Ruminants characteristically absorb a large proportion of dietary nitrogen across the portal-drained viscera as ammonia nitrogen which is detoxified by conversion to urea in the liver. In theory, ammonia can supply both nitrogen atoms of the urea molecule via mitochondrial (carbamoyl phosphate) and cytoplasmic (aspartate) precursor pathways of the ornithine cycle but the effect of amino acids on the flux of nitrogen from ammonia to each of the two urea nitrogen atoms has not been determined. We report a study designed to determine the distribution of [15N] ammonia between [15N1]urea and [15N2]urea in sheep hepatocytes in response to ammonia concentrations (0.33, 0.67 and 1.00 mM) in the presence or absence of amino acids. In the absence of amino acids, the enrichment of [15N2]urea rose more rapidly during incubations than [15N1]urea and attained enrichments of 66-88% within 5 min of incubation. At the end of 2.5 h of incubation, [15N2]urea represented 60% and 90% of the total urea molecules at low and high ammonia concentrations, respectively. The enrichments of glutamate and aspartate were similar to [15N1]urea in the cells at the end of the incubations, even in the presence of unlabelled amino acids, supporting the concept of mitochondrial ammonia being in equilibrium with cytosolic aspartate formation. In the presence of amino acids basal urea synthesis increased but ammonia uptake and 15NH4Cl conversion to urea was less than in the absence of amino acids. The rate of formation of [15N1]urea was greater in incubations containing amino acids but when ammonia concentration in the media was raised only [15N2]urea flux increased with no change in either [15N1]urea or the unlabelled species. Measurement of media amino acid concentrations after 2.5 h of incubation in the presence of amino acids revealed that arginine, glutamine, glycine and alanine were removed while there was net formation of aspartate, threonine, serine, glutamate, and the branched chain amino acids. However, less than 12% of the 15N transfer appeared in free amino acids. The increases in basal and unlabelled urea synthesis in the presence of amino acids could be numerically accounted as the sum of arginine and glutamine removal from incubations. It is concluded that in sheep hepatocytes 15NH4Cl removal leads to quantitative formation of [15N2]urea, even in the presence of a physiological mixture of amino acids. The increase in the formation of the [15N1]urea in the presence of amino acids can be explained by the preferential utilisation of the amide nitrogen of glutamine for urea synthesis.
|Number of pages||6|
|Journal||European Journal of Biochemistry|
|Publication status||Published - 15 Mar 1995|
- Amino Acids
- Ammonium Chloride
- Cells, Cultured
- Nitrogen Isotopes