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R G Shulman

Publications and source records attributed to R G Shulman.

At least 145 records · Page 8Linked to original sources
PubMed1986

Combined 1H and 31P nuclear magnetic resonance spectroscopic studies of bicuculline-induced seizures in vivo.

Using a 1.89-Tesla spectrometer, 1H and 31P nuclear magnetic resonance spectra were acquired from the brains of paralyzed rabbits ventilated with 30% oxygen in nitrous oxide. Intracellular pH and changes in lactate concentration in the cerebrum were monitored by nuclear magnetic resonance methods during and after bicuculline-induced seizures, together with the electroencephalogram, heart rate, and arterial blood pressure. During seizures lasting more than an hour, cerebral intracellular pH became acidic, the cerebral lactate level rose rapidly, and both changes persisted as long as 2 hours without signs of recovery. After less prolonged seizures, lactate elevations were no less persistent, despite nearly complete recovery of intracellular pH and the electroencephalogram.

Animals↗
PubMed1986

Hepatic gluconeogenesis from alanine: 13C nuclear magnetic resonance methodology for in vivo studies.

This paper describes an experimental protocol designed to optimize 13C NMR spectra from the liver of the living anesthetized rat at 1.9 T. The protocol involves the use of a Helmholtz NMR coil which is positioned around the liver after surgical exposure. 1H decoupling is facilitated by double tuning this coil to both the 1H and the 13C frequencies. The protocol was shown to be suitable for studying the hepatic metabolism of 13C-labeled substrates in vivo by investigating the metabolism of [3-13C]alanine. Labeled glucose, glutamate, glutamine, and aspartate were formed and detected by 13C NMR in vivo in this experiment. The labeling patterns in these metabolites provided evidence that the major flow of alanine carbon into the Krebs cycle is via the pyruvate carboxylase reaction rather than through pyruvate dehydrogenase.

Alanine↗
PubMed1986

Detection of metabolites in rabbit brain by 13C NMR spectroscopy following administration of [1-13C]glucose.

1H-decoupled 13C NMR spectra (20.2 MHz) of the living rabbit brain were collected with a surface coil following the intravenous infusion of [1-13C]glucose. Within 15 min of infusion, the alpha and beta anomers of glucose were detected and, shortly thereafter, the carbon atoms at positions C4, C3, and C2 of glutamate and(or) glutamine. After reductions of inspired oxygen from 30 to 5%, lactate C3 was detected. The intensity of the lactate resonance rose progressively during hypoxia and later fell during recovery with oxygen. The 13C fractional isotopic enrichment of arterial blood glucose was measured by 1H NMR providing information on the rate and extent of blood glucose labeling.

Animals↗
PubMed1986

Chemical and functional correlates of postischemic renal ATP levels.

Renal energy metabolism was investigated before, during, and after ischemic insults of varying durations with in vivo 31P NMR spectroscopy. The postischemic recovery of renal ATP was found to be a biphasic process regardless of the length of the ischemia. This two-stage recovery consisted of a quick initial component immediately upon reflow followed by a slower, more gradual return toward preischemic levels. To characterize the source of each phase of the recovery, kidneys were extracted with perchloric acid at the end of the different periods of ischemia (before reflow). Concentrations of adenine nucleotides and breakdown products adenosine, inosine, and hypoxanthine were determined by 1H NMR spectroscopy. Excellent correlation was found between the residual nucleotide pool and the magnitude of the initial phase of ATP recovery. Additionally, the renal ATP content after 120 min of reflow was shown to have a strong correlation with subsequent functional recovery. These experiments show that in vivo 31P NMR can provide new and dynamic information concerning the biochemical recovery from ischemia. Furthermore, this data has the potential to predict the eventual functional recovery of the organ.

Adenosine Diphosphate↗
PubMed1986

Postischemic ATP-MgCl2 provides precursors for resynthesis of cellular ATP in rats.

Postischemic administration of ATP-MgCl2 is known to enhance recovery of renal function and accelerate the restitution of cellular ATP levels. To differentiate between a direct and indirect effect of the exogenous nucleotide, rats were subjected to 45 min of bilateral renal ischemia and were infused with either ATP-MgCl2, AMP-MgCl2, or normal saline. The immediate recovery of the cellular ATP was similar in all three groups of animals, whereas the subsequent recovery was accelerated by the infusion of either nucleotide. Since ATP-MgCl2 and AMP-MgCl2 produced similar results, this study provides evidence that exogenous ATP may act by providing precursors for the resynthesis of the cellular adenine nucleotide pool rather than being a direct source of energy.

Adenosine Triphosphate↗
PubMed1985

In vivo 31P nuclear magnetic resonance saturation transfer measurements of phosphate exchange reactions in the yeast Saccharomyces cerevisiae.

