Acid Homeostasis Following Partial Ischemia in Neonatal Brain Measured In Vivo by 31P and 1H Nuclear Magnetic Resonance Spectroscopy

The purpose of this study was to investigate neonatal brain energy metabolism, acid, and lactate homeostasis in the period immediately following partial ischemia. Changes in brain buffering capacity were quantified by measuring mean intracellular brain pH, calculated from the chemical shift of P_i, in response to identical episodes of hypercarbia before and after ischemia. In addition, the relationship between brain buffer base deficit and intracellular pH was compared during and following ischemia. Thus, in vivo ³¹P and ¹H nuclear magnetic resonance spectra were obtained from the brains of seven newborn piglets exposed to sequential episodes of hypercarbia, partial ischemia, and a second episode of hypercarbia in the postischemic recovery period. For the first episode of hypercarbia, brain buffering was similar to values reported for adult animals of other species (percentage pH regulation = 54 ± 16%). During ischemia, the brain base deficit per unit change in pH was ?19 ± 5 mM/pH unit, which is similar to values reported for adult rats. By 20–35 min postischemia, brain acidosis partly resolved in spite of a net increase in lactate concentration. Therefore, the consumption of lactate could not explain acid homeostasis in the first 35 min following ischemia. We conclude that H⁺/HCO^-₃ or other proton equivalent translocation mechanisms must be sufficiently developed in piglet brain to support acid regulation. This is surprising, because a substantial body of evidence implies these processes would be less active in immature brain. The second episode of hypercarbia, from 35 to 65 min postischemia, resulted in a smaller decrease in brain pH compared with the first episode, a result indicating an increase in brain buffering capacity (percentage pH regulation = 79 ± 29%). This was associated with a parallel decrease in brain lactate content, and therefore acid regulation could be attributed to either continued ion translocation or the consumption of lactate. A mild decrease in brain pH and content of energy metabolites was observed, a finding suggesting that the metabolic consequences of severe postischemic hypercarbia are neither particularly dangerous or beneficial.     

Cerebral Acid Buffering Capacity at Different Ages Measured In Vivo by 31P and 1H Nuclear Magnetic Resonance Spectroscopy

Cerebral acidosis occurring during ischemia has been proposed as one determinant of tissue damage. Newborn animals appear to be less susceptible to ischemic tissue damage than adults. One possible component of ischemic tolerance could derive from maturational differences in the extent of acid production and buffering in newborns compared to adults. The purpose of this study was to measure the dependency of acid production on the blood plasma glucose concentrations and acid buffering capacity of piglets at different stages of development. Complete ischemia was induced in 29 piglets ranging in postconceptual age from 111 to 156 days (normal term conception, 115 days). Brain buffering capacity during the first 30 min of ischemia was quantified in vivo, via 31P and 1H nuclear magnetic resonance (NMR) spectroscopy, by measuring the change in intracellular brain pH for a given change in the concentration of compounds that contribute to the production of hydrogen ions. Animals from all four age groups showed a similar linear correlation between preischemia blood glucose concentration and intracellular pH after 30 min of ischemia. For each animal the slope of the plot of intracellular pH versus cerebral buffer base deficit was used to calculate the buffer capacity. Using data obtained over the entire 30 min of ischemia, there was no difference in the mean buffer capacity of the different age groups, nor was there a significant correlation between buffer capacity and age. However, there was a significant increase in buffer capacity for the intracellular pH range 6.6-6.0, compared to 7.0-6.6, for all age groups. No significant differences in buffer capacity for these two pH ranges were observed between any of the age groups. Acid buffering capacity was also measured by performing pH titrations on brain tissue homogenized in the presence of inhibitors of glycolysis and creatine kinase. Plots of homogenate pH versus buffer base deficit showed a nonlinear trend similar to that seen in vivo, indicating an increase in buffer capacity as intracellular pH decreases. A comparison of newborn and 1-month-old brain tissue frozen under control conditions or after 45 min of ischemia revealed no differences that could be attributed to age and a slight decrease in buffer capacity of ischemic brain compared to control brain tissue homogenates. There was no difference between the brain buffering capacity measured in vivo using 31P and 1H NMR and that measured in vitro using brain homogenates.     

