cells/cm2 of microcarrier (65% of the available area occupied). 31P NMR spectra were typically recorded on 300 X 10(6) cells continuously perfused at a flow rate of 15 ml/min in a specially designed NMR chamber in which the microcarrier beads were sequestered within the receiver coil volume. The in vivo spectrum displays a series of resonances assigned to nucleoside triphosphates (ATP and GTP), inorganic phosphate and various phosphomonoesters (mainly glucose-6-P and phosphorylcholine). Diphosphodiester resonances (DPDE, mainly UDP-N-acetyl-glucosamine and UDP-N-acetylgalactosamine) were not detected in the in vivo spectrum and were only apparent in the spectrum of the perchloric acid extract of the cells, indicating that these compounds have a restricted mobility in the intracellular compartment. The intracellular pH of HT 29 cells was 7.2 during the perfusion with a medium buffered at pH 7.3. The internal pH decreased slowly (2 X 10(-3) pH unit/min) during anoxic perfusion, but severe intracellular acidosis occurred after 40 min of ischemia (2.7 X 10(-2) pH unit/min). Sequential recording of 31P NMR spectra has shown that HT 29 cells are able to maintain their high energy phosphorylated compound levels (ATP) when subjected to 100 min of anoxia and 40 min of total ischemia.

31P nuclear magnetic resonance study of a human colon adenocarcinoma cultured cell line.

31P nuclear magnetic resonance (NMR) spectroscopy has been used to monitor the energy metabolism in a human colon adenocarcinoma cell line (HT 29). NMR spectra were recorded at 80.9 MHz on approximately 2.5 X 10(8) cells continuously perfused with culture medium within a 20-mm NMR sample tube. Typical NMR spectra display a series of well-resolved resonances assigned to nucleoside triphosphates (mainly adenosine 5'-triphosphate), uridine diphosphohexose derivatives (uridine 5'-diphosphate-N-acetylglucosamine, uridine 5'-diphosphate-N-acetylgalactosamine, uridine 5'-diphosphate-glucose), intra- and extracellular inorganic phosphate, and phosphomonoesters (mainly phosphorylcholine and glucose 6-phosphate). Measurement of phosphorylated metabolite concentrations from the intensity of NMR signals is in good agreement with the results provided by conventional biochemical assays. 31P NMR allows to follow noninvasively the effect of anoxia on HT 29 cells. The results indicate that the cells are able to maintain about 60% of their initial nucleoside triphosphate level after 2 h of anaerobic perfusion. Cells accumulate inorganic phosphate during anoxia and the intracellular-extracellular pH gradient increases from 0.5 in well-oxygenated cells to more than 1 pH unit under anoxic conditions. The value of intracellular pH of well-oxygenated HT 29 cells is 7.1. The effect of glucose starvation upon energy metabolism has also been examined in real time by NMR: a rapid decline of adenosine 5'-triphosphate down to 10% of the initial value is observed over a period of 2 h. In contrast, the level in uridine diphosphohexoses reaches a new steady state value representing 60% of the initial one. Refeeding the cells with 25 mM glucose leads to a dramatic drop of internal pH reflecting the activation of the glycolytic pathway.

Carbon-13 and phosphorus-31 NMR study of hepatic metabolism in the perfused rat liver.

Phosphorus-31 nuclear magnetic resonance (NMR) has been used to determine non-invasively absolute concentrations of phosphorylated metabolites in the perfused rat liver. It has been shown that the NMR method does detect cytoplasmic ATP and ADP (ATP:ADP ratio of 15 +/- 3) with no contribution from mitochondrial adenine nucleotides. The concentration of ATP was 7.2 +/- 0.3 mM in the cytosol of well-oxygenated liver, after two hours of perfusion with a Krebs-Ringer buffer. Other phosphorylated metabolites were detected, mainly inorganic phosphate (1.1 mumol/g liver wet weight), phosphorylcholine (1.0 mumol/g wet weight), glycerophosphorylethanolamine (0.34 mumol/g wet weight) and glycerophosphorylcholine (0.30 mumol/g wet weight). The intracellular pH measured from the position of the Pi resonance has a value of 7.2 +/- 0.1. It is likely that the detectable Pi originates from the cytosolic compartment since a pH value of 7.4-7.6 would be expected for the mitochondrial matrix. Natural abundance carbon-13 NMR has also been used to follow the glycogen breakdown in situ by measuring the intensity of the glycogen C-1 resonance in the perfused liver spectrum as a function of the perfusion time. The glycogenolytic process has been studied as a function of the glucose content of the perfusate. Rate of glycogenolysis from 2.7 to 0.16 muEq glycosyl units g wet weight-1 min-1 were found when glucose concentration in the perfusate was varied from 0 to 50 mM. The fate of 90% enriched [2-13C] acetate has been studied in the perfused rat liver by 13C-NMR in order to investigate the mitochondrial metabolism and the interrelations between cytosolic and mitochondrial pools of metabolites. Some compounds of the intermediary metabolism where found to be extensively labelled, e.g. glutamate, glutamine, acetoacetate and beta-hydroxybutyrate. Under our experimental conditions, labelling of glutamate reached a steady-state within 30 min after the onset of perfusion of 20 mM acetate. In addition, the observed incorporation of carbon-13 isotope into glutamine can be linked to the operation of the glutamate-glutamine antiporter and to the high activity of the cytosolic glutamate synthetase. The finding of both active glutaminase and glutamine synthetase activity in the same liver cells is an evidence of the existence of an active glutamine-glutamate futile cycle.

[Nuclear magnetic resonance spectroscopy. New tool for investigating cellular metabolism in living systems]. / La spectroscopie de résonance magnétique nucléaire. Un nouvel outil d'investigation du métabolisme cellulaire dans les systèmes vivants.

Nuclear magnetic resonance spectroscopy is increasingly used to study cellular metabolism in a manner respecting cell integrity. The contribution of phosphorus-31 and carbon-13 NMR is discussed and illustrated by specific examples taken from work carried out in this laboratory or from the literature. A particular emphasis is layed on metabolite identification, quantitation and fluxes as studied by phosphorus-31 NMR which provides a direct insight of energy metabolism. The analysis of perfused rat liver by natural abundance carbon-13 NMR illustrates the potential of the method of study non-invasively lipid and carbohydrate metabolism in living systems. The use of carbon-13 enriched substrates to pinpoint a specific pathway of the intermediary metabolism is described in the case of excised rat heart and liver perfused with (2-C13)-acetate. Clinical applications of NMR spectroscopy are rapidly reviewed.