Dieter Leibfritz

Multinuclear NMR studies on the energy metabolism of glial and neuronal cells

In this multinuclear NMR study myo-inositol is identified as a glia-specific marker for in vivo NMR studies. The unusually high inositol concentration may participate in the osmoregulatory system in astrocytes. Primary astrocytes also synthesize and export high amounts of hypotaurine, an intermediate of taurine synthesis. Taurine--another osmolyte--is synthesized from cysteine by astrocytes but not by primary neurons. Taurine as well as hypotaurine is accumulated by neurons from the extracellular medium. 13C NMR labelling results with 2-13C pyruvate indicate a considerable contribution of the anaplerotic pathway in primary neurons from rat. The activity is only half of the activity in primary astrocytes. The ratio of pyruvate carboxylase/malic enzyme activity versus pyruvate dehydrogenase activity reflects the degree of maturation. The 13C isotopomer ratio of glutamate and glutamine is different for pure astrocyte cultures. Therefore, the different isotopomer ratios of glutamate to glutamine obtained from intact brain studies alone do not prove TCA cycle compartimentation in the brain. Finally, the PCr/ATP ratio in primary astrocytes is 3 times higher than in primary neurons. This has to be considered in case of recovery from ischemic insults.

Evolution of regional changes in apparent diffusion coefficient during focal ischemia of rat brain: the relationship of quantitative diffusion NMR imaging to reduction in cerebral blood flow and metabolic disturbances.

Middle cerebral artery occlusion was performed in rats while the animals were inside the nuclear magnetic resonance (NMR) tomograph. Successful occlusion was confirmed by the collapse of amplitude on an electrocorticogram. The ultrafast NMR imaging technique UFLARE was used to measure the apparent diffusion coefficient (ADC) immediately after the induction of cerebral ischemia. ADC values of normal cortex and caudate-putamen were 726 ± 22 × 10−6 mm2/s and 659 ± 17 × 10−6 mm2/s, respectively. Within minutes of occlusion, a large territory with reduced ADC became visible in the ipsilateral hemisphere. Over the 2 h observation period, this area grew continuously. Quantitative analysis of the ADC reduction in this region showed a gradual ADC decrease from the periphery to the core, the lowest ADC value amounting to about 60% of control. Two hours after the onset of occlusion, the regional distribution of ATP and tissue pH were determined with bioluminescence and fluorescence techniques, respectively. There was a depletion of ATP in the core of the ischemic territory (32 ± 20% of the hemisphere) and an area of tissue acidosis (57 ± 19% of the hemisphere) spreading beyond that of ATP depletion. Regional CBF (rCBF) was measured autoradiographically with the iodo[14C]antipyrine method. CBF gradually decreased from the periphery to the ischemic core, where it declined to values as low as 5 ml 100 g−1 min−1. When reductions in CBF and in ADC were matched to the corresponding areas of energy breakdown and of tissue acidosis, the region of energy depletion corresponded to a threshold in rCBF of 18 ± 14 ml 100 g−1 min−1 and to an ADC reduction to 77 ± 3% of control. Tissue acidosis corresponded to a flow value below 31 ± 11 ml 100 g−1 min−1 and to an ADC value below 90 ± 4% of control. Thus, the quantification of ADC in the ischemic territory allows the distinction between a core region with total breakdown of energy metabolism and a corona with normal energy balance but severe tissue acidosis.

Restricted diffusion and exchange of intracellular water: theoretical modelling and diffusion time dependence of 1H NMR measurements on perfused glial cells

Intracellular diffusion properties of water in F98 glioma cells immobilized in basement membrane gel threads, are investigated with a pulsed‐field‐gradient spin‐echo NMR technique at diffusion times from 6 to 2000 ms and at different temperatures. In extended model calculations the concept of ‘restricted intracellular diffusion at permeable boundaries’ is described by a combined Tanner–Kärger formula. Signal components in a series of ct experiments (constant diffusion time) are separated due to different diffusion properties (Gaussian and restricted diffusion), and physiological as well as morphological cell parameters are extracted from the experimental data. The intracellular apparent diffusion coefficients strongly depend on the diffusion time and are up to two orders of magnitude smaller than the self diffusion constant of water. Propagation lengths are found to be in the range of 4–7 μm. Hereby intracellular signals of compartments with a characteristic diameter could be selected by an appropriate gradient strength. With cg experiments (constant gradient) a mean intracellular residence time for water is determined to be about 50 ms, and the intrinsic intracellular diffusion constant is estimated to 1 × 10−3  mm2 /s. Studying the water diffusion in glial cells provides basic understanding of the intracellular situation in brain tissue and may elucidate possible influences on the changes in the diffusion contrast during ischemic conditions.