31P saturation transfer techniques have been used to measure phosphate kinetics in the yeast Saccharomyces cerevisiae. The phosphate consumption rate observed in acetate grown mid-log cells was combined with measurements of O2 consumption to yield P/O ratios of 2.2 and 2.9, for cells respiring on glucose and ethanol, respectively. However, no phosphate consumption activity was observed in saturation transfer experiments on anaerobic glucose fed cells. The phosphate consumption rates measured by saturation transfer in cells respiring on glucose and ethanol was attributed to the unidirectional rates of mitochondrial ATP synthesis.

Adenosine Triphosphate↗
PubMed1985

Kinetics of creatine kinase in heart: a 31P NMR saturation- and inversion-transfer study.

The kinetics of the phosphate exchange by creatine kinase (CK) was studied in solution and in the Langendorff-perfused rat heart at 37 degrees C. 31P inversion-transfer (IT) and saturation-transfer (ST) methods were applied. The kinetic parameters obtained by the two magnetization transfer methods were the same, whether in solution or in the perfused heart. Inversion transfer is the more efficient method, yielding the kinetic constants for the exchange and the relaxation rates of the transferred phosphate in both substrates, in one experiment. In solution the forward (kF) and reverse (kR) pseudo-first-order rate constants for the CK reaction (kF = k1[MgADP][H+]; kR = k-1[creatine]) as well as the concentrations of phosphocreatine (PCr), MgATP, and creatine (Cr) remained constant between pH 6.9 and pH 7.8. Equilibrium at this pH region is therefore maintained by compensating changes in the concentration of MgADP. The forward and reverse fluxes in the perfused heart were equal with an average flux ratio (fluxF/fluxR) of 0.975 +/- 0.065 obtained by both methods. Average values of kF and kR were 0.725 +/- 0.077 and 1.12 +/- 0.14 s-1, respectively. These results clearly indicate that the CK reaction in the Langendorff-perfused heart is in equilibrium and its rate is not limited by the diffusion of substrates between different locations of the enzyme. There is therefore no indication of compartmentation of substrates of the CK reaction.

Animals↗
PubMed1985

Application of multipulse NMR to observe 13C-labeled metabolites in biological systems.

Limitations in resolution and sensitivity of 13C NMR spectroscopy have reduced the information obtainable from intact biological systems. With the aim of increasing the information from in vivo 13C NMR two multipulse NMR techniques, the DEPT pulse sequence and the gated spin-echo sequence, were used to obtain edited 13C NMR spectra from different 13C-labeled mammalian tissues. This allowed the separation of the 13C NMR signals from the tissues into subspectra containing either CH, CH2, or CH3 signals, thereby increasing the information obtainable from these spectra. Comparing the two techniques, the DEPT sequence gives more accurate editing than the gated spin-echo sequence but suffers from the difficulty of determining 1H pulse angles in vivo.

Acetates↗
PubMed1985

1H homonuclear editing of rat brain using semiselective pulses.

We have used a semiselective Hahn spin-echo sequence of the form (1331)-tau-(2662)-tau-AQ, delivered by a surface coil to obtain high-resolution 1H NMR spectra from the brains of intact dead rats. This sequence gave suppression of the tissue water resonance by a factor of 80,000 when tau = 68 ms. Delivery of a frequency-selective Dante pulse train to the alpha-CH resonance of lactate at 4.11 ppm, simultaneously with the 2662 refocusing pulse, altered the j-modulation in the spin-coupled beta-CH3 protons. Subtraction of this spectrum from one in which the Dante was ineffective gave an edited spectrum containing only the beta-CH3 resonance of lactate at 1.31 ppm. When the position of the Dante was shifted to 3.78 ppm to selectively invert the alpha-CH protons of alanine, an edited spectrum of alanine was obtained.

Alanine↗
PubMed1985

A 1H NMR technique for observing metabolite signals in the spectrum of perfused liver.

We have developed a 1H NMR technique to selectively edit the spectrum of perfused liver for specific resonances of metabolites that occur in low concentration. The method employs selective DANTE pulses, which avoid exciting the water signal and at the same time control the J modulation effect in the homonuclear spin-echo experiment. By difference spectroscopy, we have suppressed the background signals from lipids and water and have resolved the CH3 resonance of lactate at 1.33 ppm. Moreover, the technique is highly selective and allows us to select the CH3 resonance of alanine at 1.47 ppm in the presence of the CH3 resonance of lactate at 1.33 ppm, even though the latter was much larger before editing. We have applied this technique to study the metabolic effect of ethanol in perfused mouse liver and have observed that the rate of formation of lactate from pyruvate is increased by a factor of 2.8 when ethanol is added.