Observation of Cerebral Metabolites in an Animal Model of Acute Liver Failure In Vivo: A 1H and 31P Nuclear Magnetic Resonance Study

Acute liver failure was induced in rats by a single intragastric dose of carbon tetrachloride. This causes hepatic centrilobular necrosis, as indicated by histological examinations, and produces a large increase in the activity of serum alanine aminotransferase. The plasma NH4+ level (mean +/- SEM) was 123 +/- 10 microM in the control group and 564 +/- 41 microM in animals with acute liver failure (each n = 5). 31P nuclear magnetic resonance (NMR) was used to monitor brain cortical high-energy phosphate compounds, Pi, and intracellular pH. 1H NMR spectroscopy was utilised to detect additional metabolites, including glutamate, glutamine, and lactate. The results show that the forebrain is capable of maintaining normal phosphorus energy metabolite ratios and intracellular pH despite the metabolic challenge by an elevated blood NH4+ level. There was a significant increase in the brain glutamine level and a concomitant decrease in the glutamate level during hyperammonaemia. The brain lactate level increased twofold in rats with acute liver failure. The results indicate that 1H NMR can be used to detect cerebral metabolic changes in this model of hyperammonaemia, and our observations are discussed in relation to compartmentation of NH4+ metabolism.     

Brain Metabolism and Intracellular pH During Ischaemia and Hypoxia: An In Vivo 31P and 1H Nuclear Magnetic Resonance Study in the Lamb

Brain metabolism and intracellular pH were studied during and after episodes of ischaemia and hypoxia-ischaemia in lambs anaesthetised with sodium pentobarbitone. 31P and 1H magnetic resonance spectroscopy methods were used to monitor brain pHi and brain concentrations of Pi, phosphocreatine (PCr), beta--nucleoside triphosphate (beta NTP), and lactate. Simultaneous measurements were made of cerebral blood flow and cerebral oxygen and glucose consumption. Cerebral ischaemia sufficient to reduce oxygen delivery to 75% of control values was associated with a fall in brain pHi and increase in brain Pi. Progressively severe hypoxia-ischaemia was associated with a progressive fall in brain pHi, PCr, and beta NTP and increase in brain Pi. In two animals the increase in brain lactate during hypoxia-ischaemia measured by 1H nuclear magnetic resonance (NMR) could be quantitatively accounted for by the increased net uptake of glucose by the brain in relation to oxygen, but was insufficient to account for the concomitant acidosis according to previous estimates of brain buffering capacity. In four animals brain pHi, PCr, Pi, and beta NTP had returned to normal 1 h after the hypoxic-ischaemic episode. In one animal brain pHi had reverted to normal at a time when 1H NMR indicated persistent elevation of brain lactate.     

In Vivo Measurements of Ethanol Concentration in Rabbit Brain by 1H Magnetic Resonance Spectroscopy

In vivo 1H magnetic resonance spectroscopy was used to measure the cerebral ethanol concentration in the rabbit after both intraarterial and intragastric administration. There was good agreement between cerebral and blood ethanol concentrations at all times after administration by either route. Cerebral ethanol levels, measured using in vivo 1H spectroscopy, agreed well with those measured in perchloric acid extracts of brain, analyzed by both high-resolution 1H spectroscopy and gas chromatography. Ethanol may be useful as an indicator to measure cerebral blood flow by 1H spectroscopy and chemical shift-selective magnetic resonance imaging.     

