Combined 1H and 31P NMR studies of the rat brain in vivo: effects of altered intracellular pH on metabolism.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
K L Behar
Publications and source records attributed to K L Behar.
Detection of metabolites in rabbit brain by 13C NMR spectroscopy following administration of [1-13C]glucose.
1H-decoupled 13C NMR spectra (20.2 MHz) of the living rabbit brain were collected with a surface coil following the intravenous infusion of [1-13C]glucose. Within 15 min of infusion, the alpha and beta anomers of glucose were detected and, shortly thereafter, the carbon atoms at positions C4, C3, and C2 of glutamate and(or) glutamine. After reductions of inspired oxygen from 30 to 5%, lactate C3 was detected. The intensity of the lactate resonance rose progressively during hypoxia and later fell during recovery with oxygen. The 13C fractional isotopic enrichment of arterial blood glucose was measured by 1H NMR providing information on the rate and extent of blood glucose labeling.
Animals↗
Application of multipulse NMR to observe 13C-labeled metabolites in biological systems.
Limitations in resolution and sensitivity of 13C NMR spectroscopy have reduced the information obtainable from intact biological systems. With the aim of increasing the information from in vivo 13C NMR two multipulse NMR techniques, the DEPT pulse sequence and the gated spin-echo sequence, were used to obtain edited 13C NMR spectra from different 13C-labeled mammalian tissues. This allowed the separation of the 13C NMR signals from the tissues into subspectra containing either CH, CH2, or CH3 signals, thereby increasing the information obtainable from these spectra. Comparing the two techniques, the DEPT sequence gives more accurate editing than the gated spin-echo sequence but suffers from the difficulty of determining 1H pulse angles in vivo.
Acetates↗
1H-Observe/13C-decouple spectroscopic measurements of lactate and glutamate in the rat brain in vivo.
We have used (13C)-1H NMR spectroscopy at 360.13 MHz to resolve the 13C coupled proton resonance of glutamate and lactate in the rat brain in vivo. The time required for the 13C fractional enrichment of the 4-CH2 position of brain glutamate to reach isotopic steady state was determined during a continuous infusion of D-[1-13C]glucose. Under conditions of ischemia, measurements made of the 3-CH3 of lactate in (13C)-1H NMR spectra revealed the relative contribution of brain glucose and glycogen to lactate formation. (13C)-1H NMR was 11 times more sensitive than 13C NMR for the detection of 13C in the 3-CH3 position of lactate and 6 times more sensitive for the detection of 13C in the 4-CH2 of glutamate under similar in vivo conditions.
Animals↗
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.
Metabolic alterations in amino acids, high-energy phosphates, and intracellular pH during and after insulin hypoglycemia in the rat brain was studied in vivo by 1H and 31P nuclear magnetic resonance (NMR) spectroscopy. Sequential accumulations of 1H and 31P spectra were obtained from a double-tuned surface coil positioned over the exposed skull of a rat while the electroencephalogram was recorded continuously. The transition to EEG silence was accompanied by rapid declines in phosphocreatine, nucleoside triphosphate, and an increase in inorganic orthophosphate in 31P spectra. In 1H spectra acquired during the same time interval, the resonances of glutamate and glutamine decreased in intensity while a progressive increase in aspartate was observed. Following glucose administration, glutamate and aspartate returned to control levels (recovery half-time, 8 min); recovery of glutamine was incomplete. An increase in lactate was detected in the 1H spectrum during recovery but it was not associated with any change in the intracellular pH as assessed in the corresponding 31P spectrum. Phosphocreatine returned to control levels following glucose administration, in contrast to nucleoside triphosphate and inorganic orthophosphate which recovered to only 80% and 200% of their control levels, respectively. These results show that the changes in cerebral amino acids and high-energy phosphates detected by alternating the collection of 1H and 31P spectra allow for a detailed assessment of the metabolic response of the hypoglycemic brain in vivo.
Amino Acids↗
Cerebral metabolism in hyper- and hypocarbia: 31P and 1H nuclear magnetic resonance studies.
Paralyzed rabbits ventilated with an oxygen, nitrous oxide, and carbon dioxide mixture were subjected to hyper- and hypocarbic stress. An Oxford Instrument TMR 32-200 spectrometer was used to record phosphorus-31 and nonwater proton nuclear magnetic resonance spectra of the in vivo brain. These spectra provide measurements of cerebral pHi, phosphocreatine, orthophosphate, ATP, and lactate. The brain exhibited twice as much acute pH-regulating ability as the arterial blood. During hypercarbia, orthophosphate rose while phosphocreatine declined in a reciprocal manner, and ATP remained constant. During hypocarbia, lactate rose gradually over a period of 1 hour, while orthophosphate, phosphocreatine, and ATP remained constant and calculated values of adenosine mono- and diphosphate rose.
