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 [l-2H,]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 [l-2H,]ethanol (95 atoms% 'H 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 [l-2H,]ethanol, and that these two routes are not completely equilibrated under the experimental conditions. Metabolism of ethanol results in an increased ratio between reduced and oxidized members of several redox couples in the liver [l]. This effect is considered to be due to a changed redox state of NAD in the cytosol, resulting from the increased production of NADH in the alcohol dehydrogenase reaction together with a slow transfer of reducing equivalents over the mitochondrial membrane [2,3] or a slow oxidation of intramitochondrial NADH [3,4]. Similar but smaller effects have been noted in redox couples which are in near-equilibrium with NADP [ 5 ] . Previous studies have shown that deuterium from [1-'H2]-ethanol is used directly both in NADH-dependent

Deuterium‐Labelling of sn‐Glycerol 3‐Phosphate during Metabolism of [1‐2H2]Ethanol in Rats

“…Furthermore, intramitochondria1 pyridine nucleotides are likely to have a very low labelling since 3-hydroxybutyrate is essentially unlabelled [28]. Thus, the most likely explanation is that there is more than one pool of sn-glycerol 3-phosphate in the liver.…”

“…The hydrogens at C-3 correspond to those at C-3 of malate and oxaloacetate, and deuterium may be introduced in the combined malate dehydrogenase and fumarate hydratase reactions [6,30]. Since the deuterium excess at C-3 of cytosolic malate is 20 atoms% or more [28], and since glucose was essentially unlabelled, about 15 -25 % of hepatic sn-glycerol3-phosphate may be formed via the gluconeogenetic pathway under the present experimental conditions. This value could be slightly higher due to loss of deuterium by enolization of oxaloacetate [31].…”

Hydrogen transfer between ethanol molecules during oxidoreduction in vivo

“…With knowledge of the labelling of the free NADH pool used in binding to alcohol dehydrogenase it would be possible to calculate the rate of association of NADH to the enzyme in relation to the total rate of oxidation of ethanol. However, different values (18-37% of that in the [1,1-2H2]ethanol) have been obtained for the labelling of the NADH used in different cytosolic reductions (Cronholm et al, 1974;Cronholm & Fors, 1976;Curstedt, 1982). The highest value (37%) was observed for the hydrogen at C-2 of the glycerol moiety in newly formed phosphatidylcholines (Curstedt, 1982).…”

Deuterium Transfer from [1,1-2H]Ethanol during Metabolism of Bile Acids and Cyclohexanone in the Isolated Perfused Rat Liver

“…Although both NADH and NADPH may be cofactors of the 3a-hydroxysteroid dehydrogenase [13], the much more reduced state of the NADP couple in vivo [14] indicates that NADPH is the cofactor used. The deuterium content of NADPH is a function of the deuterium content of NADH and the extent of dilution with protium during transfer of deuterium via substrates to NADPH from NADH [15]. The deuterium content of free NADH is determined by the deuterium content of NADH bound to alcohol dehydrogenase and the rate of exchange with unlabelled substrates.…”