Intracellular pH, Lactate, and Energy Metabolism in Neonatal Brain During Partial Ischemia Measured In Vivo by 31P and 1H Nuclear Magnetic Resonance Spectroscopy

Sequential 31P and 1H nuclear magnetic resonance spectra were measured for neonatal piglets (n = 7) to determine the relationship between brain intracellular pH (pHi), lactate, and phosphorylated energy metabolites during partial ischemia. Simultaneous determinations of arterial and cerebral venous blood gases, pH, O2 content, and plasma concentrations of glucose and lactate were also made. Ischemia, induced by bilateral carotid artery ligation plus hemorrhagic hypotension for 35 min, resulted in variable reductions in ATP, phosphocreatine, and increases in Pi, H+, and lactate relative to control levels. In four piglets, whose arterial blood glucose rose above control, brain lactate exceeded 20 mumol g-1 with corresponding decreases in pHi of greater than 0.7 units compared to control levels. The extents of brain acidosis and lactosis showed a strong linear correlation with each other (r = 0.94). Maximal changes in brain lactate, pHi, and ATP at the end of ischemia showed significant positive linear correlations with the control levels of arterial blood glucose, but did not correlate with arterial glucose or arterial cerebral-venous glucose difference values during ischemia. The relationship between pHi and buffer base deficit was comparable to results reported for adult animals up to 20 mumol ml-1. However, in contrast to models proposed for adult brain, the continued linear relationship between pH and higher buffer base levels is most consistent with a theoretical model that assumes the presence of weak acid buffers with pKa values from 6.7 to 5.2.     

Nondestructive Detection of Glutamate by 1H Nuclear Magnetic Resonance Spectroscopy in Cortical Brain Slices from the Guinea Pig: Evidence for Changes in Detectability During Severe Anoxic Insults

31P and 1H nuclear magnetic resonance spectroscopy (NMR) was used to study the metabolism of intact superfused cortical brain slices during normoxia and anoxia. Attention was focused on quantification of 1H NMR-detected glutamate by a water-suppressed spin-echo method, using N-acetyl aspartate as an internal concentration reference. To quantify the 1H NMR signals, the spin-spin relaxation times and saturation effects were estimated for given metabolites. In addition, absolute concentrations of metabolites were determined by biochemical methods from acid extracts of the preparations after NMR experiments. Under aerobic conditions, 1H NMR detected 79% of the glutamate determined biochemically from the brain slice extracts. During anoxia in the absence of glucose when a severe energetic failure was evident, both 1H NMR and biochemical assays gave closely matching levels for glutamate. We conclude that in the brain cortex 21% of glutamate is located in an intracellular compartment in which this amino acid does not contribute to the 1H NMR signal. However, during severe anoxia an intracellular reorganisation occurs increasing the detectability of this amino acid neurotransmitter by NMR.     

Effects of Acute Hyperammonemia on Cerebral Amino Acid Metabolism and pHi In Vivo, Measured by 1H and 31P Nuclear Magnetic Resonance

The effects of an acute intravenous infusion of ammonium acetate on rat cerebral glutamate and glutamine concentrations, energy metabolism, and intracellular pH were measured in vivo with 1H and 31P nuclear magnetic resonance (NMR). The level of blood ammonia maintained by the infusion protocol used in this study (approximately 500 microM, arterial blood) did not cause significant changes in arterial PCO2, PO2, or pH. Cerebral glutamate levels fell to at least 80% of the preinfusion value, whereas glutamine concentrations increased 170% relative to the preinfusion controls. The fall in brain glutamate concentrations followed a time course similar to that of the rise of brain glutamine. There were no detectable changes in the content of phosphocreatine (PCr) or nucleoside triphosphates (NTP), within the brain regions contributing to the sensitive volume of the surface coil, during the ammonia infusion. Intracellular pH, estimated from the chemical shift of the inorganic phosphate resonance relative to the resonance of PCr in the 31P spectrum, was also unchanged during the period of hyperammonemia. 1H spectra, specifically edited to allow quantitation of the brain lactate content, indicated that lactate rose steadily during the ammonia infusion. Detectable increases in brain lactate levels were observed approximately 10 min after the start of the ammonia infusion and by 50 min of infusion had more than doubled. Spectra acquired from rats that received a control infusion of sodium acetate were not different from the spectra acquired prior to the infusion of either ammonium or sodium acetate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of Intracellular pH in Guinea Pig Cerebral Cortex Ex Vivo Studied by 31P and 1H Nuclear Magnetic Resonance Spectroscopy: Role of Extracellular Bicarbonate and Chloride

