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

Abstract

Isolated hepatocytes prepared from sheep were used to determine the effects of added ammonia (as NH4Cl) on the flux of 15N from 15N-labelled amino acids to [14N15N]urea and [15N15N]urea. Hepatocyte suspensions were incubated with NH4Cl (0, 0.31, 0.63 and 1.25 mM) in the presence of L-[15N]alanine, L-[15N]methionine, L-[15N]leucine or L-[15N]phenylalanine (1.0 mM final concentration in the incubation media). Following 1.5 h incubation, addition of NH4Cl increased total urea and unlabelled (14N14N) urea production in all incubations (P < 0.001). Adding NH4Cl did not affect 15N isotopic enrichment of [14N15N]urea (P = 0.705) and [15N15N]urea (P = 0.204), or production of [14N15N]urea (P = 0.279) and [15N15N]urea (P = 0.708) in isolated hepatocytes incubated with [15N]alanine. Addition of NH4Cl increased [14N15N]urea production in incubations containing [15N]methionine (quadratic effect, P = 0.019), but had no effect in incubations containing [15N]leucine (P = 0.107) or [15N]phenylalanine (P = 0.135). There was no detectable production of [15N15N]urea in incubations containing [15N]methionine, [15N]leucine and [15N]phenylalanine. In all incubations, unlabelled (14N14N) urea was the predominant form of urea, indicating that isolated sheep hepatocytes can detoxify excess NH3 by channelling NH3-N into both mitochondrial carbamoyl phosphate and cytoplasmic aspartate synthesis. Key words: Sheep, liver cells, amino acids, ureagenesis, ammonia, 15N