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Biomedical subjects

K L Behar

Publications and source records attributed to K L Behar.

At least 55 records · Page 3Linked to original sources
PubMed1995

Simultaneous determination of the rates of the TCA cycle, glucose utilization, alpha-ketoglutarate/glutamate exchange, and glutamine synthesis in human brain by NMR.

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.

Brain↗
PubMed1995

Initial observations on effect of vigabatrin on in vivo 1H spectroscopic measurements of gamma-aminobutyric acid, glutamate, and glutamine in human brain.

Recent developments involving 1H nuclear magnetic resonance (NMR) spectroscopic editing techniques have allowed noninvasive measurements of gamma-aminobutyric acid (GABA) in human cerebrum. The additional information gained from GABA and macromolecule measurements permitted more precise glutamate (Glu) and glutamine (Gln) measurements. Occipital lobe GABA in 10 nonepileptic, healthy subjects was 1.0 mumol/g brain [95% confidence interval (CI) 0.9-1.1]. Vigabatrin (VGB) is a safe and effective antiepileptic drug (AED) that irreversibly inhibits neuronal and glial GABA-transaminase. GABA levels were increased in all patients treated with VGB. With a standard dose of 3-6 g/day, GABA levels were 2.6 mumol/g (95% CI 2.3-2.8). Mean occipital GABA level measured in epileptic patients not receiving VGB was 0.9 mumol/g (95% CI 0.7-1.1). Gln was increased by 1.9 mumol/g and Glu was decreased by 0.8 mumol/g in patients receiving VGB as compared with patients receiving standard medications alone.

Adult↗
PubMed1994

Measurement of GABA following GABA-transaminase inhibition by gabaculine: a 1H and 31P NMR spectroscopic study of rat brain in vivo.

A selective 1H NMR spin-echo editing method was used to detect the 4-CH2 of GABA in rat brain in vivo before and after intravenous administration of the highly selective GABA transaminase inhibitor, gabaculine (3-amino-2,3-dihydrobenzoic acid-HCl; 100 mg/kg, intravenously). The effects of the inhibitor on high energy phosphates and pHi were determined by 31P NMR. GABA levels increased approximately linearly (r = 0.81 to 0.94; P < 0.0005) from 1.9 +/- 0.4 mumol/g (pre-gabaculine; mean +/- SD) to between 6 and 8 mumol/g after 4 hr at rates of accumulation of 1.1 to 2.9 mumol/hr/g. 1H NMR spectroscopic measurements of cerebral GABA and its rate of turnover offers a new approach in the study of GABA-mediated processes in vivo.

4-Aminobutyrate Transaminase↗
PubMed1994

Analysis of macromolecule resonances in 1H NMR spectra of human brain.

Macromolecule resonances underlying metabolites in 1H NMR spectra were investigated in temporal lobe biopsy tissue from epilepsy patients and from localized 1H spectra of the brains of healthy volunteers. The 1H NMR spectrum of brain tissue was compared with that of cytosol and dialyzed cytosol after removal of low molecular weight molecules (< 3500 daltons) at 8.4 and 2.1 Tesla. The assignment of specific resonances to macromolecules in 2.1 Tesla, short-TE, localized human brain 1H NMR spectra in vivo was made on the basis of a J-editing method using the spectral parameters (delta, J) and connectivities determined from 2D experiments in vitro. Two prominent connectivities associated with macromolecules in vitro (0.93-2.05 delta and 1.6-3.00 delta) were also detected in vivo by the J-editing method. Advantage was taken of the large difference in measured T1 relaxation times between macromolecule and metabolite resonances in the brain spectrum to acquire 'metabolite-nulled' macromolecule spectra. These spectra appear identical to the spectra of macromolecules isolated in vitro.

Adult↗
PubMed1994

Dynamic magnetic resonance imaging of the rat brain during forepaw stimulation.

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.

Animals↗
PubMed1993

Localized 1H NMR measurements of gamma-aminobutyric acid in human brain in vivo.

Localized 1H NMR spectroscopy in conjunction with J editing was used to measure the concentration of gamma-aminobutyric acid (GABA) in the occipital lobe of four control human volunteers and four epileptic volunteers who were receiving the drug vigabatrin. The GABA concentration measured in four nonepileptic subjects was 1.1 +/- 0.1 mumol/cm3 of brain, which is in good agreement with previous values measured in surgically removed human cortex. A dose-dependent elevation of GABA concentration was measured in patients receiving the GABA transaminase inhibitor vigabatrin, with the maximum measured level of 3.7 mumol/cm3 of brain measured at the highest dose (6 g per day) studied. 1H NMR measurements of GABA in those patients receiving GABA-elevating agents such as vigabatrin will be of importance in establishing the relationship between seizure suppression and the concentration of brain GABA.

