Metabolic assessment of a neuron‐enriched fraction of rat cerebrum using high‐resolution 1H and 13C NMR spectroscopy

This study explored the utility of 1H and 13C magnetic resonance spectroscopy to study a neuron‐enriched preparation made from rat cerebrum. The preparation contained high concentrations of N‐acetylaspartate and γ‐aminobutyric acid and low concentrations of glutamine, indicating that it was in fact rich in neuronal cytosol. This was confirmed by immunohistochemical studies with antibodies to neuronal and glial markers. A method of metabolite quantification based on the creatine signal yielded metabolite concentrations similar to those of rat cerebrum, whereas concentrations based on the metabolite/protein ratio were five times lower, suggesting that much protein in the preparation was not associated with functioning cytoplasm. The metabolic competence of the preparation was assessed by quantitative measurements of its ability to convert 1−13C‐glucose into lactate, glutamate, aspartate, and other metabolites under well oxygenated conditions for 30 min. Calculated from the creatine standard, the mean glycolytic rate was the same as in a synaptosomal preparation studied under similar conditions and the same as rat cerebrum in vivo. Tricarboxylic acid cycle flux occurred at half the rate observed in the synaptosomal preparation and 16% of the basal cerebral metabolic rate in vivo.

[1]  A. K. Sinha,et al.  SOME PROPERTIES OF ISOLATED NEURONAL CELL FRACTIONS , 1969, Journal of neurochemistry.

[2]  B. A. Eagles,et al.  THE CREATINE CONTENT OF BRAIN , 1924 .

[3]  D Garfinkel,et al.  A simulation study of the metabolism and compartmentation in brain of glutamate, aspartate, the Krebs cycle, and related metabolites. , 1966, The Journal of biological chemistry.

[4]  O. Petroff,et al.  Quantitative analysis of rat synaptosomes and cerebrum using high-resolution 1H magnetic resonance spectroscopy. , 1992, Clinica chimica acta; international journal of clinical chemistry.

[5]  O. H. Lowry,et al.  DIVERSITY OF METABOLIC PATTERNS IN HUMAN BRAIN TUMORS—I. HIGH ENERGY PHOSPHATE COMPOUNDS AND BASIC COMPOSITION 1 , 1977, Journal of neurochemistry.

[6]  M. Yudkoff,et al.  Glucose and Synaptosomal Glutamate Metabolism: Studies with [15N]Glutamate , 1988, Journal of neurochemistry.

[7]  M. Farooq,et al.  A MODIFIED PROCEDURE FOR ISOLATION OF ASTROCYTE‐ AND NEURON‐ENRICHED FRACTIONS FROM RAT BRAIN , 1978, Journal of neurochemistry.

[8]  R. Shulman,et al.  Proton NMR Observation of Phenylalanine and an Aromatic Metabolite in the Rabbit Brain in Vivo , 1990, Pediatric Research.

[9]  K. Mikoshiba,et al.  NEURONAL CELL BODY ENRICHED AND GLIAL CELL ENRICHED FRACTIONS FROM YOUNG AND ADULT RAT BRAINS: PREPARATION AND MORPHOLOGICAL AND BIOCHEMICAL PROPERTIES , 1974, Journal of neurochemistry.

[10]  R G Shulman,et al.  The Flux from Glucose to Glutamate in the Rat Brain in vivo as Determined by 1-Observed, 13C-Edited NMR Spectroscopy , 1990, Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism.

[11]  B. Künnecke,et al.  Cerebral metabolism of [1,2-13C2]acetate as detected by in vivo and in vitro 13C NMR. , 1990, The Journal of biological chemistry.

[12]  B. Siesjö,et al.  Glucose consumption in rat cerbral cortex in normoxia, hypoxia and hypercapnia. , 1976, Acta physiologica Scandinavica.

[13]  R. Shulman,et al.  Detection of metabolites in rabbit brain by 13C NMR spectroscopy following administration of [1‐13C]glucose , 1986, Magnetic resonance in medicine.

[14]  S. Rose,et al.  Bulk separation of neurones and glia: aa comparison of techniques. , 1971, Brain research.

[15]  O. Petroff,et al.  Effects of glutamate, quisqualate, and N-methyl-d-aspartate in neonatal brain , 1991, Experimental Neurology.

[16]  K. Behar,et al.  Assignment of resonances in the 1H spectrum of rat brain by two‐dimensional shift correlated and j‐resolved NMR spectroscopy , 1991, Magnetic resonance in medicine.

[17]  J. Brainard,et al.  13C Nuclear Magnetic Resonance Evidence for γ‐Aminobutyric Acid Formation via Pyruvate Carboxylase in Rat Brain: A Metabolic Basis for Compartmentation0 , 1989, Journal of neurochemistry.

[18]  J. S. Cohen,et al.  Differences in metabolite levels upon differentiation of intact neuroblastoma × glioma cells observed by proton NMR spectroscopy , 1983, FEBS letters.

