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D Leibfritz

Publications and source records attributed to D Leibfritz.

At least 55 records · Page 3Linked to original sources
PubMed1998

Metabolism of glycine in primary astroglial cells: synthesis of creatine, serine, and glutathione.

The metabolism of [2-13C]glycine in astroglia-rich primary cultures obtained from brains of neonatal Wistar rats was investigated using 13C NMR spectroscopy. After a 24-h incubation of the cells in a medium containing glucose, glutamate, cysteine, and [2-13C]glycine, cell extracts and incubation media were analyzed for 13C-labeled compounds. Labeled creatine, serine, and glutathione were identified in the cell extracts. If arginine and methionine were present during the incubation with [2-13C]glycine, the amount of de novo synthesized [2-13C]creatine was two-fold increased, and in addition, 13C-labeled guanidinoacetate was found in cell extracts and in the media after 24 h of incubation. A major part of the [2-13C]glycine was utilized for the synthesis of glutathione in astroglial cells. 13C-labeled glutathione was found in the cell extracts as well as in the incubation medium. The presence of newly synthesized [2-13C] serine, [3-13C]serine, and [2,3-13C]serine in the cell extracts and the incubation medium proves the capability of astroglial cells to synthesize serine out of glycine and to release serine. Therefore, astroglial cells are able to utilize glycine as a precursor for the synthesis of creatine and serine. This proves that at least one cell type of the brain is able to synthesize creatine. In addition, guanidinoacetate, the intermediate of creatine synthesis, is released by astrocytes and may be used for creatine synthesis by other cells, i.e., neurons.

Animals↗
PubMed1998

Metabolism of cysteine in astroglial cells: synthesis of hypotaurine and taurine.

The synthesis of hypotaurine and taurine was investigated in astroglia-rich primary cultures obtained from brains of neonatal Wistar rats using 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. Cell extracts of astroglial cultures analyzed by 1H NMR spectroscopy show prominent signals of hypotaurine. To identify cysteine as precursor for hypotaurine and taurine synthesis in astroglial cells, primary cultures were incubated with [3-(13)C]cysteine for 24 or 72 h. Cell extracts and incubation media were then analyzed with 13C NMR spectroscopy. Labeled hypotaurine, taurine, glutathione, and lactate were identified in the cell extracts. Within 72 h, 35.0% of the total intracellular hypotaurine and 22.5% of taurine were newly synthesized from [3-(13)C] cysteine. The presence of [1-(13)C]hypotaurine and [1-(13)C]taurine in the incubation medium proves the release of those products of cysteine metabolism into the medium. Minor amounts of the [3-(13)C]cysteine were used for the synthesis of glutathione in astroglial cells or metabolized to [3-(13)C]lactate, which was found in cell extracts and media. These results indicate that the formation of hypotaurine and taurine is a major pathway of cysteine metabolism in astroglial cells.

Animals↗
PubMed1998

Multinuclear NMR spectroscopy studies on NH4Cl-induced metabolic alterations and detoxification processes in primary astrocytes and glioma cells.

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.

Amino Acids↗
PubMed1998

Characterization of dimethylguanosine, phenylethylamine, and phenylacetic acid as inhibitors of Ca2+ ATPase in end-stage renal failure.

The activity of the plasma membrane Ca2+ ATPase of chronic renal failure patients is decreased by circulating inhibitors yet to be characterized. In this study, inhibitors of Ca2+ ATPase were isolated from ultrafiltrate of patients with end-stage renal failure. They were identified as dimethylguanosine, phenylethylamine, and phenylacetic acid by chromatography and mass spectrometry. Ca2+ ATPase activity was measured spectrophotometrically as the difference in hydrolysis of ATP in the presence and absence of Ca2+ with different concentrations of ATP and the isolated substances. All of the identified compounds are sufficiently lipophilic to penetrate the blood-brain barrier and to accumulate in cerebral tissue. The inhibitory effects of these agents were additive. The apparent K(m) values for ATP and Ca2+ were not altered by these substances, suggesting a noncompetitive mechanism of inhibition. In plasma of healthy subjects, the substances were not detectable. The Ca2+ ATPase inhibitors identified may play a role in the pathophysiology of end-stage renal failure and, potentially, in monitoring toxic effects on cellular Ca2+ metabolism in renal failure.

Adult↗
PubMed1997

Study of the interactions of cadmium and zinc ions with cellular calcium homoeostasis using 19F-NMR spectroscopy.