Structural and membrane modifying properties of suzukacillin, a peptide antibiotic related to alamethicin: Part A. Sequence and conformation

The primary structure and conformation of the polypeptide antibiotic suzukacillin A are investigated. Suzukacillin A is isolated from the Trichoderma viride strain 1037 and exhibits membrane modifying and lysing properties similar to those of alamethicin. A combined gas chromatographic mass spectrometric analysis of the trifluoroacetylated peptide methyl esters of partial hydrolysates revealed a tentative sequence of 23 residues including 10 2-methylalanines and one phenylalaninol, which shows many fragments known from alamethicin: Ac-Aib-Pro-Val-Aib-Val-Ala-Aib-Ala-Aib-Aib-Gln-Aib-Leu-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Glu(Pheol)-Gln-OH. All chiral amino acids and phenylalainol have L-configuration. Ultraviolet and infrared spectroscopy, circular dichroism in various solvents and in particular 13C nuclear magnetic resonance have been used for a comparative study of suzukacillin with alamethicin. Suzukacillin has a partially alpha-helical structure and the helix content increases largely from polar to lipophilic solvents. Suzukacillin aggregates more strongly than alamethicin in aqueous medis due to a longer alpha-helical part and higher number of aliphatic residues. A part of the alpha-helix is exceptionally stabilized due to 2-methylalanine residues shielding the peptide bonds from interactions with polar solvents. In lipophilic solvents and lecithin vesicles particularly large temperature induced reductions of the high alpha-helix content are found for alamethicin. Suzukacillin shows similar temperature coefficients in lipophilic media, however, in contrast to alamethicin a more linear change in intensity of the Cotton effects is observed.

Effects of hypothermia on brain glucose metabolism in acute liver failure: a 1H/13C-nuclear magnetic resonance study ☆

BACKGROUND & AIMS Mild hypothermia has a protective effect on brain edema and encephalopathy in both experimental and human acute liver failure. The goals of the present study were to examine the effects of mild hypothermia (35 degrees C) on brain metabolic pathways using combined (1)H and (13)C-Nuclear Magnetic Resonance (NMR) spectroscopy, a technique which allows the study not only of metabolite concentrations but also their de novo synthesis via cell-specific pathways in the brain. METHODS (1)H and (13)C NMR spectroscopy using [1-(13)C] glucose was performed on extracts of frontal cortex obtained from groups of rats with acute liver failure induced by hepatic devascularization whose body temperature was maintained either at 37 degrees C (normothermic) or 35 degrees C (hypothermic), and appropriate sham-operated controls. RESULTS At coma stages of encephalopathy in the normothermic acute liver failure animals, glutamine concentrations in frontal cortex increased 3.5-fold compared to sham-operated controls (P < 0.001). Comparable increases of brain glutamine were observed in hypothermic animals despite the absence of severe encephalopathy (coma). Brain glutamate and aspartate concentrations were respectively decreased to 60.9% +/- 7.7% and 42.2% +/- 5.9% (P < 0.01) in normothermic animals with acute liver failure compared to control and were restored to normal values by mild hypothermia. Concentrations of lactate and alanine in frontal cortex were increased to 169.2% +/- 15.6% and 267.3% +/- 34.0% (P < 0.01) respectively in normothermic rats compared to controls. Furthermore, de novo synthesis of lactate and alanine increased to 446.5% +/- 48.7% and 707.9% +/- 65.7% (P < 0.001), of control respectively, resulting in increased fractional (13)C-enrichments in these cytosolic metabolites. Again, these changes of lactate and alanine concentrations were prevented by mild hypothermia. CONCLUSIONS Mild hypothermia (35 degrees C) prevents the encephalopathy and brain edema resulting from hepatic devascularization, selectively normalizes lactate and alanine synthesis from glucose, and prevents the impairment of oxidative metabolism associated with this model of ALF, but has no significant effect on brain glutamine. These findings suggest that a deficit in brain glucose metabolism rather than glutamine accumulation is the major cause of the cerebral complications of acute liver failure.

Energy metabolism in astrocytes and neurons treated with manganese: relation among cell-specific energy failure, glucose metabolism, and intercellular trafficking using multinuclear NMR-spectroscopic analysis.

A central question in manganese neurotoxicity concerns mitochondrial dysfunction leading to cerebral energy failure. To obtain insight into the underlying mechanism(s), the authors investigated cell-specific pathways of [1–13C]glucose metabolism by high-resolution multinuclear NMR-spectroscopy. Five-day treatment of neurons with 100-μmol/L MnCl2 led to 50% and 70% decreases of ATP/ADP and phosphocreatine–creatine ratios, respectively. An impaired flux of [1-13C]glucose through pyruvate dehydrogenase, which was associated with Krebs cycle inhibition and hence depletion of [4–13C]glutamate, [2–13C]GABA, and [13C]glutathione, hindered the ability of neurons to compensate for mitochondrial dysfunction by oxidative glucose metabolism and further aggravated neuronal energy failure. Stimulated glycolysis and oxidative glucose metabolism protected astrocytes against energy failure and oxidative stress, leading to twofold increased de novo synthesis of [3–13C]lactate and fourfold elevated [4–13C]glutamate and [13C]glutathione levels. Manganese, however, inhibited the synthesis and release of glutamine. Comparative NMR data obtained from cocultures showed disturbed astrocytic function and a failure of astrocytes to provide neurons with substrates for energy and neurotransmitter metabolism, leading to deterioration of neuronal antioxidant capacity (decreased glutathione levels) and energy metabolism. The results suggest that, concomitant to impaired neuronal glucose oxidation, changes in astrocytic metabolism may cause a loss of intercellular homeostatic equilibrium, contributing to neuronal dysfunction in manganese neurotoxicity.

