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In the unstimulated brain energy is primarily supplied by the oxidation of glucose. However the oxygen-to-glucose index (OGI), which is the ratio of metabolic rates of oxygen to glucose, CMR(O2)/CMR(glc), diverges from the theoretical value of 6 as activity is increased. In vivo measurements of brain lactate show its concentration to increase with stimulation. The decreasing OGI with stimulation had led to the suggestion that activation, unlike resting activity, is supported by anaerobic glycolysis. To date a unifying concept that accommodates glucose oxidation at rest with lactate generation and OGI decrease during stimulation of brain is lacking. Furthermore, energetics that change with increasing activity are not consistent with a neuroenergetic model that has been proposed from 1-(13)C-glucose MRS experiments. That model, based upon in vivo MRS measurements and cellular studies by Pellerin and Magistretti, showed that glutamate neurotransmitter cycling was coupled to glucose oxidation over a wide range of brain activities from rest down to deep anesthesia. Here we reconcile these paradoxical observations by suggesting that anaerobic glucose consumption (which can provide energy rapidly) increases with activation to meet the power requirements of millisecond neuronal firing. It is proposed, in accord with our neuroenergetic model, that the extra glucose mobilized rapidly for glial clearance of glutamate, is not needed for the oxidative processes that are responsible for neuronal firing and glutamate release, and consequently it is effluxed as lactate. A stoichiometric relation between OGI and lactate concentration is derived from the neuroenergetic model, showing that the enhanced glucose uptake during activation is consistent with neuronal activity being energetically supported by glucose oxidation., (Copyright 2001 John Wiley & Sons, Ltd.)

Positron-emission tomography and functional MRS imaging signals can be analyzed to derive neurophysiological values of cerebral blood flow or volume and cerebral metabolic consumption rates of glucose (CMR(Glc)) or oxygen (CMR(O(2))). Under basal physiological conditions in the adult mammalian brain, glucose oxidation is nearly complete so that the oxygen-to-glucose index (OGI), given by the ratio of CMR(O(2))/CMR(Glc), is close to the stoichiometric value of 6. However, a survey of functional imaging data suggests that the OGI is activity dependent, moving further below the oxidative value of 6 as activity is increased. Brain lactate concentrations also increase with stimulation. These results had led to the concept that brain activation is supported by anaerobic glucose metabolism, which was inconsistent with basal glucose oxidation. These differences are resolved here by a proposed model of glucose energetics, in which a fraction of glucose is cycled through the cerebral glycogen pool, a fraction that increases with degree of brain activation. The "glycogen shunt," although energetically less efficient than glycolysis, is followed because of its ability to supply glial energy in milliseconds for rapid neurotransmitter clearance, as a consequence of which OGI is lowered and lactate is increased. The value of OGI observed is consistent with passive lactate efflux, driven by the observed lactate concentration, for the few experiments with complete data. Although the OGI changes during activation, the energies required per neurotransmitter release (neuronal) and clearance (glial) are constant over a wide range of brain activity.

The aims of this study were twofold: (i) to determine quantitatively the contribution of glutamate/glutamine cycling to total astrocyte/neuron substrate trafficking for the replenishment of neurotransmitter glutamate; and (ii) to determine the relative contributions of anaplerotic flux and glutamate/glutamine cycling to total glutamine synthesis. In this work in vivo and in vitro (13)C NMR spectroscopy were used, with a [2-(13)C]glucose or [5-(13)C]glucose infusion, to determine the rates of glutamate/glutamine cycling, de novo glutamine synthesis via anaplerosis, and the neuronal and astrocytic tricarboxylic acid cycles in the rat cerebral cortex. The rate of glutamate/glutamine cycling measured in this study is compared with that determined from re-analysis of (13)C NMR data acquired during a [1-(13)C]glucose infusion. The excellent agreement between these rates supports the hypothesis that glutamate/glutamine cycling is a major metabolic flux ( approximately 0.20 micromol/min/g) in the cerebral cortex of anesthetized rats and the predominant pathway of astrocyte/neuron trafficking of neurotransmitter glutamate precursors. Under normoammonemic conditions anaplerosis was found to comprise 19-26% of the total glutamine synthesis, whilst this fraction increased significantly during hyperammonemia ( approximately 32%). These findings indicate that anaplerotic glutamine synthesis is coupled to nitrogen removal from the brain (ammonia detoxification) under hyperammonemic conditions.