The effects of the heavy-metal ions Cd2+ and Zn2+ on the homoeostasis of intracellular free Ca2+ in E367 neuroblastoma cells were examined using 19F-NMR spectroscopy with the fluorinated chelator probe 1,2-bis-(2-amino-5-fluorophenoxy)ethane-N,N,N', N'-tetra-acetic acid (5F-BAPTA). First, the technique was used to quantify the uptake and intracellular free concentrations of the heavy metals after treatment of the cells with 20 microM CdCl2 or 100 microM ZnCl2. Secondly, metal-induced transients in intracellular free Ca2+ were recorded. Addition of 20 microM CdCl2, but not 100 microM ZnCl2, evoked a transient increase in Ca2+ from a resting level of 84 nM to approx. 190 nM within 15 min after addition of the metal. Zn2+ at 20 microM completely prevented the induction of a Ca2+ transient by Cd2+. Ca2+ was mobilized by Cd2+ from intracellular organelles, since depletion of these stores by thapsigargin abolished the effect of the toxic metal. Furthermore, 20 microM Cd2+ evoked a transient rise in cellular Ins(1,4,5)P3, reaching a maximum level within 5 min after addition of the metal. These results demonstrate that perturbation of the Ins(1,4,5)P3/Ca2+ messenger system is an early and discrete cellular effect of Cd2+.

Animals↗
PubMed1997

Determination of de novo synthesized amino acids in cellular proteins revisited by 13C NMR spectroscopy.

13C nuclear magnetic resonance spectroscopy was used to determine the absolute amounts to de novo synthesized amino acids in both the perchloric acid extracts and the hydrolyzed protein fractions of F98 glioma cells incubated for 2 h with 5 mmol/l [U-13C]glucose. 13C NMR spectra of the hydrolyzed protein fraction revealed a marked incorporation of 13C-labelled alanine, aspartate and glutamate into the proteins of F98 cells within the incubation period. Additionally, small amounts of 13C-labelled glycine, proline and serine could unambiguously be identified in the protein fraction. Astonishingly, approximately equal amounts of 13C-labelled glutamate and aspartate were incorporated into the cellular proteins, although the cytosolic steady-state concentration of aspartate was below 13C NMR detectability. Hypertonic stress decreased the incorporation of 13C-labelled amino acids into the total protein, albeit their cytosolic concentrations were increased, which reflects an inhibition of protein synthesis under these conditions. On the other hand, hypotonic stress increased the amount of 13C-labelled proline incorporated into the cellular proteins even though the cytosolic concentration of 13C-labelled proline was largely decreased. Apparently, hypoosmotic conditions stimulate the synthesis of proteins or peptides with a high proline content. The results show that already after 2 h of incubation with [U-13C]glucose there is a pronounced flux of 13C label into the cellular proteins, which is usually disregarded if cytosolic fluids are examined only. This means that calculations of metabolic fluxes based on 13C NMR spectroscopic data obtained from perchloric acid extracts of cells or tissues and also from in vivo measurements consider only the labelled 'NMR visible' cytosolic metabolites, which may have to be corrected for fast label flowing off into other compartments.

Amino Acids↗
PubMed1997

Adverse effects of reverse triiodothyronine on cellular metabolism as assessed by 1H and 31P NMR spectroscopy.

Effects of 3,3',5'-triiodothyronine (rT3) in connection with 3,3',5-triiodothyronine (T3) on 3T3 cells were studied in vitro by means of 1H and 31P NMR spectroscopy. In the cells incubated with 5 nM T3 for 3 h at pH 7.4, the ATP/ADP ratio was elevated from 6.9 to 8.4, whereas it was reduced to 6.1 in cells incubated with rT3. When the cells were incubated at pH 6.7, the ATP/ADP ratio was reduced to 6.6 and 5.2 at 1 and 2 h, respectively. In the presence of 5 nM of T3, however, the ratio was maintained above the control level. A 1-h preincubation with rT3 dramatically augmented the reductions caused by elevated acidity. These reductions were completely reversed when the cells were incubated with T3.

3T3 Cells↗
PubMed1997

Assessment of the mechanism of astrocyte swelling induced by the macrolide immunosuppressant sirolimus using multinuclear nuclear magnetic resonance spectroscopy.