Animals↗
PubMed1985

23Na and 39K NMR studies of ion transport in human erythrocytes.

Ion transport in human erythrocytes was studied by 23Na and 39K NMR with an anionic paramagnetic shift reagent, Dy(P3O10)2(7-). The intra- and extracellular 23Na and 39K NMR signals were well separated (over 10 ppm) at 5 mM concentration of the shift reagent. The NMR visibility of the intracellular Na+ and K+ was determined to be 100% in human and duck erythrocytes. The intracellular ion concentrations were 8.1 +/- 0.8 mM Na+ (n = 7) and 110 +/- 12 mM K+ (n = 4) for fresh human erythrocytes. The ouabain-sensitive net Na+ efflux was 1.75 +/- 0.08 mmol/hr per liter of cells at 37 degrees C (n = 3). The gramicidin-induced ion transport in human erythrocytes was also studied by 23Na and 39K NMR or by simultaneous measurements of 23Na NMR and a K+-selective electrode. The time courses of the Na+ and K+ transport induced by the ionophore were biphasic. The initial rapid fluxes were due to an exchange of Na+ for K+, which were found to occur with a 1:1 stoichiometry. The subsequent slow components were the net Na+ and K+ effluxes rate-limited by the Cl- permeability and accompanied by a reduction in cell volume. The Cl- permeability determined from the NMR measurements of these slow fluxes was 3.2 +/- 0.5 X 10(-8) cm/sec at 25 degrees C (n = 4).

Biological Transport, Active↗
PubMed1985

1H-Observe/13C-decouple spectroscopic measurements of lactate and glutamate in the rat brain in vivo.

We have used (13C)-1H NMR spectroscopy at 360.13 MHz to resolve the 13C coupled proton resonance of glutamate and lactate in the rat brain in vivo. The time required for the 13C fractional enrichment of the 4-CH2 position of brain glutamate to reach isotopic steady state was determined during a continuous infusion of D-[1-13C]glucose. Under conditions of ischemia, measurements made of the 3-CH3 of lactate in (13C)-1H NMR spectra revealed the relative contribution of brain glucose and glycogen to lactate formation. (13C)-1H NMR was 11 times more sensitive than 13C NMR for the detection of 13C in the 3-CH3 position of lactate and 6 times more sensitive for the detection of 13C in the 4-CH2 of glutamate under similar in vivo conditions.

Animals↗
PubMed1985

Effect of hypoglycemic encephalopathy upon amino acids, high-energy phosphates, and pHi in the rat brain in vivo: detection by sequential 1H and 31P NMR spectroscopy.

Metabolic alterations in amino acids, high-energy phosphates, and intracellular pH during and after insulin hypoglycemia in the rat brain was studied in vivo by 1H and 31P nuclear magnetic resonance (NMR) spectroscopy. Sequential accumulations of 1H and 31P spectra were obtained from a double-tuned surface coil positioned over the exposed skull of a rat while the electroencephalogram was recorded continuously. The transition to EEG silence was accompanied by rapid declines in phosphocreatine, nucleoside triphosphate, and an increase in inorganic orthophosphate in 31P spectra. In 1H spectra acquired during the same time interval, the resonances of glutamate and glutamine decreased in intensity while a progressive increase in aspartate was observed. Following glucose administration, glutamate and aspartate returned to control levels (recovery half-time, 8 min); recovery of glutamine was incomplete. An increase in lactate was detected in the 1H spectrum during recovery but it was not associated with any change in the intracellular pH as assessed in the corresponding 31P spectrum. Phosphocreatine returned to control levels following glucose administration, in contrast to nucleoside triphosphate and inorganic orthophosphate which recovered to only 80% and 200% of their control levels, respectively. These results show that the changes in cerebral amino acids and high-energy phosphates detected by alternating the collection of 1H and 31P spectra allow for a detailed assessment of the metabolic response of the hypoglycemic brain in vivo.

Amino Acids↗
PubMed1985

NMR measurements of intracellular sodium in the rabbit proximal tubule.

The present study evaluated the use of nuclear magnetic resonance (NMR) spectroscopy to monitor directly and continuously intracellular sodium levels in rabbit renal cortical tubule suspensions. When the paramagnetic shift reagent dysprosium tripolyphosphate was added to the extracellular medium it was possible to resolve signals from intracellular and extracellular sodium without adversely affecting cellular viability. An efflux of intracellular sodium against a significant concentration gradient was observed when sodium-loaded cells were warmed from 4 to 37 degrees C. At 37 degrees C in steady state, inhibition of Na+-K+-ATPase activity by ouabain increased intracellular sodium content in a dose-dependent and time-dependent manner. A biphasic time course of increased intracellular sodium following ouabain (10(-3) M) suggested that the sodium permeability of the plasma membrane may decrease following pump inhibition, thus limiting sodium influx. Nystatin, an agent known to facilitate sodium entry across cell membranes, increased intracellular sodium fivefold. In another series of experiments several maneuvers were performed to ascertain the fraction of intracellular sodium that was NMR visible. Quantitative assessment of either an efflux or influx of sodium indicated that the NMR visibility of the transported sodium was 100%. Furthermore, disruption of the cell membranes with Triton X-100 showed that the entire pool of intracellular sodium was 100% NMR visible.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Mechanism of liver glycogen repletion in vivo by nuclear magnetic resonance spectroscopy.