Objective: The imaging of brain activity with positron emission tomography (PET) and functional magnetic resonance imaging has assumed a central position in psychiatry. Functional imaging signals arise from changes in the neurophysiological parameters of glucose and oxygen consumption mediated by blood flow., Method: Recent in vivo (13)C nuclear magnetic resonance (NMR) neurochemical studies have established a quantitative coupling between the rates of glucose oxidation and glutamate neurotransmitter flux in rats and humans, thereby linking measured neurophysiological parameters to brain function., Results: These results show that in the awake, resting, and unstimulated states, 70%-80% of brain energy consumption is devoted to the same glutamate/glutamine neurotransmitter signaling as are the small percentages stimulated by tasks. Furthermore, in anesthetized animals, in which unstimulated activity is reduced, the total signal rather than a particular increment is required for a response., Conclusions: On this basis, the total signal, as well as the difference in the signal, measures cortical neurotransmitter flux. The total signal in a region therefore contains valuable information about required brain activity. Although signal change is often more easily measured, certain PET and (13)C NMR methods can quantify total regional signal activity and thereby provide another measure of neurotransmitter activity.

The study of intermediary metabolism in biomolecules has been given new directions by recent experiments in human muscle and brain by 13C NMR. Labeled substrates, generally glucose, have enabled the fluxes to be determined in vivo, whereas the naturally abundant 13C has enabled concentrations to be measured. In muscle the glycogen synthesis pathway has been measured and the flux control determined by metabolic control analysis of data, which shows that this pathway is mainly responsible for insulin-stimulated glucose disposal and that a deficiency in the glucose transporter in the pathway is responsible for hyperglycemia in non-insulin-dependent diabetics. From a physiological point of view the most surprising result was that the heavily regulated allosteric enzyme, glycogen synthase, does not control flux but is needed to maintain homeostasis during flux changes. This novel role for a phosphorylated allosteric enzyme is proposed to be a general phenomenon in metabolic and signaling pathways, which physiologically link different cellular activities. In human and rat brains 13C NMR measurements of the flow of labeled glucose into glutamate and glutamine simultaneously determine the rate of glucose oxidation and glutamate neurotransmitter cycling and reveal a 1:1 stoichiometry between the two fluxes. Implications for the interpretation of functional imaging studies and for psychology are discussed. These results demonstrate how intermediary metabolism serves to connect biochemistry with systemic physiology when measured and analyzed by in vivo NMR methods.

Focal changes in the cerebral metabolic rate of glucose utilization (CMRglc) are small (10-40%) during sensory activation in awake humans, as well as in awake rodents and primates (20-50%). They are significantly larger (50-250%) in sensory activation studies of anesthetized rats and cats. Our data, in agreement with literature values, show that in the resting anesthetized state values of CMRglc are lower than in the resting nonanesthetized state whereas the final state values, reached upon activation, are similar for the anesthetized and nonanesthetized animals. The lower resting anesthetized state values of CMRglc explain why the increments upon activation from anesthesia are larger than when starting from the nonanesthetized conditions. Recent 13C NMR measurements in our laboratory have established a quantitative relationship between the energetics of glucose oxidation, CMRglc (oxidative), and the flux of the glutamate/gamma-aminobutyric acid/glutamine neurotransmitter cycle, Vcycle. In both the resting awake value of CMRglc(oxidative), and its increment upon stimulation, a large majority (approximately 80%) of the brain energy consumption is devoted to Vcycle. In the differencing methods of functional imaging, it is assumed that the incremental change in the measured signal represents the modular activity that supports the functional response. However, the same amount of activity must be present during the response to stimulation, irrespective of the initial basal state of the cortex. Thus, whereas the incremental signals of DeltaCMRglc can localize neurotransmitter activity, the magnitude of such activity during the response is represented by the total localized CMRglc, not the increment.