[19]  A. Howseman,et al.  1H-[13C] NMR measurements of [4-13C]glutamate turnover in human brain. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[20]  G. Shulman,et al.  Cerebral Lactate Turnover after Electroshock: In vivo Measurements by 1H/13C Magnetic Resonance Spectroscopy , 1992, Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism.

[21]  R G Shulman,et al.  High-resolution 1H nuclear magnetic resonance study of cerebral hypoxia in vivo. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[22]  R. Busto,et al.  The dissociation of cerebral blood flow, metabolism, and function in the early stages of developing cerebral infarction , 1980, Annals of neurology.

[23]  B. Siesjö,et al.  THE EFFECT OF HYPERCAPNIA UPON INTRACELLULAR pH IN THE BRAIN, EVALUATED BY THE BICARBONATE‐ CARBONIC ACID METHOD AND FROM THE CREATINE PHOSPHOKINASE EQUILIBRIUM , 1972, Journal of neurochemistry.

[24]  J. Alger,et al.  High‐field proton magnetic resonance spectroscopy of human cerebrum obtained during surgery for epilepsy , 1989, Neurology.

[25]  R. C. Collins,et al.  Metabolic anatomy of brain: A comparison of regional capillary density, glucose metabolism, and enzyme activities , 1989, The Journal of comparative neurology.

[26]  Jimmy D Bell,et al.  1H NMR study of cerebral development in the rat , 1989, NMR in biomedicine.

[27]  J. Peeling,et al.  1H NMR properties of N‐acetylaspartylglutamate in extracts of nervous tissue of the rat , 1992, NMR in biomedicine.

[28]  J. Neale,et al.  Enhanced carbodiimide fixation for immunohistochemistry: application to the comparative distributions of N-acetylaspartylglutamate and N-acetylaspartate immunoreactivities in rat brain. , 1993, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[29]  G. Lnenicka,et al.  Morphological transformation of synaptic terminals of a phasic motoneuron by long-term tonic stimulation , 1986, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[30]  A. Blamire,et al.  Spectroscopic imaging of stroke in humans , 1992, Neurology.

[31]  A. Schousboe,et al.  1H NMR study of cortex neurons and cerebellar granule cells on microcarriers and their PCA extracts: Lactate production under hypoxia , 1992, Magnetic resonance in medicine.

[32]  W. Norton,et al.  The isolation of cerebral neurons with partial retention of processes , 1977, Brain Research.

[33]  W. Norton,et al.  Neuronal Soma and Whole Neuroglia of Rat Brain: A New Isolation Technique , 1970, Science.

[34]  David G. Gadian,et al.  Proton MR Spectroscopy of Intracranial Tumours: In Vivo and In Vitro Studies , 1990, Journal of computer assisted tomography.

[35]  H. Bachelard,et al.  Cerebral metabolism of acetate and glucose studied by 13C-n.m.r. spectroscopy. A technique for investigating metabolic compartmentation in the brain. , 1990, The Biochemical journal.

[36]  R. Shulman,et al.  Localized 1H NMR spectra of glutamate in the human brain , 1992, Magnetic resonance in medicine.

[37]  M. Rumsby,et al.  Preparation of astrocytes, neurones and oligodendrocytes from the same rat brain , 1977, Brain Research.

[38]  M. Erecińska,et al.  Energy Metabolism in Rat Brain Synaptosomes from Nembutal‐Anesthetized and Nonanesthetized Animals , 1980, Journal of neurochemistry.

[39]  M Noble,et al.  Specific Expression of N‐Acetylaspartate in Neurons, Oligodendrocyte‐Type‐2 Astrocyte Progenitors, and Immature Oligodendrocytes In Vitro , 1992, Journal of neurochemistry.

[40]  M. Bárány,et al.  Natural‐abundance 13C NMR of brain , 1985, Magnetic resonance in medicine.

[41]  S. Brenner,et al.  The structure of the nervous system of the nematode Caenorhabditis elegans. , 1986, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[42]  G. Dutton Isolation, Culture, and Use of Viable Central Nervous System Perikarya , 1990 .

[43]  E. Novotny,et al.  In Vivo Measurements of Ethanol Concentration in Rabbit Brain by 1H Magnetic Resonance Spectroscopy , 1990, Journal of neurochemistry.

[44]  K. Uğurbil,et al.  Studies of anaerobic and aerobic glycolysis in Saccharomyces cerevisiae. , 1986, Biochemistry.

[45]  A. Defalco,et al.  THE SYNTHESIS OF CREATINE BY THE BRAIN OF THE INTACT RAT * , 1961, Journal of neurochemistry.

[46]  R. Kauppinen,et al.  Cerebral energy metabolism and intracellular pH during severe hypoxia and recovery: A study using 1H, 31P, and 1H [13C] nuclear magnetic resonance spectroscopy in the guinea pig cerebral cortex in vitro , 1990, Journal of neuroscience research.

[47]  R. Kauppinen,et al.  Brain metabolites as 1H NMR markers of neuronal and glial disorders , 1989, NMR in biomedicine.

[48]  E. Novotny,et al.  Neonatal Excitotoxic Brain Injury , 1990 .

