THE EFFECT OF AMINO-ACIDS ON THE METABOLIC-FATE OF (NH4CL)-N-15 IN ISOLATED SHEEP HEPATOCYTES

Ruminants characteristically absorb a large proportion of dietary nitr ogen across the portal-drained viscera as ammonia nitrogen which is de toxified by conversion to urea in the liver. In theory, ammonia can su pply both nitrogen atoms of the urea molecule via mitochondrial (carba moyl 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 deter mined. We report a study designed to determine the distribution of [N- 15] ammonia between [N-15(1)]urea and [N-15(2)]urea in sheep hepatocyt es in response to ammonia concentrations (0.33, 0.67 and 1.00 mM) in t he presence or absence of amino acids. In the absence of amino acids, the enrichment of [N-15(2)]urea rose more rapidly during incubations t han [N-15(1)]urea and attained enrichments of 66-88% within 5 min of i ncubation. At the end of 2.5 h of incubation, [N-15(2)]urea represente d 60% and 90% of the total urea molecules at low and high ammonia conc entrations, respectively. The enrichments of glutamate and aspartate w ere similar to [N-15(1)]urea in the cells at the end of the incubation s, even in the presence of unlabelled amino acids, supporting the conc ept of mitochondrial ammonia being in equilibrium with cytosolic aspar tate formation. In the presence of amino acids basal urea synthesis in creased but ammonia uptake and (NH4Cl)-N-15 conversion to urea was les s than in the absence of amino acids. The rate of formation of [N-15(1 )]urea was greater in incubations containing amino acids but when ammo nia concentration in the media was raised only [N-15(2)]urea flux incr eased with no change in either [N-15(1)]urea or the unlabelled species . Measurement of media amino acid concentrations after 2.5 h of incuba tion in the presence of amino acids revealed that arginine, glutamine, glycine and alanine were removed while there was net formation of asp arate, threonine, serine, glutamate, and the branched chain amino acid s. However, less than 12% of the N-15 transfer appeared in free amino acids. The increases in basal and unlabelled urea synthesis in the pre sence of amino acids could be numerically accounted as the sum of argi nine and glutamine removal from incubations. It is concluded that in s heep hepatocytes (NH4Cl)-N-15 removal leads to quantitative formation of [N-15(2)]urea, even in the presence of a physiological mixture of a mino acids. The increase in the formation of the [N-15(1)]urea in the presence of amino acids can be explained by the preferential utilisati on of the amide nitrogen of glutamine for urea synthesis.