Functional imaging experiments, in particular positron-emission tomography and functional magnetic resonance imaging, can be analyzed either in psychological terms or on the basis of neuroscience. In the usual psychological interpretation, stimulations are designed to activate specific mental processes identified by cognitive psychology, which are then localized by the signals in functional imaging experiments. An alternate approach would be to analyze experiments in terms of the neurobiological processes responsible for the signals. Recent in vivo 13C NMR measurements of the glutamate-to-glutamine neurotransmitter cycling in rat and human brains facilitate a neuroscientific interpretation of functional imaging data in terms of neurobiological processes since incremental neurotransmitter flux showed a 1:1 stoichiometry with the incremental rate of glucose oxidation. Because functional imaging signals depend on brain energy consumption, a quantitative relationship can be established between the signal (S) and the specific neurochemical cerebral neurotransmitter activity (N) of glutamate-to-glutamine neurotransmitter cycling. The quantitation of neuronal activity proposed has implications for the psychological design and interpretation of functional imaging experiments. Measurements of the neurotransmitter cycling flux at rest in functional imaging experiments suggest that performing cognitive tasks and sensory stimulations increases neurotransmitter cycling by only 10-20%. Therefore it cannot be assumed that reference state activities are negligible, nor that they are constant during stimulation.

Ammonia transport and glutamine synthesis were studied in the hyperammonaemic rat brain in vivo using 15N-NMR spectroscopy at a plasma ammonia level of approximately 0.39 mM raised via an intravenous [15N]-ammonium acetate infusion. The initial slope of the time course of the summed cerebral 15N-labelled metabolites was used to determine the rate of ammonia net transport during hyperammonemia as 0.13 +/- 0.02 micromol/min/g (mean +/- SD; n = 5). Based on the total accumulation of glutamine and the 1:2 stoichiometric relationship between fluxes of four-carbon skeletons and nitrogen atoms, the rate of de novo glutamine synthesis through anaplerosis and subsequent glutamate dehydrogenase action was calculated to be 0.065 +/- 0.01 micromol/min/g. The rate of total glutamine synthesis was estimated to be 0.20 +/- 0.06 micromol/min/g (n = 5) by fitting the [5-15N]glutamine time course to a previously described model of glutamate-glutamine cycling between astrocytes and neurones. A large dilution was also observed in [2-15N]glutamine, which supports the glutamate-glutamine cycle as being an important pathway for neuronal glutamate repletion in vivo.

The use of in vivo 13C nuclear magnetic resonance spectroscopy (NMR) has established the pathways of functional interaction between neurons and astrocytes in the mammalian brain and enabled quantitation of these fluxes. A mathematical model of glutamate, glutamine and ammonia metabolism in the brain has been developed, under the constraints of carbon and nitrogen mass balance, allowing the direct and quantitative comparison of in vivo 13C- and 15N-NMR data. Using this model and 13C-NMR data, the authors have separated the neurotransmitter cycling and detoxification components of glutamine synthesis by measuring the rate of glutamine synthesis under normal and hyperammonaemic conditions in the rat brain cortex in vivo. In addition, the simultaneous measurement of the rates of oxidative glucose metabolism and glutamate neurotransmitter cycling in the rat brain cortex has shown that over a range of EEG activity (from isoelectric up to near-resting levels) the stoichiometry between glucose metabolism and glutamate cycling is close to 1:1. Under mild anesthesia, cortical glucose oxidation coupled to glutamatergic synaptic activity accounts for over 80% of total glucose oxidation. Previously, changes in cerebral glucose metabolism have been taken to indicate alterations in functional activity. These recent in vivo results demonstrate, however, that those changes are, in fact, quantitatively coupled to the crux of functional activity, neurotransmitter release. These findings bear upon a number of hypotheses concerning the neurophysiological basis of brain functional imaging methods.

