Stimulation of the urea cycle in endotoxemic rats

Purchase, Kym (1998) Stimulation of the urea cycle in endotoxemic rats. Masters thesis, Memorial University of Newfoundland.

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Abstract

Muscle wasting is common in infection. There is increased muscle proteolysis without concomitant increased muscle protein synthesis so negative nitrogen balance results. Much work has focused on this aspect of catabolic states. However, increased release of amino acids into circulation leads to elevated amino nitrogen levels. The liver is the site for detoxification of amino nitrogen wastes, particularly ammonia. The urea cycle, located only within the periportal hepatic acinus, converts neurotoxic ammonia to urea which may be excreted in the urine. The response of the urea cycle subsequent to endotoxin administration was of specific interest to this work. -- In this study, a single intraperitoneal injection of E. coli lipopolysaccharide (LPS,0127 : B8) at a sublethal dose (3 mg/kg) resulted in a nearly three-fold increase in urea excretion from fasted rats between 12-24 hours after injection, compared to saline- injected controls. Substrates for urea synthesis were also measured. Ammonia levels were not changed in liver, muscle, blood or plasma 18 hours after endotoxin injection. There were significant changes in the total concentrations of amino acids in both arterial plasma and in the liver. Levels of alanine and glutamine, the primary glucogenic amino acids, were also affected subsequent to LPS injection. Alanine was significantly increased in all sample groups (skeletal muscle, liver, blood and plasma from hepatic portal vein and abdominal aorta)in response to LPS. Glutamine concentrations were not increased in the liver nor arterial blood but were significantly elevated in hepatic portal venous plasma and blood and in arterial plasma. There was, however, a significant decrease in muscle glutamine which is in agreement with findings in the literature. There appears to be an increase in circulating ureagenic substrates as well as an increase in the hepatic content of amino acids between 12-24 hours after LPS-injection in rats. -- The urea cycle exhibits compartmentation of metabolism. The first two enzymes (carbamyl phosphate synthetase I, CPS I; ornithine transcarbamylase, OTC) are located within the mitochondrion while the remaining enzymes (argininosuccinate synthetase, AS; argininosuccinate lyase, AL; arginase) are found in the cytosol. Assays of the enzymes of the urea cycle were carried out 24 hours after injection with LPS or saline. There was no increase in total activity, in vitro, of any of the enzymes post-LPS treatment. A previous study in our lab has found that liver protein increases by 18% in response to LPS but this increase is not sufficient to account for the three-fold increase of urea excretion from rats. -- Regulation of flux through the urea cycle enzymes has been previously reported to be limited by the activity of CPS I. In isolated intact rat liver mitochondria, the rate of citrulline synthesis was doubled in rats 24 hours after injection of LPS compared to the saline controls. The mitochondrial portion of the urea cycle, therefore, was stimulated in response to LPS. N-acetylglutamate (NAG), an allosteric activator of CPS I, was hypothesized to play a role in the increased citrulline-synthesizing capacity of mitochondria isolated from endotoxemic rats. NAG levels were measured in quick frozen mitochondrial samples from endotoxemic and saline control rats in which citrulline synthesis had also been measured in a separate aliquot. GC-MS analysis by Dr. Kratz (Baltimore, MD) determined that mitochondria from endotoxemic animals contained significantly more NAG (50%) compared to saline controls. Analysis of the relationship between citrulline synthesis from these mitochondria and the NAG content showed a significant positive correlation between NAG and the rate of citrulline synthesis. Finally, to assess the effect of NAG upon citrulline synthesis, we carried out experiments utilizing uncoupled mitochondria in the presence of varying concentrations of exogenous NAG. At 24 hours post-injection, mitochondria from LPS-injected animals synthesized citrulline at double the rate of mitochondria from the saline control animals (as seen in intact, coupled mitochondrial studies) but as the concentration of exogenous NAG reached 2.0-10.0 mM the difference in citrulline synthesizing capacity between the two groups was abolished. We propose, therefore, that by 24 hours after injection with bacterial lipopolysaccharide the increased urea synthesis in rats involves both increased ureagenic substrate load (amino acids) and CPS I activation by increased mitochondrial NAG levels.

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