AMMONIA METABOLISM IN REYE'S SYNDROME

  • Hale, Daniel (PI)
  • Watkins, John (PI)
  • HUMMELER, KLAUS (PI)
  • COATES, PAUL (PI)
  • YUDKOFF, MARC (PI)
  • STANLEY, CHARLES (PI)
  • HALE, DANIEL (PI)
  • CORKEY, BARBARA (PI)
  • DOUGLAS, STEVEN DANIEL (PI)
  • COATES, PAUL (PI)
  • PLEASURE, DAVID (PI)
  • COATES, PAUL (PI)
  • Watkins, John (PI)
  • Watkins, John (PI)

Project: Research project

Project Details

Description

This project involves the study of the relationships between
nitrogen metabolism and that of fatty acids and ketoacids in the
following in vitro systems: (1) primary cultures of astrocytes;
(2) primary cultures of neurons; and (3) cultured skin fibroblasts
taken from patients with inherited disorders of fatty acid
oxidation, particularly medium chain and long chain acyl-CoA
dehydrogenase deficiencies. The core hypothesis with regard to the
work in the brain cells is that excessive levels of fatty acids
impair normative metabolism of glutamine, glutamate and aspartate
and that the encephalopathy characteristic of patients with
defective lipid oxidation of inherited or acquired (e.g., Reye's
syndrome) etiology is in part referable to excessive levels of such
neurotoxic compounds as glutamate and aspartate. The core
hypothesis underlying the research involving cultured skin
fibroblasts is that the hyperammonemia in patients with acquired
or primary defects of fatty acid oxidation is secondary not only
to impaired hepatic ureagenesis, but also to excessive ammonia
production brought about by a shift in the glutamate dehydrogenase
reaction toward the oxidative deamination of glutamate rather than
the reductive amination of alpha-ketoglutarate. This shift occurs
because the failure of lipid oxidation leads to a relative
depletion of intra-mitochondrial acetyl-CoA and a consequent
reduction in the intra-mitochondrial concentration of alpha-
ketoglutarate. We have identified a similar, though not identical,
adaptation to explain the augmentation of renal ammoniagenesis
observed in metabolic acidosis.

These hypotheses will be tested by incubating highly purified
cultures of astrocytes or neurons as well as cultured skin
fibroblasts from dehydrogenase-deficient patients with 15N labelled
precursors, e.g., (2-15N)glutamine, and utilizing gas
chromatography-mass spectrometry to quantitate isotopic abundance
in such products as (15N)aspartate, (15N)glutamate, 15NH3,
(15N)glutamic acid, etc. Our prior work has shown the stable
isotope 15N to be an extremely useful metabolic tracer for the
description of the nitrogen metabolism in in vitro systems. By
utilizing gas chromatography-mass spectrometry for the quantitation
of isotopic enrichment in the indicated products, precursor-product
relationships and net rates of nitrogen flux can be determined with
a rigor which would otherwise not be possible.
StatusFinished
Effective start/end date1/1/016/30/92

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