In the alpha-chloralose-anesthetized rat during single forepaw stimulation, a spatially localized 1H[13C] nuclear magnetic resonance spectroscopic method was used to measure the rate of cerebral [C4]-glutamate isotopic turnover from infused [1,6-(13)C]glucose. The glutamate turnover data were analyzed using a mathematical model of cerebral glucose metabolism to evaluate the tricarboxylic acid (TCA) cycle flux (V(TCA)). During stimulation the value of V(TCA) in the sensorimotor region increased from 0.47 +/- 0.06 (at rest) to 1.44 +/- 0.41 micromol x g(-1) x min(-1) (P < 0.01) in the contralateral hemispheric compartment (24 mm3) and to 0.65 +/- 0.10 micromol x g(-1) x min(-1) (P < 0.03) in the ipsilateral side. Each V(TCA) value was converted to the cerebral metabolic rates of glucose oxidation (oxidative-CMR(glc)) and oxygen consumption (CMR(O2)). These rates were corrected for partial-volume based on activation maps obtained by blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI). The percent increase and the absolute value of oxidative-CMR(glc) in the activated regions are similar to values reported previously for total-CMR(glc) using the same activation paradigm. This indicates that the large majority of energy required for brain activation, in going from the resting to an activated state, is supplied by glucose oxidation. The level of activity during stimulation is relevant to awake animals because the oxidative-CMR(glc) (1.05 +/- 0.28 micromol x g(-1) x min(-1); current study) is in the range of total-CMR(glc) previously reported for awake rats undergoing physiologic activation (0.7-1.4 micromol x g(-1) x min(-1)). It is concluded that oxidative glycolysis is the main source of energy for increased brain activity and a positive BOLD fMRI signal-change occurs in conjunction with a large increase in CMR(O2).

The cerebral tricarboxylic acid (TCA) cycle rate and the rate of glutamine synthesis were measured in rats in vivo under normal physiological and hyperammonemic conditions using 13C NMR spectroscopy. In the hyperammonemic animals, blood ammonia levels were raised from control values of approximately 0.05 mM to approximately 0.35 mM by an intravenous ammonium acetate infusion. Once a steady-state of cerebral metabolites was established, a [1-13C]glucose infusion was initiated, and 13C NMR spectra acquired continuously on a 7-tesla spectrometer to monitor 13C labeling of cerebral metabolites. The time courses of glutamate and glutamine C-4 labeling were fitted to a mathematical model to yield TCA cycle rate (V(TCA)) and the flux from glutamate to glutamine through the glutamine synthetase pathway (V(gln)). Under hyperammonemia the value of V(TCA) was 0.57 +/- 0.16 micromol/min per g (mean +/- SD, n = 6) and was not significantly different (unpaired t test; P > 0.10) from that measured in the control animals (0.46 +/- 0.12 micromol/min per g, n = 5). Therefore, the TCA cycle rate was not significantly altered by hyperammonemia. The measured rate of glutamine synthesis under hyperammonemia was 0.43 +/- 0.14 micromol/min per g (mean +/- SD, n = 6), which was significantly higher (unpaired t test; P < 0.01) than that measured in the control group (0.21 +/- 0.04 micromol/ min per g, n = 5). We propose that the majority of the glutamine synthetase flux under normal physiological conditions results from neurotransmitter substrate cycling between neurons and glia. Under hyperammonemia the observed increase in glutamine synthesis is comparable to the expected increase in ammonia transport into the brain and reported measurements of glutamine efflux under such conditions. Thus, under conditions of elevated plasma ammonia an increase in the rate of glutamine synthesis occurs as a means of ammonia detoxification, and this is superimposed on the constant rate of neurotransmitter cycling through glutamine synthetase.

NMR spectroscopy was used to test recent proposals that the additional energy required for brain activation is provided through nonoxidative glycolysis. Using localized NMR spectroscopic methods, the rate of C4-glutamate isotopic turnover from infused [1-(13)C]glucose was measured in the somatosensory cortex of rat brain both at rest and during forepaw stimulation. Analysis of the glutamate turnover data using a mathematical model of cerebral glucose metabolism showed that the tricarboxylic acid cycle flux [(V(TCA)] increased from 0.49 +/- 0.03 at rest to 1.48 +/- 0.82 micromol/g/min during stimulation (P < 0.01). The minimum fraction of C4-glutamate derived from C1-glucose was approximately 75%, and this fraction was found in both the resting and stimulated rats. Hence, the percentage increase in oxidative cerebral metabolic rate of glucose use (CMRglc) equals the percentage increases in V(TCA) and cerebral metabolic rate of oxygen consumption (CMRO2). Comparison with previous work for the same rat model, which measured total CMRglc [Ueki, M., Linn, F. & Hossman, K. A. (1988) J. Cereb. Blood Flow Metab. 8, 486-4941, indicates that oxidative CMRglc supplies the majority of energy during sustained brain activation.