In order to quantitate the pathways by which liver glycogen is repleted, we administered [1-13C]glucose by gavage into awake 24-h fasted rats and examined the labeling pattern of 13C in hepatic glycogen. Two doses of [1-13C]glucose, 1 and 6 mg/g body wt, were given to examine whether differences in the plasma glucose concentration altered the metabolic pathways via which liver glycogen was replenished. After 1 and 3 h (high-dose group) and after 1 and 2 h (low-dose group), the animals were anesthetized and the liver was quickly freeze-clamped. Liver glycogen was extracted and the purified glycogen hydrolyzed to glucose with amyloglucosidase. The distribution of the 13C-label was subsequently determined by 13C-nuclear magnetic resonance spectroscopy. The percent 13C enrichment of the glucosyl units in glycogen was: 15.1 +/- 0.8%(C-1), 1.5 +/- 0.1%(C-2), 1.2 +/- 0.1%(C-3), 1.1 +/- 0.1%(C-4), 1.6 +/- 0.1%(C-5), and 2.2 +/- 0.1%(C-6) for the high-dose study (n = 4, at 3 h); 16.5 +/- 0.5%(C-1), 2.0 +/- 0.1%(C-2), 1.3 +/- 0.1%(C-3), 1.1 +/- 0.1%(C-4), 2.2 +/- 0.1%(C-5), and 2.4 +/- 0.1%(C-6) in the low-dose study (n = 4, at 2 h). The average 13C-enrichment of C-1 glucose in the portal vein was found to be 43 +/- 1 and 40 +/- 2% in the high- and low-dose groups, respectively. Therefore, the amount of glycogen that was synthesized from the direct pathway (i.e., glucose----glucose-6-phosphate----glucose-1-phosphate----UDP-glucose---- glycogen) was calculated to be 31 and 36% in the high- and low-dose groups, respectively. The 13C-enrichments of portal vein lactate and alanine were 14 and 14%, respectively, in the high-dose group and 11 and 8%, respectively, in the low-dose group. From these enrichments, the minimum contribution of these gluconeogenic precursors to glycogen repletion can be calculated to be 7 and 20% in the high- and low-dose groups, respectively. The maximum contribution of glucose recycling at the triose isomerase step to glycogen synthesis (i.e., glucose----triose-phosphates----glycogen) was estimated to be 3 and 1% in the high- and low-dose groups, respectively. In conclusion, our results demonstrate that (a) only one-third of liver glycogen repletion occurs via the direct conversion of glucose to glycogen, and that (b) only a very small amount of glycogen synthesis can be accounted for by the conversion of glucose to triose phosphates and back to glycogen; this suggests that futile cycling between fructose-6-phosphate and fructose-1,6-diphosphate under these conditions is minimal. Our results also show that (c) alanine and lactate account for a minimum of between 7 and 20% of the glycogen synthesized, and that (d) the three pathways through which the labeled flux is measured account for a total of only 50% of the total glycogen synthesized. These results suggest that either there is a sizeable amount of glycogen synthesis via pathway(s) that were not examined in the present experiment or that there is a much greater dilution of labeled alanine/lactate in the oxaloacetate pool than previously appreciated, or some combination of these two explanations.

Alanine↗
PubMed1985

Cerebral metabolism in hyper- and hypocarbia: 31P and 1H nuclear magnetic resonance studies.

Paralyzed rabbits ventilated with an oxygen, nitrous oxide, and carbon dioxide mixture were subjected to hyper- and hypocarbic stress. An Oxford Instrument TMR 32-200 spectrometer was used to record phosphorus-31 and nonwater proton nuclear magnetic resonance spectra of the in vivo brain. These spectra provide measurements of cerebral pHi, phosphocreatine, orthophosphate, ATP, and lactate. The brain exhibited twice as much acute pH-regulating ability as the arterial blood. During hypercarbia, orthophosphate rose while phosphocreatine declined in a reciprocal manner, and ATP remained constant. During hypocarbia, lactate rose gradually over a period of 1 hour, while orthophosphate, phosphocreatine, and ATP remained constant and calculated values of adenosine mono- and diphosphate rose.

Adenosine Diphosphate↗