4-Aminobutyrate Transaminase↗
PubMed1993

Characterization of macromolecule resonances in the 1H NMR spectrum of rat brain.

The 1H NMR spectrum of the macromolecule fraction of rat brain cytosol was investigated following centrifugation and dialysis to remove low molecular weight metabolites and peptides (< 3500 daltons). At least seven well resolved resonances were detected between 0.9 and 3.0 ppm in the 1H NMR spectrum of rat brain cytosol after dialysis, several of which cannot be observed in vivo due to overlap with N-acetylaspartate, glutamate, glutamine, creatine, and gamma-aminobutyric acid. Several cross-peaks detected in 2D COSY spectra of the cytosolic macromolecule fraction coincided with those measured in a previous study of rat brain tissue in vitro and in situ (K. L. Behar, T. Ogino, Magn. Reson. Med. 17, 285 (1991)). Treatment of the cytosolic macromolecule fraction with a nonspecific protease permitted partial assignments of resonances in the 1H NMR spectrum to specific amino acids. Fractionation of the dialyzed cytosol of rat brain by gel filtration yielded qualitatively similar 1H NMR spectra for elution volumes corresponding to molecular masses from 12.5 kDa to over 100 kDa. The results indicate that many of the background nonmetabolite resonances observed in the 1H NMR spectrum of normal brain tissue arise from cytosolic proteins.

Aging↗
PubMed1993

Cerebral metabolic studies in vivo by combined 1H/31P and 1H/13C NMR spectroscopic methods.

Intracellular pH and ammonium ion concentration are potent modulators of cerebral amino acid metabolism. Furthermore, intracellular acidosis and hyperammonemia accompany conditions such as ischemic encephalopathy and seizures and may contribute to the pathological sequelae observed. In vivo NMR spectroscopy permits multiple, non-destructive measurements of important cerebral metabolic intermediates in the same animal. We describe here the use of 1H, and 31P NMR spectroscopy to investigate the effects of acute changes in intracellular pH and ammonium ions on cerebral glutamate, glutamine, and lactate levels in vivo. We then show how 1H NMR can be used to indirectly follow the flow of 13C label from [1-13C] glucose into the cerebral glutamate pool, allowing us to measure cerebral TCA activity in normal and chronically hyperammonemic rats. Male Sprague-Dawley rats (160-210 gm), fasted 24-hours, were tracheotomized, paralyzed and ventilated on 30% O2/70% N2O. NMR spectroscopy was performed at a field strength of 8.4 Tesla using a Bruker AM-360 wide bore spectrometer. An elliptical surface-coil (8 x 12 mm) was double-tuned to either the 1H and 31P or 1H and 13C frequencies. After retraction of extracranial tissues, the coil was positioned over the skull 2 mm posterior to the bregma. Tail arteries and veins were cannulated allowing periodic measurements of PO2, pCO2, pH and glucose in arterial blood and intravenous infusions. Respiratory acidosis was induced in rats by the addition of CO2 to the ventilation gas mixture. Arterial pCO2 increased within 5 min from a pre-hypercarbic value of 36.4 +/- 6.1 mm Hg to 200-220 mm Hg and was maintained at this level for over 1 hour. Hypercarbia led to rapid cerebral acidification. Intracellular pH decreased from 7.18 +/- 0.08 (pre-hypercarbic period) to 6.68 +/- 0.06 (n = 4) at 10 min and remained stable throughout the NMR observation period. Glutamate decreased to 53 +/- 4% of control after 60 min of hypercarbia, while glutamine increased to 126 +/- 7% of control. Acute hyperammonemia was produced by a programmed intravenous infusion of 250 mM ammonium acetate, which rapidly raised and maintained the concentration of ammonium ions in the blood at approximately 500 microM. Shortly after the start of the infusion (10-20 min), the levels of glutamine and lactate rose continuously throughout the experiment, reaching levels of 170 +/- 25% and 260 +/- 60% of control, respectively (n = 12) after 50 min. Glutamate decreased during the same time interval to 80 +/- 4% of control (n = 12).(ABSTRACT TRUNCATED AT 400 WORDS)

Acid-Base Equilibrium↗
PubMed1993

NMR-spectroscopic investigation of cerebral reanimation after prolonged ischemia.