The difference between 1H nuclear magnetic resonance (NMR) spectra obtained from the human brain during euglycemia and during hyperglycemia is depicted as well-resolved glucose peaks. The time course of these brain glucose changes during a rapid increase in plasma glucose was measured in four healthy subjects, aged 18-22 years, in five studies. Results demonstrated a significant lag in the rise of glucose with respect to plasma glucose. The fit of the integrated symmetric Michaelis-Menten model to the time course of relative glucose signals yielded an estimated plasma glucose concentration for half maximal transport, Kt, of 4.8 +/- 2.4 mM (mean +/- SD), a maximal transport rate, Tmax, of 0.80 +/- 0.45 micromol g-1 min-1, and a cerebral metabolic glucose consumption rate (CMR)glc of 0.32 +/- 0.16 micromol g-1 min-1. Assuming cerebral glucose concentration to be 1.0 micromol/g at euglycemia as measured by 13CMR, the fit of the same model to the time course of brain glucose concentrations resulted in Kt = 3.9 +/- 0.82 mM, Tmax = 1.16 +/- 0.29 micromol g-1 min-1, and CMRglc = 0.35 +/- 0.10 micromol g-1 min-1. In both cases, the resulting time course equaled that predicted from the determination of the steady-state glucose concentration by 13C NMR spectroscopy within the experimental scatter. The agreement between the two methods of determining transport kinetics suggests that glucose is distributed throughout the entire aqueous phase of the human brain, implying substantial intracellular concentration.

13C isotopic tracer data previously obtained by 13C nuclear magnetic resonance in the human brain in vivo were analyzed using a mathematical model to determine metabolic rates in a region of the human neocortex. The tricarboxylic acid (TCA) cycle rate was 0.73 +/- 0.19 mumol min-1 g-1 (mean +/- SD; n = 4). The standard deviation reflects primarily intersubject variation, since individual uncertainties were low. The rate of alpha-ketoglutarate/glutamate exchange was 57 +/- 26 mumol min-1 g-1 (n = 3), which is much greater than the TCA cycle rate; the high rate indicates that alpha-ketoglutarate and glutamate are in rapid exchange and can be treated as a single combined kinetic pool. The rate of synthesis of glutamine from glutamate was 0.47 mumol min-1 g-1 (n = 4), with 95% confidence limits of 0.139 and 3.094 mumol min-1 g-1; individual uncertainties were biased heavily toward high synthesis rates. From the TCA cycle rate the brain oxygen consumption was estimated to be 2.14 +/- 0.48 mumol min-1 g-1 (5.07 +/- 1.14 ml 100 g-1 min-1; n = 4), and the rate of brain glucose consumption was calculated to be 0.37 +/- 0.08 mumol min-1 g-1 (n = 4). The sensitivity of the model to the assumptions made was evaluated, and the calculated values were found to be unchanged as long as the assumptions remained near reported physiological values.

Recent advances in magnetic resonance imaging and spectroscopy make it possible to measure localized changes in human brain activity and metabolism in single subjects during sensory stimulation and cognition. Differences between stimulated and unstimulated subjects can be visualized to a resolution of mm3 in less than 1s, a significant improvement over the more established method, positron emission tomography. Magnetic resonance spectroscopy of the human brain, measuring fluxes in several cm3, has followed changes in metabolic rates during visual stimulation.