Abstract: The role of transmembrane processes that are dependent on external anions in the regulation of cerebral intracellular pH (pH_i), high-energy metabolites, and lactate was investigated using ³¹P and ¹H NMR spectroscopy in an ex vivo brain slice preparation. During oxygenated superfusion, removal of external HCO₃^?/CO₂ in the presence of Na⁺ led to a sustained split of the inorganic phosphate (P_i) peak so that the pH_i indicated by one part of the peak was 0.38 pH units more alkaline and by the other part 0.10 pH units more acidic at 5 min than in the presence of HCO₃^?. The pH in the compartment with a higher pH_i value returned to 7.29 ± 0.04 by 10.5 min of superfusion in a HCO₃^?-free medium, whereas the pH_i in an acidic compartment was reduced to 7.02. In the presence of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid or the absence of external Cl^?, removal of HCO₃^? caused alkalinization without split of the P_i peak. Both treatments reduced the rate of pH_i normalization following alkalinization. Simultaneous omission of external HCO₃^? and Na⁺ did not inhibit alkalinization of the pH_i following CO₂ exit. All these data show that the acid loading mechanism at neutral pH_i is mediated by an Na⁺-independent anion transport. During severe hypoxia, pH_i dropped from 7.29 ± 0.05 to 6.13 ± 0.16 and from 7.33 ± 0.03 to 6.67 ± 0.05 in the absence and presence of HCO₃^?, respectively, in Na⁺-containing medium. Lactate accumulated to 18.7 ± 2.8 and 19.6 ± 1.5 mmol/kg under the respective conditions. In the HCO₃^?-free medium supplemented with 1 mM amiloride, the pH_i fell only to 6.94 ± 0.08 despite the lactate concentration of 18.9 ± 2.4 mmol/kg. Acidification caused by hypoxia was also small in the slice preparations superfused in the absence of both HCO₃^? and Cl^?, as the pH_i was 7.01 ± 0.12 at a lactate concentration of 24.5 ± 2.4 mmol/kg. These data indicate that apart from anaerobic glucose metabolism, separate acidifying mechanisms are functioning during hypoxia under these conditions. Recovery of phosphocreatine levels following reoxygenation was >75% relative to the prehypoxic level in the slice preparations superfused in the absence of HCO₃^? but <47% in those preparations superfused without HCO₃^? and Cl^?. This indicates that either neutral pH_i or absence of Cl^? during hypoxia was deleterious to the energy metabolism. The present data indicate that Cl^?/HCO₃^? exchange mechanisms have distinct roles in cerebral H⁺ homeostasis depending on the level of pH_i and energy state.     

Glucose Metabolism in the Developing Cerebral Cortex as Detected by 1H{13C} Nuclear Magnetic Resonance Spectroscopy Ex Vivo