The toxic effect of the macrolide immunosuppressant sirolimus on cell metabolism of primary astrocytes was studied by multinuclear NMR spectroscopy of viable cells and perchloric acid (PCA) extracts and compared to the effects of the immunosuppressant cyclosporine. The addition of 5 mg/L sirolimus (5.5 mumol/L) induced swelling of primary astrocytes to 110% of the original volume. Alteration in astrocyte volume in the presence of sirolimus was accompanied by reduction of the following important cell osmolytes and amino acid metabolites: myo-inositol, -58 +/- 12% (mean +/- standard deviation, n = 5); taurine, -44 +/- 5%; glutamine, -13 +/- 2%; compared with control. Sirolimus altered glucose metabolism and partially inhibited the tricarboxylic acid (TCA) cycle: sigma TCA/sigma glycolyse = 1.36 +/- 0.09 (control, n = 3), 0.96 +/- 0.08 (with sirolimus). The increased concentration of phosphodiesters by sirolimus addition (glycerophosphoethanolamine, 52 +/- 18%; glycerophosphocholine, 61 +/- 14%; compared with control, n = 5) indicated disorders in phospholipid metabolism of cellular membranes. Addition of sirolimus led to a decline of the energy state in astrocytes: the concentration of phosphocreatine (PCr) decreased to 75% of control value within 60 min of perfusion with sirolimus and the nucleotide triphosphate (NTP) concentration to 85% within 90 min (n = 3). The effect of sirolimus on the cell metabolism of astrocytes equals that of the immunosuppressants cyclosporine and tacrolimus, the neurotoxicity of which is well-established in clinical studies. The results of this in vitro study indicate that sirolimus possesses neurotoxic potential as well.

Animals↗
PubMed1997

1H NMR ganglioside ceramide resonance region on the differential diagnosis of low and high malignancy of brain gliomas.

1. The high-resolution 1H NMR (MRS) spectra of human brain tumor homogenates revealed a broad resonance at 5.3-5.4 ppm in glioblastoma multiforme (N = 16) and brain metastases (N = 3). The broad resonance was identified as ceramide, a sphingosine-fatty acid combination portion of ganglioside, indicating an elevated abundance of monounsaturated fatty acids. GLC analysis of gangliosides in the highly malignant glioblastoma multiforme revealed that the elevated monounsaturated fatty acid is oleic acid (C18:1). The resonance at 5.3-5.4 ppm region was not detectable in normal human brain (N = 2), in meningiomas (N = 2), or in low-grade astrocytomas (N = 12). In normal human brain the abundance of monounsaturated fatty acid is minimal. 2. This investigation was made possible because the method of producing homogenate resulted in (i) no loss of lipids during the process and (ii) a well-homogenised sample, with (iii) no loss in chemical integrity. 3. The properties of tumor gangliosides include antigenic specificity and immunosuppressive activity and the ceramide, a sphingosine-fatty acid combination, noticeably influences the ganglioside immunosuppressive activity. 4. The observation of 1H NMR ceramide resonance in high-malignant brain tumors emphasizes the dramatic role of aberant gangliosides and ceramide precursors on the grade of malignancy and invasiveness. 5. Further insight into the specific nature of the ceramide portion of gangliosides in grading the malignancy of brain tumors should be investigated further.

Astrocytoma↗
PubMed1997

Interactions of triethyltin-chloride (TET) with the energy metabolism of cultured rat brain astrocytes: studies by multinuclear magnetic resonance spectroscopy.

The effect of triethyltin-chloride (TET), a highly neurotoxic compound, on the cellular metabolism of rat brain astrocytes in vitro was examined by nuclear magnetic resonance (NMR) spectroscopy. 5-week-old cultures were exposed to TET (0.2-40 microM) either for (1) acute (3h), (2) 24 h, or (3) chronic treatment (8 d). Cells were labeled with 1-(13)C-glucose, cell extracts were prepared and 31P, 1H, and 13C spectra were analyzed. Cytotoxic effects of TET were assessed by vital dye uptake assay using neutral red (NR) and by exclusion of trypan blue (TB). Cells were examined ultrastructurally by electron microscopy. The data show that the major target of TET at concentrations already causing morphological effects on cultured astrocytes is not the energy metabolism, but that TET rather alters the intracellular concentrations of organic osmolytes, such as myo-inositol, taurine and hypotaurine, which are part of the control of ion and volume regulation and osmotic balance in astrocytes.

Animals↗
PubMed1997

Metabolism of acetate in rat brain neurons, astrocytes and cocultures: metabolic interactions between neurons and glia cells, monitored by NMR spectroscopy.