Adenosine Diphosphate↗
Cerebral intracellular pH by 31P nuclear magnetic resonance spectroscopy.
We determined cerebral intracellular pH in living rabbits and rats under physiologic conditions, using phosphorus NMR spectroscopy and new titration curves thought to be appropriate for brain. Mean values for the two species were, respectively, 7.14 +/- 0.04 (SD) and 7.13 +/- 0.03. These are toward the alkaline end of the range of values obtained by other methods, as values reported by other NMR workers also tend to be.
Animals↗
In vivo phosphorus nuclear magnetic resonance spectroscopy in status epilepticus.
Bicuculline-induced status epilepticus was studied in paralyzed rabbits ventilated with an oxygen and nitrous oxide mixture. An Oxford Instruments TMR 32-200 spectrometer was used to record phosphorus 31 nuclear magnetic resonance spectra of the in vivo brain. An array of conventional physiological variables including the electroencephalogram was simultaneously recorded. Several features were consistently observed during status epilepticus: (1) Phosphocreatine levels fell to about two-thirds of their control values and remained at that level despite a gradual decline in seizure activity; (2) intracellular pH declined and then remained constant, whereas seizure discharges declined; (3) adenosine triphosphate levels remained constant at their control values. These new, lower levels of brain phosphocreatine and intracellular pH were largely unaffected by increases in seizure activity brought about by elevation of blood pressure from levels too low to support adequate cerebral perfusion, by waning of anticonvulsant drug effect, or by repeated doses of bicuculline.
Acid-Base Equilibrium↗
Homonuclear 1H double-resonance difference spectroscopy of the rat brain in vivo.
We have used 1H homonuclear double-resonance difference spectroscopy at 360.13 MHz to resolve specific metabolite resonances in the brains of intact rats. Metabolite resonances resolved include previously obscured proton resonances of alanine, gamma-aminobutyric acid (GABA), glutamate, and taurine. The gamma-aminobutyric acid alpha- and gamma-CH2 proton resonances were observed in the living rat in the difference spectrum obtained upon irradiation of the beta-CH2 proton resonance at 1.91 ppm. A 3-fold increase in the intensity of the alpha- and gamma-CH2 resonances of gamma-aminobutyric acid was observed 30 min after death. The alpha-CH and gamma-CH2 resonances of glutamate were also resolved in vivo by selective irradiation of the beta-CH2 protons to which they are spin-coupled. In addition, this technique was used to observe the beta-CH3 protons of lactate through the intact scalp of a rat. Large lipid signals arising from scalp tissue were eliminated in the difference spectrum, revealing the lactate beta-CH3 resonance.
Alanine↗
Detection of cerebral lactate in vivo during hypoxemia by 1H NMR at relatively low field strengths (1.9 T).
1H NMR was used to monitor lactate production and clearance during hypoxemia and recovery in the rabbit brain at the relatively low magnetic-field strength of 1.89 T. An array of conventional physiological variables were recorded simultaneously with spectrum acquisition, including the electroencephalogram and electrocardiogram. The sensitivity and spectral resolution achieved at this field strength should be applicable to studies of human brain pathophysiology in the large-bore magnets now available.
Anaerobiosis↗
High-resolution 1H nuclear magnetic resonance study of cerebral hypoxia in vivo.
1H NMR spectra at 360.13 MHz were obtained of the rat brain in vivo by using a surface coil placed over the skull. Resonances of numerous metabolites were identified by comparison with the 1H NMR spectra of excised rat brain tissue and acid extracts of the tissue. Changes in cerebral lactate levels resulting from the administration of gas mixtures low in oxygen were monitored in the in vivo brain spectra with a time resolution of 2.3 min. The electroencephalogram and electrocardiogram were recorded simultaneously during the NMR experiment. Reversibility of the hypoxic response was documented when, upon oxygen administration, cerebral lactate returned to its prehypoxic level. These experiments demonstrate the applicability of high-resolution 1H NMR to monitor pathophysiology of brain metabolism in real time.
Animals↗
Cerebral metabolic studies in vivo by 31P NMR.
31P NMR studies on the brains of living rabbits were carried out at 32 MHz in a spectrometer having a 200-mm clear bore. Paralyzed pump-ventilated animals under nitrous oxide analgesia were inserted into the 1.89-T field and signals were focused in the brain by using a 4-cm surface coil. Several conventional physiological variables were monitored together with 31P spectra during induction and reversal of insulin shock and hypoxic hypoxia sufficient to abolish the electroencephalogram and during status epilepticus. A reversible decrease in phosphocreatine stores accompanied by an increase in Pi was detected during hypoglycemia and hypoxia. Similar changes were observed in prolonged status epilepticus but were not reversed. ATP levels fell about 50% in hypoglycemia but only slightly in the other two metabolic stresses. Intracellular pH rose in hypoglycemia; in status epilepticus and hypoxia it fell, but only when cardiovascular function was severely impaired. From the measured NMR parameters and the assumptions (i) that creatine kinase was at equilibrium and (ii) that the creatine/phosphocreatine pool was constant, it was possible to calculate the relative changes in cytoplasmic ADP levels associated with these metabolic disturbances.