Abstract: Metabolism of [1-¹³C]glucose was monitored in superfused cerebral cortex slice preparations from 1-, 2-, and 5-week-old rats using ¹H-observed/¹³C-edited (¹H{¹³C}) NMR spectroscopy. The rate of label incorporation into glutamate C-4 did not differ among the three age groups: 0.52–0.67% of total ¹H NMR-detected glutamate/min. This was rather unexpected, as oxygen uptake proceeded at 1.1 ± 0.1, 1.9 ± 0.1, and 2.0 ± 0.1 µmol/min/g wet weight in brain slices prepared from 1-, 2-, and 5-week-old animals, respectively. Steady-state glutamate C-4 fractional enrichments in the slice preparations were ∼23% in all age groups. In the acid extracts of slices glutamate C-4 enrichments were smaller, however, in 1- and 2-week-old (17.8 ± 1.7 and 16.8 ± 0.8%, respectively) than in 5-week-old rats (22.7 ± 0.7%) after 75 min of incubation with 5 m M [1-¹³C]glucose. We add a new assignment to the ¹H{¹³C} NMR spectroscopy, as acetate C-2 was detected in slice preparations from 5-week-old animals. In the acid extracts of slice preparations acetate C-2 was labeled by ∼30% in 5-week-old rats but by 15% in both 1- and 2-week-old animals, showing that the turnover rate was increased in 5-week-old animals. In the extracts 3–4% of the C-6 of N -acetyl-aspartate (NAA; CH₃ of the acetyl group) contained label as determined by both NMR and mass spectrometry, which indicated that there was no significant labeling to other carbons in NAA. NAA accumulated label from [1-¹³C]glucose but not from [2-¹³C]acetate, and the rate of label incorporation increased by threefold on cerebral maturation.     

Detection of Mobile Proteins by Proton Nuclear Magnetic Resonance Spectroscopy in the Guinea Pig Brain Ex Vivo and Their Partial Purification

Abstract: Proton nuclear magnetic resonance (¹H NMR) spectroscopy was used to study metabolites of the brain cortex ex vivo. The superfused brain cortex preparation was judged to be metabolically viable on the basis of the ³¹P NMR spectrum (intracellular pH of 7.23 ± 0.03 and phosphocreatine/ ATP ratio of 1.21 ± 0.09). Using'H NMR a group of previously unidentified signals was detectable at 0.94, 1.22, and 1.40 ppm with a water-suppressed spin-echo sequence. These signals had shorter spin-spin relaxation times (51-54 ms) than TV-acetylaspartate and lactate (84-93 ms) and also smaller saturation factors, an indication of shorter spin-lattice relaxation times than the latter two low-molecular-weight metabolites. The unidentified signals also displayed homo-nuclear coupling to other spins in the methine region of the spectrum. Acid extraction of the brain slices or cortex from animals that were killed yielded a mixture of proteins that exhibited NMR properties matching the ¹H NMR signals in the brain cortex. The molecular mass of these thermoresistant, "mobile' proteins, which contained proline plus hydroxy-proline (9-16% of all amino acids), ranged between 8 and 40 kDa. These "new' assigμMents of¹H NMR-detectable compounds may influence interpretation of NMR data of some metabolites, as their signals are in the vicinity of the -CH3 ¹H NMR peaks of lactate and alanine.     

Noninvasive Demonstration of In Vivo 3-Fluoro-3-Deoxy-D-Glucose Metabolism in Rat Brain by 19F Nuclear Magnetic Resonance Spectroscopy: Suitable Probe for Monitoring Cerebral Aldose Reductase Activities

The metabolism of 3-fluoro-3-deoxy-D-glucose (3-FDG) in rat brain in vivo was investigated noninvasively using ¹⁹F nuclear magnetic resonance (NMR) Spectroscopy. Following an intravenous infusion of 3-FDG, 400 mg/kg, four resonances assigned to the α and β anomers of 3-FDG, 3-fluoro-3-deoxy-D-sorbitol, and 3-fluoro-3-deoxy-D-fructose were clearly resolved in brain, a result indicating that 3-FDG is metabolized primarily into the aldose reductase sorbitol (ARS) pathway. An orally administered aldose reductase inhibitor, sorbinil, caused reduction of the flux of 3-FDG into the ARS, an observation suggesting that the method can be applied in quantitative studies of ARS path way activities. Studies of 24-h urine specimens showed that in addition to the two metabolites observed in brain, F^-was excreted into the urine. 3-FDG appears to be a suitable metabolic probe for assessing glucose metabolism in the ARS pathway by in vivo ¹⁹F NMR Spectroscopy.     