The existence of metabolic compartmentation is generally accepted for the brain because of the specific differences in glial and neuronal metabolism. Extracellularly supplied acetate is believed to serve as a substrate only for glia cells, but not for neurons. To further test this hypothesis, primary rat brain cultures of neurons, astrocytes, and cocultures of both cell types were used to investigate the metabolic fate of [2-13C]acetate by means of nuclear magnetic resonance (NMR) spectroscopy. Acetate was metabolized by both cell types, neurons and astrocytes in culture. While glutamine was the main product of glial metabolism, labelled glutamate and aspartate were detected in neuron cultures. Astrocytes and neurons in cocultures showed a very different picture, which is similar to results obtained from brain slices, or in vivo studies. The analysis of the isotopomer pattern of glutamate and glutamine confirm an active glutamate-glutamine-cycle between neurons and astrocytes in cocultures. Furthermore, the analysis of unlabelled metabolites revealed a metabolic coupling of hypotaurine and taurine metabolism in astrocytes and neurons, which provides both cell types with one of the most important organic osmolytes.

Acetates↗
PubMed1996

Evaluation of the effects of immunosuppressants on neuronal and glial cells in vitro by multinuclear magnetic resonance spectroscopy.

The use of the undecapeptide cyclosporine and the macrolide tacrolimus as immunosuppressants in transplantation medicine and for the therapy of immune diseases often provokes side effects, among the most important one is neurotoxicity. Changes in the cellular metabolism of glial cells (C6 rat glioma), neuronal cells (N1E-115 mouse neuroblastoma) and primary glia cells (isolated from rats) after addition of cyclosporine and tacrolimus were investigated using 1H-, 13C- and 31P-NMR spectroscopy in vitro. Cells were exposed to various concentrations of the drugs from 3 h to 42 days. The immunosuppressants (cyclosporine IC50 : 55 mumol/l; tacrolimus IC50 : 47 mumol/l) inhibited cell proliferation in a concentration- and time-dependent fashion. Multinuclear NMR studies of PCA extracts of drug-treated cells showed a significant deterioration in the energy status (a decreasing level of PCr : -46 +/- 11%; an increasing NDP/NTP ratio: +136 +/- 4% and an increasing level of Pi : +248 +/- 15%; mean +/- standard deviation). It also showed decreasing concentrations of major cell metabolites like NAA (-59 +/- 12%) in neuroblastoma cells and myo-inositol (-47 +/- 6%) in glia cells compared with untreated controls. Immunosuppressive treatment caused a large reduction of taurine (-36 +/- 12%) and glutamate (-68 +/- 10%) in all cell cultures, whereas intermediates of phospholipid biosynthesis (PE: +59 +/- 13%; PC: +127 +/- 27%;) and breakdown (GPE: +215 +/- 24%; GPC: +245 +/- 17%) increased. No significant differences were observed between the two immunosuppressants. The toxic effects of immunosuppressants on cell cultures are in line with MRI studies of brain oedema observed in patients under immunosuppressive treatment.

Animals↗
PubMed1996

Status of the neonatal rat brain after NMDA-induced excitotoxic injury as measured by MRI, MRS and metabolic imaging.

Intrastriatal injection of the excitotoxin N-methyl-D-aspartate (NMDA) in neonatal rat brain resulted in an acute ipsilateral decrease of the apparent diffusion coefficient (ADC) of brain tissue water, as measured with diffusion-weighted MRI. The early diffusion changes were accompanied by only mild changes in the overall metabolic status as measured by in vivo 1H MRS and 31P MRS and metabolic imaging of brain sections. Minimal decreases in the high-energy phosphate levels and a small hemispheric acidosis were observed in the first 6 h after NMDA administration. In addition, there was very modest lactate accumulation. Twenty-four hours after the induction of the excitotoxic injury the tissue energy status was still only moderately affected, whereas an overall decrease of 1H MRS-detected brain metabolites was found. Treatment with the non-competitive NMDA-antagonist MK-801 given within 90 min after NMDA injection rapidly reversed the NMDA-induced changes in the entire ipsilateral hemisphere. The effect of the competitive NMDA-antagonist D-CPPene was restricted to the cortical areas and was accomplished on a slower time scale. Our results indicate that; (i) early excitotoxicity in the neonatal rat brain does not lead to profound changes in the metabolic status; and (ii) brain tissue water ADC changes are not necessarily associated with a metabolic energy failure.