Cerebral metabolism of D[1-13C]glucose was studied with localized 13C NMR spectroscopy during intravenous infusion of enriched [1-13C]glucose in four healthy subjects. The use of three-dimensional localization resulted in the complete elimination of triacylglycerol resonance that originated in scalp and subcutaneous fat. The sensitivity and resolution were sufficient to allow 4 min of time-resolved observation of label incorporation into the C3 and C4 resonances of glutamate and C4 of glutamine, as well as C3 of aspartate with lower time resolution. [4-13C]Glutamate labeled rapidly reaching close to maximum labeling at 60 min. The label flow into [3-13C]glutamate clearly lagged behind that of [4-13C]-glutamate and peaked at t = 110-140 min. Multiplets due to homonuclear 13C-13C coupling between the C3 and C4 peaks of the glutamate molecule were observed in vivo. Isotopomer analysis of spectra acquired between 120 and 180 min yielded a 13C isotopic fraction at C4 glutamate of 27 +/- 2% (n = 4), which was slightly less than one-half the enrichment of the C1 position of plasma glucose (63 +/- 1%), p < 0.05. By comparison with an external standard the total amount of [4-13C]glutamate was directly quantified to be 2.4 +/- 0.1 mumol/ml-brain. Together with the isotopomer data this gave a calculated brain glutamate concentration of 9.1 +/- 0.7 mumol/ml, which agrees with previous estimates of total brain glutamate concentrations. The agreement suggests that essentially all of the brain glutamate is derived from glucose in health human brain.

A magnetic resonance (MR) imaging brain mapping method was used to localize an activated volume of brain tissue in chloralose-anesthetized rats during electrical stimulation of the forepaw. Physiologically-induced changes are characterized by alterations of the magnetic properties of blood as determined by the oxygenation state of hemoglobin. Stimulation of the left forepaw led to an increase in MR signal intensity of the contralateral frontal and parietal cortices, which corresponded to forelimb motor and somatosensory areas. The activation was contiguous in coronal planes between +5 and +2 mm anterior to the bregma, and its volume was calculated to be 20-30 mm3. Each activated region was revealed using a paired t-test statistical analysis method and the activated volume was calculated from regions exposed by thresholding at p < 0.005. Physiologically-induced fractional signal changes, delta S/S, in the motor and somatosensory areas were 0.06 +/- 0.04 and 0.17 +/- 0.06, respectively.

Spatially localized 1H NMR spectroscopy has been applied to measure changes in brain glucose concentration during 8-Hz photic stimulation. NMR spectroscopic measurements were made in a 12-cm3 volume centered on the calcarine fissure and encompassing the primary visual cortex. The average maximum change in glucose levels was 0.34 mumol.g-1 (n = 5) at 15 min; glucose level had turned toward resting level at 25 min. The glucose change was used to calculate the increase of glucose cerebral metabolic rate in the visual cortex region for individual subjects by using the Michaelis-Menten model of glucose transport on the assumption of constant transport kinetics. The glucose cerebral metabolic rate was calculated to increase over the nonstimulated rate by 22% during the first 15 min of photic stimulation. A model in which the glucose metabolic rate gradually decreases during stimulation was proposed as a possible explanation for the recovery of brain glucose and previously measured lactate concentrations to prestimulus values after 15 min.

The techniques of in vivo magnetic resonance (MR) imaging and spectroscopy have been established over the past two decades. Recent applications of these methods to study human brain function have become a rapidly growing area of research. The development of methods using standard MR contrast agents within the cerebral vasculature has allowed measurements of regional cerebral blood volume (rCBV), which are activity dependent. Subsequent investigations linked the MR relaxation properties of brain tissue to blood oxygenation levels which are also modulated by consumption and blood flow (rCBF). These methods have allowed mapping of brain activity in human visual and motor cortex as well as in areas of the frontal lobe involved in language. The methods have high enough spatial and temporal sensitivity to be used in individual subjects. MR spectroscopy of proton and carbon-13 nuclei has been used to measure rates of glucose transport and metabolism in the human brain. The steady-state measurements of brain glucose concentrations can be used to monitor the glycolytic flux, whereas subsequent glucose metabolism--i.e., the flux into the cerebral glutamate pool--can be used to measure tricarboxylic acid cycle flux. Under visual stimulation the concentration of lactate in the visual cortex has been shown to increase by MR spectroscopy. This increase is compatible with an increase of anaerobic glycolysis under these conditions as earlier proposed from positron emission tomography studies. It is shown how MR spectroscopy can extend this understanding of brain metabolism.