Evaluation of Potential Effectors of Agonal Glycolytic Rate in Developing Brain Measured In Vivo by 31P and 1H Nuclear Magnetic Resonance Spectroscopy

Abstract: Previously we have shown that hypercarbia produces a larger decrease in agonal glycolytic rate in 1-month-old swine than in newborns. In an effort to understand the mechanism responsible for this difference, we tested the hypothesis that hypercarbia produces age-related changes in the concentration of one or more effectors of phosphofructokinase activity. Specifically, in vivo ³¹P and ¹H NMR spectroscopy was used to compare changes in lactate levels, intracellular pH, free magnesium concentration, and content of phosphorylated metabolites for these two age groups at three intervals during the first 1.5 min of complete ischemia in the presence or absence of hypercarbia (P_aco ₂ = 102–106 mm Hg). Hypercarbia produced the same drop in intracellular brain pH for both age groups, but the decrease in phosphocreatine level and increase in inorganic phosphate content were greater in 1-month-olds compared with newborns. During ischemia there was no difference between the magnitude of change in intracellular pH and levels of phosphocreatine and inorganic phosphate in hypercarbic 1-month-olds versus newborns. Under control conditions, i.e., normocarbia and normoxia, the free Mg²⁺ concentration was lower and the fraction of magnesium-free ATP was higher for newborns than 1-month-olds. However, there was no change in these variables for either age group during hypercarbia and early during ischemia. Thus, age-related differences in the relative decrease in agonal glycolytic rate during hypercarbia could not be explained by differences in intracellular pH, inorganic phosphate content, or free magnesium concentration. The [ADP]_free at control was higher in newborns compared with 1-month-olds, and there was no age-related difference in [AMP]_free. These variables did not change for newborns when exposed to hypercarbia, but for 1-month-olds [ADP]_free and [AMP]_free increased during hypercarbia relative to control values. High-energy phosphate utilization during ischemia for hypercarbic 1-month-olds was reduced by 74% compared with normocarbic 1-month-olds during ischemia, whereas the reduction in energy utilization (14%) was not significant for hypercarbic versus normocarbic newborns during ischemia. Because hypercarbia reduces the rate of ATP depletion during ischemia in 1-month-olds to a greater extent than in newborns, the increase in [ADP]_free and [AMP]_free will be slower in the former age group. It follows therefore that for 1-month-olds, the agonal glycolytic rate would not be accelerated by ADP and AMP to the same degree during hypercarbia plus ischemia compared with normocarbic plus ischemia, whereas for newborns hypercarbia has relatively little impact on agonal glycolytic rate.     

Traumatic Brain Injury in the Rat: Alterations in Brain Lactate and pH as Characterized by 1H and 31P Nuclear Magnetic Resonance

Application of both phosphorus (31P) and proton (1H) magnetic resonance spectroscopy (MRS) to the study of brain metabolism permits the noninvasive measurement of intracellular pH and brain lactate level. We have used water-suppression 1H MRS with novel lactate-editing techniques, together with 31P MRS, to characterize sequential changes in brain lactate level and pH in vivo over an 8-h period following fluid-percussion brain injury of graded severity in the rat. A transient fall in intracellular pH (from 7.09 +/- 0.07 at baseline to 6.88 +/- 0.09 at 40 min postinjury) occurred in animals subjected to moderate- (1.5-2.2 atm) and high- (2.5-3.3 atm) but not low-level (0.1-1.2 atm) injury; intracellular pH returned to baseline by 90 min postinjury. Transient elevations in brain lactate level were observed that temporally paralleled and were significantly correlated with the pH changes for all injury levels (r = 0.93, p less than 0.001). Postinjury alterations in intracellular brain pH and lactate level were identical in magnitude in animals subjected to either moderate or high-level injury. However, animals subjected to moderate injury had a moderate chronic neurological deficit that persisted up to 4 weeks postinjury, whereas animals subjected to a high level of injury showed greater histopathological damage and a more severe chronic neurological deficit. These data suggest that the extent of posttraumatic intracellular cerebral acidosis in our model of experimental head injury is not directly related to the severity of functional neurological deficit.     

