We propose to re-explore the regulation of cerebral glutamine, glutamate and gamma-aminobutyric acid (GABA) metabolism in order to elucidate the mechanisms of neurotoxicity in hepatic encephalopathy (HE). The new impetus is the development of non-invasive assays of enzyme activity and metabolite flux in the brain of living rats, using novel techniques of 15N magnetic resonance (MR) and the more sensitive method of 1H-15N heteronuclear multiple quantum coherence (HMQC) MR spectroscopy (MRS). Both methods depend upon enrichment of metabolic pools with the stable isotope 15N. Specifically, in vivo activities of glutamine synthetase (GS), an astrocyte marker enzyme, and of glutamate decarboxylase (GAD), a marker enzyme of pre-synaptic nerve-terminals, will be measured in the brain of hyperammonemic rats as an animal model of HE, and in thioacetamide-treated rats as a model of fulminant hepatic failure. The GS activity will indicate the in vivo rate of ammonia incorporation into glutamine and of glutamate depletion from the astrocytes. The GAD activity will reflect glutamate concentration in nerve terminals and indicate the rate of GABA synthesis. Finally, the rate of replenishment of the total cerebral glutamate pool will be determined from the rate of transamination from 15N-enriched essential amino acids that readily cross the blood-brain barrier. These experiments will clarify the effect of hyperammonemia on neuronal vs astrocytic pools of glutamate and GABA and contribute to evaluation of three leading hypotheses on the mechanism of neurotoxicity in HE: 1) ammonia per se is neurotoxic; 2) glutamine accumulation contributes to the pathogenesis; and 3) hyperammonemia causes HE through depletion of the neurotransmitter pool of glutamate. In addition, the relative importance of benzodiazepines and GABA in the etiology of brain dysfunction in fulminant hepatic failure will be explored. These experiments will shed light on an increasingly important group of neurological diseases, including hepatic encephalopathy.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS029048-02
Application #
2267364
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1994-08-01
Project End
1997-07-01
Budget Start
1995-08-01
Budget End
1996-07-31
Support Year
2
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Huntington Medical Research Institutes
Department
Type
DUNS #
077978898
City
Pasadena
State
CA
Country
United States
Zip Code
91101
Kanamori, Keiko; Ross, Brian D (2004) Quantitative determination of extracellular glutamine concentration in rat brain, and its elevation in vivo by system A transport inhibitor, alpha-(methylamino)isobutyrate. J Neurochem 90:203-10
Kanamori, K; Kondrat, R W; Ross, B D (2003) 13C enrichment of extracellular neurotransmitter glutamate in rat brain--combined mass spectrometry and NMR studies of neurotransmitter turnover and uptake into glia in vivo. Cell Mol Biol (Noisy-le-grand) 49:819-36
Kondrat, Richard W; Kanamori, Keiko; Ross, Brian D (2002) In vivo microdialysis and gas-chromatography/mass-spectrometry for 13C-enrichment measurement of extracellular glutamate in rat brain. J Neurosci Methods 120:179-92
Kanamori, Keiko; Ross, Brian D; Kondrat, Richard W (2002) Glial uptake of neurotransmitter glutamate from the extracellular fluid studied in vivo by microdialysis and (13)C NMR. J Neurochem 83:682-95
Kanamori, K; Ross, B D (2001) The first in vivo observation of (13)C-(15)N coupling in mammalian brain. J Magn Reson 153:193-202
Kanamori, K; Ross, B D (1999) In vivo detection of (15)N-coupled protons in rat brain by ISIS localization and multiple-quantum editing. J Magn Reson 139:240-9
Kanamori, K; Ross, B D (1999) Localized 15N NMR spectroscopy of rat brain by ISIS. Magn Reson Med 41:456-63
Kanamori, K; Ross, B D; Kondrat, R W (1998) Rate of glutamate synthesis from leucine in rat brain measured in vivo by 15N NMR. J Neurochem 70:1304-15
Bluml, S; Zuckerman, E; Tan, J et al. (1998) Proton-decoupled 31P magnetic resonance spectroscopy reveals osmotic and metabolic disturbances in human hepatic encephalopathy. J Neurochem 71:1564-76
Kanamori, K; Ross, B D (1997) Glial alkalinization detected in vivo by 1H-15N heteronuclear multiple-quantum coherence-transfer NMR in severely hyperammonemic rat. J Neurochem 68:1209-20

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