A limitation of previous methods for studying human brain glucose metabolism, such as positron emission tomography, is that metabolic steps beyond glucose uptake cannot be studied. Nuclear magnetic resonance (NMR) has the advantage of allowing the nondestructive measurement of 13C distribution in specific carbon positions of metabolites. In this study 1H-[13C] NMR spectroscopy in conjunction with volume localization was used to measure the rate of incorporation of 13C isotope from infused enriched [1-13C]glucose to human brain [4-13C]glutamate. In three studies C4 glutamate turnover time constants of 25, 20, and 17 min were measured in a 21-cm3 volume centered in the region of the visual cortex. Based on an analysis of spectrometer sensitivity the spatial resolution of the method can be improved to < 4 cm3. In conjunction with metabolic modeling and other NMR measurements this method can provide a measure of regional rates of the brain tricarboxylic acid cycle and other metabolic pathways.

Natural abundance 13C NMR spectra obtained from 144-cm3 volumes in the human brain contained well-resolved resonances of myo-inositol after 60 min of data accumulation. A mean concentration of 7.2 +/- 0.5 mumol/g (+/- SE, n = 7) was calculated from the comparison with phantoms. 13C NMR spectroscopy thus provides a complementary role in the quantitation of metabolites also observed in the crowded 1H spectrum.

Background and Purpose: Proton magnetic resonance spectroscopy can measure in vivo brain lactate and other metabolites noninvasively. We measured the biochemical changes accompanying stroke in 16 human subjects with cortical or deep cerebral infarcts within the first 3 weeks after symptom onset, and performed follow-up studies on six., Methods: One-dimensional proton spectroscopic imaging encompassing the infarct region was performed with a 2.1-T whole-body magnet using the stimulated echo pulse sequence and an echo time of 270 msec., Results: All but one of the cortical stroke patients had increased lactate within or near the infarct. Persistently elevated cerebral lactate was documented in five of six cases studied serially as long as 251 days after infarction. N-acetylaspartate levels were decreased in most cortical strokes. Elevated lactate, accompanied by minimal reduction in N-acetylaspartate, was recorded in two of four patients in the first week following a small subcortical infarct., Conclusions: Long-term elevation of lactate commonly occurs after stroke. This lactate may arise from ongoing ischemia or infiltrating leukocytes, or it may be a residual of the lactate formed during the initial insult. The ability to observe stroke-elevated lactate pools at any time after lesion onset provides an approach to distinguishing among these possibilities in the future.

Proton NMR editing techniques were utilized to study the pharmacology of 13C-labeled ethanol in the cerebrum of the living rabbit at 4.7 T. The sensitivity of these proton spectroscopic methods was compared to direct carbon spectroscopy and a 14-fold improvement in sensitivity of 1H over 13C NMR spectroscopy was observed in vivo. This increase in sensitivity permitted the observation of the time course of the influx and afflux of this 13C-labeled compound with a time resolution of approximately 2 min per spectrum.

1H nuclear magnetic resonance (NMR) was used to detect directly the signal from the aromatic protons of phenylalanine (phe) in the brains of rabbits made hyperphenylalaninemic by administration of a diet high in phe and containing 0.4% alpha-methylphenylalanine. In addition to those resonances found in the region between 6.5 and 8.5 ppm in the 1H NMR spectra of control rabbits, a resonance centered at 7.37 ppm was observed in the spectra obtained from the brains of hyperphenylalaninemic rabbits in vivo or in situ postmortem. The chemical shift of this additional resonance was that expected for protons of the phenyl ring of phe. Its intensity correlated well with measurements of brain phe levels made on postmortem samples by amino acid analyzer. Both of these measurements correlated poorly with amino acid analyzer measurements of serum phe, especially at high values of the latter. High-resolution 1H NMR spectra of the brain extracts showed that in most animals an unidentified aromatic compound, possibly gamma-glutamyl-phe, was present in addition to phe. This study demonstrates the feasibility of measuring the concentration of brain phenyl and its metabolites noninvasively by 1H NMR. The method can be used for similar measurements in human brain.