Noninvasive In Vivo Demonstration of 2-Fluoro-2-Deoxy-d-Glucose Metabolism Beyond the Hexokinase Reaction in Rat Brain by 19F Nuclear Magnetic Resonance Spectroscopy

The metabolism of 2-fluoro-2-deoxy-D-glucose (FDG) in vivo was observed noninvasively in rat brain using 19F nuclear magnetic resonance (NMR) spectroscopy following an intravenous injection of FDG (400 mg/kg). At 3 h after infusion, four resonances with discrete chemical shifts were resolved. Chemical shift analysis of these resonances suggested the chemical identity of two of the resonances to be FDG and/or FDG-6-phosphate and 2-fluoro-2-deoxy-delta-phosphogluconolactone and/or 2-fluoro-2-deoxy-6-phosphogluconate. The chemical identities of the other two resonances remain to be elucidated. The present study indicates that the metabolism of FDG in vivo is more extensive than is previously recognized and demonstrates the feasibility of using 19F NMR spectroscopy to follow the 19F-containing metabolites of FDG in vivo.     

Age-Dependent Neurotoxicity of Striatal Lesions Produced by Aminooxyacetic Acid: Quantitative In Vitro 1H NMR Spectroscopic Studies

Abstract: Aminooxyacetic acid, a potent inhibitor of the mitochondrial malate-aspartate shunt, was used to assess the role of mitochondrial energy metabolism in damaged brain of rats of different age groups. Three age groups—juvenile (<1 month), young adult (3–4 months), and adult (7 months)—were subjected to examine the age-related aminooxyacetic acid effect. We measured the absolute concentrations of metabolites in perchloric acid extracts of rat striatum after intraperitoneal administration of aminooxyacetic acid using ¹H NMR spectroscopy. Among aminooxyacetic acid-treated groups, a twofold increase of GABA concentration was observed in juveniles and young adults, and a threefold elevation of GABA level was observed in adults, whereas there were no significant differences of alanine and glutamate levels in all three aminooxyacetic acid-treated groups as compared with the age-matched controls. Statistically significant decreases of N -acetylaspartate, glutamine, and creatine levels were detected in treated adults. A statistically significant elevation of lactate concentration was found in the adult treated group. These data have demonstrated that the impairment of energy metabolism in rat striatum induced by aminooxyacetic acid can be revealed by quantitative in vitro ¹H NMR spectroscopy and that aminooxyacetic acid produces age-dependent striatal abnormalities.     

Conformations of P2 Protein of Peripheral Nerve Myelin by Nuclear Magnetic Resonance Spectroscopy

High-resolution 1H NMR spectra of P2 protein from bovine peripheral nerve myelin indicate that the protein contains a high degree of tertiary structure in aqueous solution. Denaturation of the protein in urea solutions is a multi-step process. Binding of lysophosphatidylcholine micelles to the protein causes a conformational change and a broadening of NMR peaks from side chains of aromatic amino acid and methionine residues, with much less effect on upfield methyl resonances.     

