title: Cerebral metabolic studies in vivo by combined 1H/31P and 1H/13C NMR spectroscopic methods.
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 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)
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven.
authors.forename: K L
authors.initials: KL
authors.lastname: Behar
authors.forename: S M
authors.initials: SM
authors.lastname: Fitzpatrick
authors.forename: H P
authors.initials: HP
authors.lastname: Hetherington
authors.forename: R G
authors.initials: RG
authors.lastname: Shulman
mesh_terms: D000136:Acid-Base Equilibrium / Q000502:physiology*; D000596:Amino Acids / Q000097:blood; D000641:Ammonia / Q000097:blood*; D000818:Animals; D001921:Brain / Q000503:physiopathology; D001925:Brain Damage, Chronic / Q000473:pathology / Q000503:physiopathology*; D002545:Brain Ischemia / Q000473:pathology / Q000503:physiopathology; D004734:Energy Metabolism / Q000502:physiology*; D006801:Humans; D007773:Lactates / Q000097:blood; D019344:Lactic Acid; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D000596:Amino Acids; D007773:Lactates; D019344:Lactic Acid; D000641:Ammonia