The rate of incorporation of carbon from [1-13C]glucose into the [4-CH2] and [3-CH2] of cerebral glutamate was measured in the rat brain in vivo by 1H-observed, 13C-edited (POCE) nuclear magnetic resonance (NMR) spectroscopy. Spectra were acquired every 98 s during a 60-min infusion of [1-13C]glucose. Complete time courses were obtained from six animals. The measured intensity of the unresolved [4-13CH2] resonances of glutamate and glutamine increased exponentially during the infusion and attained a steady state in approximately 20 min with a first-order rate constant of 0.130 +/- 0.010 min-1 (t1/2 = 5.3 +/- 0.5 min). The appearance of the [3-13CH2] resonance in the POCE difference spectrum lagged behind that of the [4-13CH2] resonance and had not reached steady state at the end of the 60-min infusion (t1/2 = 26.6 +/- 4.1 min). The increase observed in 13C-labeled glutamate represented isotopic enrichment and was not due to a change in the total glutamate concentration. The glucose infusion did not affect the levels of high-energy phosphates or intracellular pH as determined by 31P NMR spectroscopy. Since glucose carbon is incorporated into glutamate by rapid exchange with the tricarboxylic acid (TCA) cycle intermediate alpha-ketoglutarate, the rate of glutamate labeling provided an estimate of TCA cycle flux. We have determined the flux of carbon through the TCA cycle to be approximately 1.4 mumols g-1 min-1. These experiments demonstrate the feasibility of measuring metabolic fluxes in vivo using 13C-labeled glucose and the technique of 1H-observed, 13C-decoupled NMR spectroscopy.

The kinetics of the CK reaction in the living rabbit brain was studied by three 31P magnetization transfer methods: inversion recovery (IR), inversion transfer (IT), and steady-state saturation transfer (SSST). Longitudinal relaxation rates (tau -1) were determined from IR and IT experiments. The values were 0.88 +/- 0.08 s-1 for PCr and 2.45 +/- 0.39 s-1 for gamma-ATP. Analysis of the results of SSST experiments in which gamma-ATP was saturated yielded a forward rate constant kF of 0.53 +/- 0.07 s-1. Upon saturation of PCr no change in the gamma-ATP signal could be detected in SSST. An average reverse rate constant (kR +/- S.D.) of 0.52 +/- 0.04 s-1 was estimated by analyzing IT data from three experiments with selective inversion of gamma-ATP. The standard error of kR was 50%. The average [PCr]/[ATP] of 1.21 +/- 0.16 together with the values of kF and kR yielded a forward-to-reverse flux ratio of 1.23. Within our limits of accuracy this ratio was not significantly different from 1.0, thus suggesting that in the brain the forward and reverse CK fluxes are equal.

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.

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.

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.

Using a surface coil, we have obtained 1H NMR spectra from metabolites in the human brain. Localization was achieved by combining depth pulses with image-selected in vivo spectroscopy magnetic field gradient methods. 1H spectra in which total creatine (3.03 ppm) has a signal/noise ratio of 95:1 were obtained in 4 min from 14 ml of brain. A resonance at 2.02 ppm consisting predominantly of N-acetylaspartate was measured relative to the creatine peak in gray and white matter, and the ratio was lower in the white matter. The spin-spin relaxation times of N-acetylaspartate and creatine were measured in white and gray matter and while creatine relaxation times were the same in both, the N-acetylaspartate relaxation time was longer in white matter. Lactate was detected in the normoxic brain and the average of three measurements was approximately equal to 0.5 mM from comparison with the creatine plus phosphocreatine peak, which was assumed to be 10.5 mM.

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.

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.

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)

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.

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.

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.

1H magnetic resonance spectroscopic measurements of cerebral lactate accumulation were used to estimate maximum agonal cerebral glycolytic rate (AGR) after cardiac arrest in 10 rabbits, six of which had received cortical electroshock. In the four control rabbits, mean AGR was 3.1 mumol glucose equivalents/g wet weight/min (standard error of the mean, 0.6), a figure in close agreement with earlier studies by workers using other techniques. AGR depended much more on carbohydrate availability as expressed by terminal blood glucose than on the shock-conditioned state of the glycolytic system reflected by individual rate constants of lactate accumulation. Regardless of shock history, AGR rose with blood glucose as though it were limited only by substrate availability. The unique capability of magnetic resonance spectroscopy to obtain chemically specific time course data noninvasively made these observations possible. The method has considerable potential for further analysis of normal and deranged cerebral metabolism.

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.