Cerebral Metabolic Compartmentation as Revealed by Nuclear Magnetic Resonance Analysis of D-[1-13C]Glucose Metabolism

Abstract: Nuclear magnetic resonance (NMR) was used to study the metabolic pathways involved in the conversion of glucose to glutamate, γ-aminobutyrate (GABA), glutamine, and aspartate. d -[1^-13C]Glucose was administered to rats intraperitoneally, and 6, 15, 30, or 45 min later the rats were killed and extracts from the forebrain were prepared for ¹³C-NMR analysis and amino acid analysis. The absolute amount of ¹³C present within each carbon-atom pool was determined for C-2, C-3, and C-4 of glutamate, glutamine, and GABA, for C-2 and C-3 of aspartate, and for C-3 of lactate. The natural abundance ¹³C present in extracts from control rats was also determined for each of these compounds and for N-acetylaspartate and taurine. The pattern of labeling within glutamate and GABA indicates that these amino acids were synthesized primarily within compartments in which glucose was metabolized to pyruvate, followed by decarboxylation to acetyl-CoA for entry into the tricarboxylic acid cycle. In contrast, the labeling pattern for glutamine and aspartate indicates that appreciable amounts of these amino acids were synthesized within a compartment in which glucose was metabolized to pyruvate, followed by carboxylation to oxaloacetate. These results are consistent with the concept that pyruvate carboxylase and glutamine synthetase are glia-specific enzymes, and that this partially accounts for the unusual metabolic compartmentation in CNS tissues. The results of our study also support the concept that there are several pools of glutamate, with different metabolic turnover rates. Our results also are consistent with the concept that glutamine and/or a tricarboxylic acid cycle intermediate is supplied by astrocytes to neurons for replenishing the neurotransmitter pool of GABA. However, a similar role for astrocytes in replenishing the transmitter pool of glutamate was not substantiated, possibly due to difficulties in quantitating satellite peaks arising from ¹³C-¹³C coupling.     

Fingerprinting Analysis of Rhizoma Chuanxiong of Commercial Types using 1H Nuclear Magnetic Resonance Spectroscopy and High Performance Liquid Chromatography Method

The ¹H nuclear magnetic resonance (¹H NMR) fingerprints of fractionated non-polar extracts (control substance for a plant drug (CSPD) A) from Rhizoma chuanxiong, the rhizomes of Ligusticum chuanxiong Hort., of seven specimens from different sources were measured on Fourier Transform (FT)-NMR spectrometer and assigned by comparing them with the ¹H NMR spectra of the isolated pure compounds. The ¹H NMR fingerprints showed exclusively characteristic resonance signals of the major special constituents of the plant. Although the differences in the relative intensity of the ¹H NMR signals due to a discrepancy in the ratio of the major constituents among these samples could be confirmed by high performance liquid chromatography analysis, the general features of the ¹H NMR fingerprint established for an authentic sample of the rhizomes of L. chuanxiong exhibited exclusive data from those special compounds and can be used for authenticating L. Chuanxiong species.     

L-[3H]Glutamate Binding to a Membrane Preparation from Crayfish Muscle

The binding of L-[3H]glutamate to an isolated membrane preparation from crayfish tail muscle has been studied. The muscle homogenate was osmotically shocked, frozen and thawed, and thoroughly washed before incubation with L-[3H]glutamate. The preparation showed high specific binding of L-glutamate with a KD of 0.12 microM and Bmax of 4.7 pmol/mg protein measured in Tris/HCl pH 7.3 and at 4 degrees C. Nonspecific binding was 5-10% of total binding. The glutamate binding was highly stereospecific [K0.5 (D-glutamate), 270 microM] and showed a high degree of discrimination between L-glutamate and L-aspartate [K0.5 (L-aspartate), 54 microM]. In mammalian CNS preparations potent agonists of L-glutamate such as kainate and N-methyl-D-aspartate had no effect at 1 mM, and quisqualate was a weak inhibitor of L-glutamate binding [K0.5 (quisqualate), 162 microM]. Ibotenate was the most potent inhibitor [K0.5 (ibotenate), 0.27 microM], and various esters of L-glutamate were of intermediate potency as displacers of L-[3H]glutamate binding (K0.5 values from 6 to 60 microM). The glutamate binding site from crayfish muscle is clearly different from any of the subclasses of glutamate receptors in mammalian CNS. A possible physiological function of the binding site is a postsynaptic receptor for glutamate, either an extra-junctional or a junctional receptor.