The severity of brain injury following interruption of blood flow depends on a number of ischemic and post-ischemic variables. The most important ischemic variables are the duration of ischemia, the amount of residual blood flow, the type and depth of anesthesia, brain glucose content and temperature. Among the post-ischemic factors the no-reflow phenomenon, edema and a variety of biochemical disturbances are of particular importance. Due to the complex interaction of these factors irreversible brain injury usually occurs after less than 10 min cerebrocirculatory arrest in normothermia. However, the safe ischemia time of the brain can be substantially extended when appropriate therapeutic measures are used to alleviate post-ischemic injury. NMR-spectroscopy is particularly suited for the analysis of this process. Recording of 31P, 1H and 19F spectra allow the continuous non-invasive assessment of such basic parameters as brain energy state, tissue pH, the content of lactate and blood flow (using Freon-23 as an inert tracer). In addition, information is obtained about changes in the content of phosphomonoesters and -diesters, glutamate, glutamine, aspartate and N-acetyl aspartate. These measurements can be combined with in vivo electrophysiological and post-mortem biochemical investigations for the further refinement of functional/metabolic monitoring. We have used this approach to study the potentials of post-ischemic resuscitation after one hour complete ischemia of the normothermic cat brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid-Base Equilibrium↗
PubMed1993

Glycolysis-citric acid cycle interrelation: a new approach and some insights in cellular and subcellular compartmentation.

In the development of an integrated approach to study metabolic compartmentation and regulation in brain, we have emphasized the importance, versatility, and need to exploit the recent methodological advances in (1) NMR spectroscopy, (2) primary cultures of neurons and glial cells, and (3) subcellular fractionation (especially brain mitochondrial isolation). The integrated approach has the advantage of being able to draw data and inferences based on some combination of results derived from in vivo, cellular, and subcellular studies. For example, some in vivo NMR data may suggest that an enzymatic step may be rate-limiting in a particular pathway. This information may be used to frame testable hypotheses and questions that can be investigated in experiments involving primary cultures of neural cells and subcellular fractions. Subsequently, the data from such in vitro studies could serve as the bases for constructing the hypothetical framework for predicting the regulatory role, in vivo, of the enzyme in the pathway. We have discussed the known as well as the as yet ill-defined facets of the cellular and subcellular aspects of the glycolysis-citric acid cycle interrelation and have attempted to illustrate how such an integrated approach could be applied to generate testable hypotheses for investigating the mechanisms concerned with metabolic compartmentation and regulation in brain. In the process of the illustration, we discuss some of the evidence in support of the general hypothesis that the transfer of reducing equivalents across the inner mitochondrial membrane plays a major role in mediating the coupling of the glycolytic flux to that of the citric acid cycle. We have given some indications as to how this hypothesis could be further investigated employing our approach. Moreover, we hope that other workers will find this integrated approach useful in designing multidisciplinary studies to investigate mechanistic issues related to this important theme.

Animals↗
PubMed1992

NMR determination of the TCA cycle rate and alpha-ketoglutarate/glutamate exchange rate in rat brain.

A mathematical model of cerebral glucose metabolism was developed to analyze the isotopic labeling of carbon atoms C4 and C3 of glutamate following an intravenous infusion of [1-13C]glucose. The model consists of a series of coupled metabolic pools representing glucose, glycolytic intermediates, tricarboxylic acid (TCA) cycle intermediates, glutamate, aspartate, and glutamine. Based on the rate of 13C isotopic labeling of glutamate C4 measured in a previous study, the TCA cycle rate in rat brain was determined to be 1.58 +/- 0.41 mumol min-1 g-1 (mean +/- SD, n = 5). Analysis of the difference between the rates of isotopic enrichment of glutamate C4 and C3 permitted the rate of exchange between alpha-ketoglutarate (alpha-KG) and glutamate to be assessed in vivo. In rat brain, the exchange rate between alpha-KG and glutamate is between 89 +/- 35 and 126 +/- 22 times faster than the TCA cycle rate (mean +/- SD, n = 4). The sensitivity of the calculated value of the TCA cycle rate to other metabolic fluxes and to concentrations of glycolytic and TCA cycle intermediates was tested and found to be small.

Animals↗
PubMed1992

NMR determination of intracerebral glucose concentration and transport kinetics in rat brain.

