Effects of in vitro addition of ammonia on the metabolism of N-15-labelledamino acids in isolated sheep hepatocytes

Isolated hepatocytes prepared from sheep were used to determine the effects of added ammonia las NH,CI) on the flux of N-15 from N-15-labelled amino a cids to [(NN)-N-14-N-15]urea and [(NN)-N-15-N-15]urea. Hepatocyte suspensio ns were incubated with NH4CI (0, 0.31, 0.63 and 1.25 mM) in the presence of L-[N-15]alanine, L-[N-15]methionine, L-[N-15]leucine or L-[N-15]phenylalan ine (1.0 mM final concentration in the incubation media). Following 1.5 h i ncubation, addition of NH4Cl increased total urea and unlabelled ((NN)-N-14 -N-14) urea production in all incubations (P < 0.001). Adding NH4Cl did not affect N-15 isotopic enrichment of [(NN)-N-14-N-15]urea (P = 0.705) and [( NN)-N-15-N-15]urea (P 0.204), or production of [(NN)-N-14-N-15]urea (P = 0. 279) and [(NN)-N-15-N-15]urea (P = 0.708) in isolated hepatocytes incubated with [N-15]alanine. Addition of NH4Cl increased [(NN)-N-14-N-15]urea produ ction in incubations containing [N-15]methionine (quadratic effect, (P = 0. 019), but had no effect in incubations containing [N-15]leucine (P = 0.107) or [N-15]phenylalanine (P = 0.135). There was no detectable production of [(NN)-N-15-N-15]urea in incubations containing [N-15]methionine, [N-15]leuc ine and [N-15]phenylalanine. In all incubations, unlabelled ((NN)-N-14-N-14 ) urea was the predominant form of urea, indicating that isolated sheep hep atocytes can detoxify excess NH3 by channelling NH3-N into both mitochondri al carbamoyl phosphate and cytoplasmic aspartate synthesis.