A MASS ISOTOPOMER STUDY OF UREA AND GLUTAMINE SYNTHESIS FROM N-15-LABELED AMMONIA IN THE PERFUSED-RAT-LIVER

This study examines the incorporation of N-15 from (NH4Cl)-N-15 into u rea and glutamine, predicts the pattern of isotopomers produced as a f unction of the N-15 enrich ment of the relevant precursor pools, and p resents a means of determining the isotopic enrichment of these pools, Rat livers were perfused, in the nonrecirculating mode, with 0.3 mM ( NH4Cl)-N-15, and the isotopomers of urea and of glutamine produced wer e determined by gas chromatography-mass spectrometry methodology, Thre e different nitrogen mass isotopomers of urea were found, containing n o, one, or two atoms of N-15. Four glutamine isotopomers were found, c ontaining no N-15, one atom of N-15 in either the amino or amide posit ion, or two N-15 atoms, A mathematical relationship was deduced that p redicts that the relative proportions of the urea isotopomers depends not only on the relative enrichment of N-15 in the two precursor pools of urea nitrogen (mitochondrial ammonia and cytoplasmic aspartate) bu t on their absolute enrichment, This relationship was validated in exp eriments in which the isotopic enrichment of the substrate, (NH4Cl)-N- 15, was varied, The proportions of the urea isotopomers produced can b e predicted if one knows the N-15 enrichment in the two precursor pool s, We found that when the N-15 enrichment of citrulline and aspartate in the perfusate were used as proxies for that in the mitochondrial am monia and cytoplasmic aspartate pools we could accurately predict the relative proportion of the three isotopomers. The production of the fo ur nitrogen isotopomers of glutamine could be used to determine the N- 15 enrichment in the two precursor pools of glutamine nitrogen, the cy toplasmic ammonia and glutamate pools of the perivenous hepatocytes.