Animals↗
PubMed1996

Fast proton spectroscopic imaging employing k-space weighting achieved by variable repetition times.

A k-space weighted spectroscopic imaging (SI) method is presented that allows a reduction in the total data acquisition time by up to 55% compared with standard SI. The k-space weighting is achieved by varying the repetition time, thus realizing an inherent apodization that corresponds to a circularly symmetric generalized Hamming filter. The flip angle is varied with the repetition time to enhance the signal-to-noise ratio. These techniques were employed using a short echo time of 10 ms. In vivo measurements on healthy rat brain at 4.7 T were conducted, obtaining two-dimensional spectroscopic imaging data from a 25 x 25 circularly reduced k-space area in as little as 5 min. The signal-to-noise ratio is sufficiently high to detect J-coupled resonances such as myo-inositol or glutamate/glutamine, demonstrating the ability to combine short acquisition times with comprehensive metabolic information. The T1 dependency of the apodization and the corresponding point spread function was evaluated by computer simulations. The achievable signal-to-noise ratio per unit time was compared with standard SI giving a parameter-dependent advantage of approximately 20% of the standard SI method.

Animals↗
PubMed1996

Adaptation of cellular metabolism to anisosmotic conditions in a glial cell line, as assessed by 13C-NMR spectroscopy.

13C-NMR spectroscopy of perchloric acid and lipid extracts of F98 glioma cells showed that volume-regulatory processes under anisosmotic conditions were accompanied by marked alterations in cellular metabolism. Production of alanine, glutamate, and glycine from [U-13C]-glucose is decreased under hypotonic stress and is oppositely increased under hypertonic stress. In contrast, degradation of these molecules is raised under hypotonic conditions and reduced under hypertonic conditions. Furthermore, phospholipid synthesis is decreased under hypertonic stress and increased under hypotonic stress. Obviously, glial metabolism is directed under hypertonic conditions to maintain a high level of small, osmotically active molecules, whereas under hypotonic conditions molecular fragments are increasingly incorporated into the phospholipids and so do not contribute to the osmotic pressure. The latter is evoked by the activation of membrane synthesis process to compensate for stretching and/or damaging of the membranes due to cell swelling.

Adaptation, Physiological↗
PubMed1996

Immobilization of primary astrocytes and neurons for online monitoring of biochemical processes by NMR.

Immobilization of primary astrocytes and cerebellar granule neurons in basement membrane gel threads and on porous microcarriers, respectively, is described. Five different porous microcarriers were examined for their capabilities to support maturation of neurons, as evaluated by determinations of protein content and glucose uptake. These systems proved suitable for perfusion in the NMR tube for long periods of time. The energetic status of the cells was monitored by 31P-NMR, and a very good temporal resolution (less than 30 min) was achieved with superfused astrocytes; thus, this provides a valuable method for real time NMR investigation of biochemical processes in primary astrocytes. Significantly longer acquisition times (approximately 8 h) were required in order to obtain a spectrum of neurons with a reasonably good signal to noise ratio, even so, the results presented here and obtained with this difficult system are very encouraging.

Animals↗
PubMed1995

On the use of two-dimensional-J NMR measurements for in vivo proton MRS: measurement of homonuclear decoupled spectra without the need for short echo times.

The potential of two-dimensional (2D)-J NMR for in vivo proton MRS is examined. Single voxel measurements on the rat brain were performed at 4.7 T using point-resolved spectrocopy localization with a voxel size of 64 microliter and total measuring times of 10-15 min. It is shown that a series of measurements with only 16 or fewer different echo times (TE) enables good signal localization in the f1 axis corresponding to the coupling patterns. For data evaluation, the 2D-J NMR spectrum as well as cross-sections at given f1 values and projections onto the f2 axis are used. A comparison between cross-section spectra taken at different f1 values may help to solve problems of peak assignment. The projection of the 2D magnitude spectrum onto the f2 axis corresponds to a homonuclear decoupled 1D proton spectrum. Because the T2 relaxation times of several coupled resonances (e.g., myo-inositol and glutamate) are rather long, only minor losses in the quality of the projection spectra occur if the measurements with short TE (< or = 50 ms) are not used for data processing. Thus, homonuclear decoupled proton spectra detecting uncoupled and several coupled resonances can be measured with high quality in vivo, even on MR systems that are not equipped with actively shielded gradients, prohibiting data acquisition with TEs of 50 ms or less.

Animals↗