[49]  R. Saneto Preparation of Highly Purified Populations of Neurons, Astrocytes, and Oligodendrocytes , 1990 .

[50]  R. Shulman,et al.  Measurement of ethanol in the human brain using NMR spectroscopy. , 1990, Journal of studies on alcohol.

[51]  T. Yoshimine,et al.  Immunohistochemical localization of creatine kinase BB-isoenzyme in human brain: Comparison with tubulin and astroprotein , 1983, Brain Research.

[52]  J. Korf,et al.  In vivo Identification and Quantitative Evaluation of Carrier-Mediated Transport of Lactate at the Cellular Level in the Striatum of Conscious, Freely Moving Rats , 1988, Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism.

[53]  R. Shulman,et al.  Lactate rise detected by 1H NMR in human visual cortex during physiologic stimulation. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[54]  A. Howseman,et al.  Localized proton NMR observation of [3‐13C] lactate in stroke after [1‐13C] glucose infusion , 1991, Magnetic resonance in medicine.

[55]  J. Clark,et al.  Studies on the mitochondrially bound form of rat brain creatine kinase. , 1978, The Biochemical journal.

[56]  A. Schousboe,et al.  First direct demonstration of preferential release of citrate from astrocytes using [13C]NMR spectroscopy of cultured neurons and astrocytes , 1991, Neuroscience Letters.

[57]  H. Bachelard,et al.  Neuronal-glial metabolism under depolarizing conditions. A 13C-n.m.r. study. , 1992, The Biochemical journal.

[58]  J. Coyle,et al.  Immunocytochemical localization of N-acetyl-aspartate with monoclonal antibodies , 1991, Neuroscience.

[59]  B. Ross,et al.  Carbohydrate metabolism of the rat c6 glioma. an in vivo 13c and in vitro 1h magnetic resonance spectroscopy study , 1988, NMR in biomedicine.

[60]  K. Iqbal,et al.  Isolation of neurons and glial cells from normal and pathological human brains. , 1972, Brain research.

[61]  J. Alger,et al.  High‐Resolution Proton Magnetic Resonance Spectroscopy of Rabbit Brain: Regional Metabolite Levels and Postmortem Changes , 1988, Journal of neurochemistry.

[62]  H. Bradford,et al.  DEPOLARIZING STIMULI AND THE RELEASE OF PHYSIOLOGICALLY ACTIVE AMINO ACIDS FROM SUSPENSIONS OF MAMMALIAN SYNAPTOSOMES , 1973, Journal of neurochemistry.

[63]  S. Rose THE COMPARTMENTATION OF GLUTAMATE AND ITS METABOLITES IN FRACTIONS OF NEURON CELL BODIES AND NEUROPIL; STUDIED BY INTRAVENTRICULAR INJECTION OF [U‐14C]GLUTAMATE , 1970, Journal of neurochemistry.

[64]  Sheila M. Cohen,et al.  Carbon-13 and phosphorus-31 NMR study of gluconeogenesis: utilization of carbon-13-labeled substrates by perfused liver from streptozotocin-diabetic and untreated rats , 1987 .

[65]  M. Erecińska,et al.  Relationships Among ATP Synthesis, K+ Gradients, and Neurotransmitter Amino Acid Levels in Isolated Rat Brain Synaptosomes , 1987, Journal of neurochemistry.

[66]  R. Shulman,et al.  Effect of Hypoglycemic Encephalopathy upon Amino Acids, High‐Energy Phosphates, and pHi in the Rat Brain In Vivo: Detection by Sequential 1H and 31P NMR Spectroscopy , 1985, Journal of neurochemistry.

[67]  S. Rose,et al.  Preparation of enriched fractions from cerebral cortex containing isolated, metabolically active neuronal and glial cells. , 1967, The Biochemical journal.

[68]  M. M. Bradford A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[69]  E M Chance,et al.  Mathematical analysis of isotope labeling in the citric acid cycle with applications to 13C NMR studies in perfused rat hearts. , 1983, The Journal of biological chemistry.

[70]  David F Wilson,et al.  The Effect of Acute Hypoxia on Synaptosomes from Rat Brain , 1980, Journal of neurochemistry.

[71]  J. Clark,et al.  A rapid method for the preparation of relatively pure metabolically competent synaptosomes from rat brain. , 1978, The Biochemical journal.

[72]  R. Veech,et al.  MEASUREMENT OF THE RATE OF GLUCOSE UTILIZATION BY RAT BRAIN IN VIVO , 1974, Journal of neurochemistry.

[73]  R. Shulman,et al.  NMR Determination of the TCA Cycle Rate and α-Ketoglutarate/Glutamate Exchange Rate in Rat Brain , 1992 .

[74]  H A Krebs,et al.  Cytosolic phosphorylation potential. , 1979, The Journal of biological chemistry.

[75]  R G Shulman,et al.  1H-Observe/13C-decouple spectroscopic measurements of lactate and glutamate in the rat brain in vivo. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[76]  A. Hamberger,et al.  NEURONAL AND GLIAL SYSTEMS FOR γ‐AMINOBUTYRIC ACID METABOLISM , 1975 .