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Cerebral Metabolic Studies in vivo by Combined 1H/31P and 1H/13C NMR Spectroscopic Methods

  • Kevin L. Behar
Part of the Acta Neurochirurgica book series (NEUROCHIRURGICA, volume 57)

Abstract

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 ⃗ 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 µM. 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). These changes were not observed in animals infused with sodium acetate (control group, n = 6). No changes were observed in intracellular pH or high energy phosphates in the 31P spectrum as a result of either the ammonium acetate or sodium acetate infusions.

In the final study we used 1H-observed, 13C-decoupled spectroscopy to measure the flow of 13C label from [1-13C] glucose into cerebral glutamate (and glutamine) in vivo in normal and chronically hyperammonemic (4-week portacaval shunted) rats. The flow of label into glutamate-C4 allowed us to measure the steady-state cerebral TCA cycle activity in vivo. An infusion protocol was developed to rapidly increase and maintain the enrichment of [1-13C] glucose in arterial blood. The metabolism of [1-13C] glucose through glycolysis and the TCA cycle results initially in the incorporation of the label in the C4 of α-ketoglutarate (α-KG) and glutamate, with which a-KG is in rapid equilibrium. Subsequent turns of the TCA cycle will lead to 13C label incorporation in C2, C3 and later in C1 of glutamate. In control animals the rate of incorporation of 13C into glutamate-C4 followed first order kinetics with a rate constant of 0.130 ± 0.012 min-1; glutamate-C3 was much slower (0.026 ± 0.004 min-1). In contrast, the rate constant for glutamate-C4 labeling in 4-week portacaval shunted rats was only 0.038 ± 0.008 min-1, suggesting a significant reduction in TCA cycle activity in chronic hyperammonemia.

These studies indicate
  1. 1)

    acute cerebral acidosis leads to a large reduction in cerebral glutamate and a smaller increase in glutamine;

     
  2. 2)

    acute hyperammonemia does not alter cerebral pH, but leads to large increases in glutamine and lactate, and a smaller decrease in glutamate;

     
  3. 3)

    chronic hyperammonemia causes a significant reduction in the rate of label incorporation from [1-13C] glucose into glutamate, probably due to a fall in cerebral TCA cycle activity.

     

Keywords

Intracellular pH hyperammonemia 1H-observed 13C decoupled spectroscopy label flow 

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Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • Kevin L. Behar
    • 1
  1. 1.Department of Molecular Biophysics and BiochemistryYale UniversityNew HavenUSA

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