13C isotopomer analysis of glucose and alanine metabolism reveals cytosolic pyruvate compartmentation as part of energy metabolism in astrocytes

After incubation of glial cells with both 13C‐labeled and unlabeled glucose and alanine, 13C isotopomer analysis indicates two cytosolic pyruvate compartments in astrocytes. One pyruvate pool is in an exchange equilibrium with exogenous alanine and preferentially synthesizes releasable lactate. The second pyruvate pool, which is of glycolytic origin, is more closely related to mitochondrial pyruvate, which is oxidized via tri carbonic acid (TCA) cycle activity. In order to provide 2‐oxoglutarate as a substrate for cytosolic alanine aminotransferase, glycolytic activity is increased in the presence of exogenous alanine. Furthermore, in the presence of alanine, glutamate is accumulated in astrocytes without subsequent glutamine synthesis. We suggest that the conversion of alanine to releasable lactate proceeds at the expense of flux of glycolytic pyruvate through lactate dehydrogenase, which is used for ammonia fixation by alanine synthesis in the cytosol and for mitochondrial TCA cycle activity. In addition, an intracellular trafficking occurs between cytosol and mitochondria, by which these two cytosolic pyruvate pools are partly connected. Thus, exogenous alanine modifies astrocytic glucose metabolism for the synthesis of releasable lactate disconnected from glycolysis. The data are discussed in terms of astrocytic energy metabolism and the metabolic trafficking via a putative alanine‐lactate shuttle between astrocytes and neurons. GLIA 34:200–212, 2001.

NMR spectroscopic study on the metabolic fate of [3-13C]alanine in astrocytes, neurons, and cocultures: Implications for glia-neuron interactions in neurotransmitter metabolism

Nuclear magnetic resonance (NMR) spectroscopy and biochemical assays were used to study the fate of [3‐13C]alanine in astrocytes, neurons, and cocultures. 1H‐ and 13C‐NMR analysis of the media demonstrated a high and comparable uptake of [3‐13C]alanine by the cells. Thereafter, alanine is transaminated predominantly to [3‐13C]pyruvate, from which the 13C‐label undergoes different metabolic pathways in astrocytes and neurons: Lactate is almost exclusively synthesized in astrocytes, while in neurons and cocultures labeled neurotransmitter amino acids are formed, i.e., glutamate and γ‐aminobutyric acid (GABA). A considerable contribution of the anaplerotic pathway is observed in cocultures, as concluded from the ratio (C‐2–C‐3)/C‐4 of labeled glutamine. Analysis of the multiplet pattern of glutamate isotopomers indicates carbon scrambling through the TCA cycle and the use of alanine also as energy substrate in neurons. In cocultures, astrocyte‐deduced lactate and unlabeled exogenous carbon substrates contribute to glutamate synthesis and dilute the [2‐13C]acetyl‐CoA pool by 30%. The coupling of neuronal activity with shuttling of tricarboxylic acid (TCA) cycle‐derived metabolites between astrocytes and neurons is concluded from the use of [4‐13C]‐monolabeled glutamate leaving the first TCA cycle turn already for glutamine and GABA synthesis, as well as from the labeling pattern of extracellular glutamine. Further evidence of a metabolic interaction between astrocytes and neurons is obtained, as alanine serves as a carbon and nitrogen carrier through the synthesis and regulated release of lactate from astrocytes for use by neurons. Complementary to the glutamine‐glutamate cycle in the brain, a lactate‐alanine shuttle between astrocytes and neurons would account for the nitrogen exchange of the glutamatergic neurotransmitter cycle in mammalian brain. GLIA 32:286–303, 2000.

Separation of phospholipid classes by hydrophilic interaction chromatography detected by electrospray ionization mass spectrometry.

A new isocratic separation method was developed for separation of phospholipid (PL) classes based on a silica hydrophilic interaction liquid chromatography (HILIC) column with electrospray ionization (ESI) mass spectrometric detection. Although HILIC is typically used for polar compounds, also amphiphilic molecules like phospholipids can be separated very well. Compared to normal-phase (NP) chromatography, which is usually used for PL class separation, HILIC has the advantage to use on-line ESI-MS detection because its eluents are ESI compatible. Furthermore, this HILIC method is isocratic and hence less time consuming than most (gradient) NP HPLC methods. A chromatographic baseline separation of a standard mixture containing phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), sphingomyelin (SM) and lysophosphatidylcholine (LPC) was achieved within a total run time of 17 min using a mobile phase consisting of acetonitrile, methanol and ammonium acetate 10 mM. The new method was subsequently tested on phospholipid fractions of a body fluid (human blood plasma) and a tissue extract (swine brain) whereby it achieved nearly the same baseline separation of the PL classes. The detected classes in both cases were PE, PC, SM and LPC.

Metabolism of Glycine in Primary Astroglial Cells: Synthesis of Creatine, Serine, and Glutathione

Abstract: The metabolism of [2‐13C]glycine in astrogliarich 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.