Transfer of Deuterium from [1‐2H2]Ethanol to Krebs Cycle and Related Acids of Rat Liver in vivo

Abstract

The utilization in intermediary metabolism of hydrogens derived from C‐1 in ethanol was studied with the aid of [1‐2H2]ethanol. A gas chromatographic‐mass spectrometric method was developed and used to measure the deuterium content in individual positions of lactate, malate, 3‐hydroxybutyrate, citrate, succinate and fumarate isolated from freeze‐clamped livers of rats metabolizing [1‐2H2]ethanol (95 atoms%2H excess) at a maximal rate. A steady state of 2H‐labelling was reached within a few minutes. 3‐Hydroxybutyrate contained only 1–2 atoms% of deuterium which was interpreted to indicate that intramitochondrial NADH was essentially unlabelled due to exchange with protons in the NADH dehydrogenase reaction. A low labelling of lactate, 17 atoms%, indicated that the exchange of hydrogens in cytosolic NADH with unlabelled substrates and water was 4 times higher than the rate of ethanol oxidation. Since intramitochondrial oxidation of NADH is not rate limiting, the presence of labelled NADH in the cytosol may indicate that transfer of hydrogen through the mitochondrial membrane is a rate‐limiting step. As expected from the reversible reactions catalyzed by fumarate hydratase and malate dehydrogenase the deuterium excess at C‐2 and C‐3 of malate and fumarate were about the same, 20 atoms% Citrate had a deuterium excess of about 9 atoms%, assuming that only one hydrogen was labelled. Succinate contained very little deuterium. The labelling of citrate may thus be due either to incorporation of oxaloacetate labelled at C‐3 or to labelling of the same hydrogen in a reaction catalyzed by isocitrate dehydrogenase. The results indicate that both malate and isocitrate are potential sources of labelled NADPH during oxidation of [1‐2H2]ethanol, and that these two routes are not completely equilibrated under the experimental conditions. Copyright © 1974, Wiley Blackwell. All rights reserved