1H-2H exchange in the perfused rat liver metabolizing [3-13C]alanine and 2H2O as detected by multinuclear NMR spectroscopy

Abstract

The exchange of individual protons of hepatic metabolites against the solvent deuterons has been investigated in perfused rat liver. Livers from starved rats were perfused for 20 min with a 10 mM solution of unlabeled or 3-13C-labeled L-alanine in Krebs Ringer bicarbonate buffer, with or without 50% deuterium oxide (2H2O). High resolution 13C NMR analysis of deuterium-induced isotopic shifts and of 2H-13C couplings revealed a differential 1H-2H exchange depending on the chemical nature of the metabolite and on the site of 13C labeling. [3-13C]Aspartate isotopomers showed similar 2H/1H ratios in the C3 and in the C2 carbons while [2-13C]aspartate isotopomers had much smaller 2H/1H ratios in the C2 than in the C3 carbons. Similarly, [2-13C]glutamate isotopomers had 2H/1H ratios significantly smaller in the C2 than in the C3 carbon. These results suggest that the hydration-dehydration reactions of the citric acid cycle, which result in exchange at the C3 carbons of aspartate and glutamate, approach equilibrium with the perfusate faster than the aminotransferases of aspartate and alanine, which induce exchange at the C2 carbons of these amino acids. Taken together, the results obtained are consistent with a heterogeneous solvent exchange environment in the perfused liver.