NMR Studies of Metabolism of Cells and Perfused Organs

Kaplan, Ofer; Cohen, Peter C. M. van; Cohen, Jack S.

doi:10.1007/978-3-642-77218-4_1

Cited by 13 publications

(9 citation statements)

References 309 publications

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“…For the same purpose, transaminase reactions were inhibited by aminooxyacetate to investigate the role of transaminases in ammonia metabolism. Multinuclear magnetic resonance spectroscopy (NMR) is a method well-suited to the analysis of cell metabolism [37,38]. 1 H and 31 P-NMR spectra of perchloric acid cell extracts were recorded to quantify water-soluble metabolites and to monitor the energy status of the cells.…”

Section: Introductionmentioning

confidence: 99%

Multinuclear NMR Spectroscopy Studies on NH<sub>4</sub>Cl-Induced Metabolic Alterations and Detoxification Processes in Primary Astrocytes and Glioma Cells

et al. 1998

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Glutamine synthesis, the major pathway of ammonia detoxification, and the intracellular concentration of organic osmolytes in primary astrocytes and F98 glioma cells were investigated with multinuclear magnetic resonance spectroscopy. Acute exposure to ammonia (3 h incubation with NH₄Cl) raised the concentration of glutamine and other amino acids, such as glutamate and aspartate, and decreased myo-inositol, hypotaurine, and taurine concentrations. The loss of these osmolytes was partially reversed by co-treatment with the glutamine synthetase inhibitor, methionine sulphoximine. Glutamate, the precursor of glutamine, is provided by stimulated anaplerotic flux via pyruvate carboxylase and glutamate dehydrogenase activity. Thus, the glutamine increase and myo-inositol decrease observed by in vivo magnetic resonance spectroscopy on patients with hepatic encephalopathy may be due to the disturbed osmoregulation in astrocytes caused by accumulation of glutamine and the subsequent loss of organic osmolytes.

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Section: Introductionmentioning

confidence: 99%

Multinuclear NMR Spectroscopy Studies on NH<sub>4</sub>Cl-Induced Metabolic Alterations and Detoxification Processes in Primary Astrocytes and Glioma Cells

et al. 1998

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“…Perhaps, this could be done using hydrophilic hollow-fiber artificial capillary systems (18,19). These bioreactors consist of a large number of porous capillaries, thus creating large surface areas (2000 cm 2 /cartridge).…”

Section: Discussionmentioning

confidence: 99%

Intermolecular Dipole–Dipole Relaxation of 129Xe Dissolved in Water

Dimitrov

Reddy

Leigh

2000

Journal of Magnetic Resonance

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“…The gradient ( gz ) values used were generally 30 G/cm, so that we could observe the slow‐moving components. The duration of the field gradient pulses was 0.002 s, and Δ was 0.1 s, with a spin‐echo delay of 1.5 s. The spectra of cells (500 transients) were determined as described previously using the gel diffusion method, with matrigel (prepared by M. Sterin in our laboratory) or alginate beads (14), an 8‐mm NMR probe with a z ‐magnetic field gradient, and an 8‐mm screwtop tube, as described previously (15). Cells (6–7 × 10 8 ) in ∼1.5 ml matrigel were perfused at 0.5 ml/min in their medium with a closed system of ∼100 ml with 95% CO 2 /5% O 2 bubbled, containing 5% D 2 O for the lock signal.…”

Section: Methodsmentioning

confidence: 99%

Determination of intracellular pH and compartmentation using diffusion‐weighted NMR spectroscopy with pH‐sensitive indicators

Cohen¹,

Motiei²,

Carmi³

et al. 2004

Magnetic Resonance in Med

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The intracellular pH (pHi) of a series of cancer cell lines was determined using the pH-sensitive indicators imidazole (Im) or histidine (His) and diffusion-weighted (DW) proton NMR spectroscopy. The DW method allows the observation at high magnetic field gradient values of only the slow-moving (intracellular) components, thus ensuring complete separation between intra-and extracellular components. Using the chemical shift difference (⌬␦) between the imidazole ring C2-H and C4(5)-H peaks, we were able to measure the pHi independently of chemical shift standardization. With His, the cell lines gave pHi values of ϳ6.5-7.0, whereas with Im, a second, more acidic compartment (pHi ‫؍‬ 5. Intracellular (pHi) and extracellular (pHe) pH values are important parameters in cell biology, particularly in relation to cancer. Tumors tend to have acidic pHe due to the overproduction of lactate, while the cells themselves may remain neutral or become alkaline due to compensation mechanisms. Also, pHi can be affected by drugs and other cellular processes, such as apoptosis (1,2).There are many methods available to determine pHi (3), including the use of 31 P NMR in many biological systems (4 -6), and 1 H NMR with imidazole-based compounds (7-9). It is often difficult to determine pHi using a pH-sensitive indicator because of interference from the indicator signals in the (often larger) extracellular space. We have developed a method for the efficient separation of the signals of intra-and extracellular pH indicators using diffusion-weighted (DW) NMR spectroscopy that enables the separation of signals from slow-and fast-moving molecules (10,11).In this study we used the pH-sensitive indicators imidazole (Im) and histidine (His). We followed the methods of Gasparovic et al. (8), who used conventional proton NMR spectroscopy. We were able to observe the intracellular signals of the indicators much more readily than Gasparovic et al. (8), and our results confirm their findings. We applied this method to several cancer cell lines. MATERIALS AND METHODS Cell LinesCells were obtained from various sources and grown in different media. Malignant sarcoma cells were provided by Dr. Tali Siegal (Department of Oncology, Hadassah Medical School) and grown in RPMI supplemented with 1% pen-strep (penicillin 1000 U/ml) ϩ 1% L-glutamine ϩ 10% fetal calf serum. OC-238 human ovarian carcinoma cells were provided by Dr. Reuven Reich (Department of Pharmacology, Hebrew University) and grown in DMEM ϩ 10% fetal calf serum ϩ 1% pyruvate ϩ 1% pen-strep (penicillin 1000 U/ml) ϩ 1% MEM non-essential amino acids ϩ 1% glutamate ϩ 1% MEM vitamin solution. HaCaT keratinocytes were grown in DMEM ϩ 1% penstrep ϩ 1% L-glutamate ϩ 10% fetal calf serum following the procedures of Boukamp et al. (17). K562 lymphocytes were obtained from Dr. Amnon Peled (Department of Genetic Therapy, Hadassah Medical School) and grown in RPMI with 1% pen-strep ϩ 1% L-glutamine ϩ 10% fetal bovine serum ϩ 1% non-essential amino acids ϩ 1% sodium pyruvate. NMR SpectroscopyAll NMR sp...

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NMR Studies of Metabolism of Cells and Perfused Organs

Cited by 13 publications

References 309 publications

Multinuclear NMR Spectroscopy Studies on NH<sub>4</sub>Cl-Induced Metabolic Alterations and Detoxification Processes in Primary Astrocytes and Glioma Cells

Multinuclear NMR Spectroscopy Studies on NH<sub>4</sub>Cl-Induced Metabolic Alterations and Detoxification Processes in Primary Astrocytes and Glioma Cells

Intermolecular Dipole–Dipole Relaxation of 129Xe Dissolved in Water

Determination of intracellular pH and compartmentation using diffusion‐weighted NMR spectroscopy with pH‐sensitive indicators

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