The concentration of intracerebral glucose as a function of plasma glucose concentration was measured in rats by 13C NMR spectroscopy. Measurements were made in 20-60 min periods during the infusion of [1-13C]D-glucose, when intracerebral and plasma glucose levels were at steady state. Intracerebral glucose was found to vary from 0.7 to 19 mumol g-1 wet weight as the steady-state plasma glucose concentration was varied from 3 to 62 mM. A symmetric Michaelis-Menten model was fit to the brain and plasma glucose data with and without an unsaturable component, yielding the transport parameters Km, Vmax, and Kd. If it is assumed that all transport is saturable (Kd = 0), then Km = 13.9 +/- 2.7 mM and Vmax/Vgly = 5.8 +/- 0.8, where Vgly is the rate of brain glucose consumption. If an unsaturable component of transport is included, the transport parameters are Km = 9.2 +/- 4.7 mM, Vmax/Vgly = 5.3 +/- 1.5, and Kd/Vgly = 0.0088 +/- 0.0075 ml mumol-1. It was not possible to distinguish between the cases of Kd = 0 and Kd greater than 0, because the goodness of fit was similar for both. However, the results in both cases indicate that the unidirectional rate of glucose influx exceeds the glycolytic rate in the basal state by 2.4-fold and as a result should not be rate limiting for normal glucose utilization.

Animals↗
PubMed1991

Assignment of resonance in the 1H spectrum of rat brain by two-dimensional shift correlated and J-resolved NMR spectroscopy.

Two-dimensional shift-correlated (COSY) and J-resolved NMR spectroscopy was used to identify and assign resonance in the aliphatic region (0.8 to 4.5 ppm) of the 1H spectrum of acid extracts and tissue of rat brain. The chemical shift and spin-spin coupling constants of several resonances, which could not be resolved in one-dimensional spectra of tissue, were determined. These properties, together with the appropriate multiplet structure and scalar coupling patterns observed in 2D J-resolved and COSY spectra, were used to assign the resonances of lactate, threonine, alanine gamma-amino butyrate, N-acetyl aspartate, aspartate, glutamate, glutamine, taurine, and myo-inositol. The single peak observed at 1.33 ppm in the 1D 1H spectrum of excised brain tissue, which is generally assigned to lactate, was shown instead to consist of two overlapping peaks in 2D COSY contour plots. The intensity of both resonances increased considerably after death. The second resonance was assigned to threonine and the assignment was confirmed by ion-exchange chromatography. Several cross peaks were identified in 2D COSY spectra of intact brain tissue that were not present in similar spectra of the acid extract. The nonmetabolite resonances were assigned to macromolecules. Analysis of 2D COSY contour plots in conjunction with 1D spectra revealed that the total creatinine (creatinine + phosphocreatine) resonance at 3.0 ppm was overlapped by both GABA and macromolecules. This macromolecule resonance was present in a 2D COSY spectrum of the rat brain obtained in situ with a surface coil following cardiac arrest, indicating that its presence in the 2D spectrum of excised brain tissue did not arise from tissue disruption.

Alanine↗
PubMed1990

The flux from glucose to glutamate in the rat brain in vivo as determined by 1H-observed, 13C-edited NMR spectroscopy.

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.

Animals↗
PubMed1989

NMR spectroscopic investigation of the recovery of energy and acid-base homeostasis in the cat brain after prolonged ischemia.

The effects of 1 h of complete global ischemia on the recovery of high-energy phosphates, intracellular pH (pHi), and lactate in the cat brain in vivo was investigated by 31P and 1H NMR spectroscopy. Ischemia led to a decrease in creatine phosphate (CrP), nucleoside triphosphates (NTP), and pHi, while inorganic phosphate and lactate increased. Intracellular pH decreased from a control value of 7.07 +/- 0.04 to 6.17 +/- 0.12 after 1 h of ischemia (N = 7). The degree of metabolic recovery after recirculation was variable. In three animals CrP and NTP were detected within 4 min and NTP increased to greater than or equal to 90% of control within 1 h; these levels were maintained for the 3 h of observation. In four other animals, CrP and NTP reached only 20 to 80% of control; however, high-energy phosphates decreased and lactate increased spontaneously between 1 and 2.5 h. Immediately following recirculation, pHi decreased further by an average of 0.3 units. The rate of recovery of cerebral pHi was slower than that of PCr and NTP for the majority of animals. Recovery of pHi was not detected for an average of 32 min after recirculation--by this time, NTP had attained 80 +/- 10% of their preischemic level. Recovery of pHi (and lactate) was not observed in two animals where PCr and NTP recovered transiently to only 30-43% of the preischemic level. Recovery of cerebral pHi was markedly heterogeneous in one animal, since two Pi peaks were detected shortly after recirculation.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid-Base Equilibrium↗
PubMed1989

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)

Acetates↗