Animals↗
In vivo carbon-13 nuclear magnetic resonance studies of mammals.
Natural abundance carbon-13 nuclear magnetic resonances (NMR) from human arm and rat tissues have been observed in vivo. These signals arise primarily from triglycerides in fatty tissue. Carbon-13 NMR was also used to follow, in a living rat, the conversion of C-1-labeled glucose, which was introduced into the stomach, to C-1-labeled liver glycogen. The carbon-13 sensitivity and resolution obtained shows that natural abundance carbon-13 NMR will be valuable in the study of disorders in fat metabolism, and that experiments with substrates labeled with carbon-13 can be used to study carbohydrate metabolism in vivo.
Adipose Tissue↗
13C NMR study of transamination during acetate utilization by Saccharomyces cerevisiae.
13C NMR was used to follow the metabolism of [2- 13C]acetate and [1- 13C]acetate in aerobic suspensions of Saccharomyces cerevisiae. In the experiment with [2- 13C]acetate, the 13C label appeared first in glutamate C4 and subsequently in glutamate C2 and C3. After exhaustion of the acetate, the glutamate signals diminished and the aspartate C2 and C3 peaks increased. During a subsequent chase experiment with unlabeled acetate, the aspartate peaks decreased and the glutamate C2 and C3 peaks increased in intensity. These observations are interpreted in terms of an interplay between the glutamic-oxalacetic transaminase and Krebs cycle activity. This interpretation was confirmed by an experiment with the transaminase inhibitor 2-amino oxyacetate. During all of these experiments, we observed the formation of trehalose. The NMR gives a direct measurement of the label distribution and from that information it followed that the flows through the glyoxylate and the Krebs cycles are comparable. The intermediates citrate, succinate, fumarate, malate, phosphoenolpyruvate, 3-phosphoglycerate, and glucose 6-phosphate were identified in a 13C NMR spectrum of a perchloric acid extract taken during the metabolism of [2- 13C]acetate. Enrichment of the glutamate C5 position shows the existence of a futile cycle in which phosphoenolpyruvate, formed from oxaloacetate, returns to the Krebs cycle through pyruvate and acetyl CoA
Acetates↗
Quantitative functional imaging of the brain: towards mapping neuronal activity by BOLD fMRI.
Quantitative magnetic resonance imaging (MRI) and spectroscopy (MRS) measurements of energy metabolism (i.e. cerebral metabolic rate of oxygen consumption, CMR(O2)), blood circulation (i.e. cerebral blood flow, CBF, and volume, CBV), and functional MRI (fMRI) signal over a wide range of neuronal activity and pharmacological treatments are used to interpret the neurophysiologic basis of blood oxygenation level dependent (BOLD) image-contrast at 7 T in glutamatergic neurons of rat cerebral cortex. Multi-modal MRI and MRS measurements of CMR(O2), CBF, CBV and BOLD signal (both gradient-echo and spin-echo) are used to interpret the neuroenergetic basis of BOLD image-contrast. Since each parameter that can influence the BOLD image-contrast is measured quantitatively and separately, multi-modal measurements of changes in CMR(O2), CBF, CBV, BOLD fMRI signal allow calibration and validation of the BOLD image-contrast. Good agreement between changes in CMR(O2) calculated from BOLD theory and measured by (13)C MRS, reveals that BOLD fMRI signal-changes at 7 T are closely linked with alterations in neuronal glucose oxidation, both for activation and deactivation paradigms. To determine the neurochemical basis of BOLD, pharmacological treatment with lamotrigine, which is a neuronal voltage-dependent Na(+) channel blocker and neurotransmitter glutamate release inhibitor, is used in a rat forepaw stimulation model. Attenuation of the functional changes in CBF and BOLD with lamotrigine reveals that the fMRI signal is associated with release of glutamate from neurons, which is consistent with a link between neurotransmitter cycling and energy metabolism. Comparisons of CMR(O2) and CBF over a wide dynamic range of neuronal activity provide insight into the regulation of energy metabolism and oxygen delivery in the cerebral cortex. The current results reveal the energetic and physiologic components of the BOLD fMRI signal and indicate the required steps towards mapping neuronal activity quantitatively by fMRI at steady-state. Consequences of these results from rat brain for similar calibrated BOLD fMRI studies in the human brain are discussed.
Animals↗