Transfer of the 1-pro-R and the 1-pro-S hydrogen atoms of ethanol into Krebs-cycle and related acids in vivo [Female Sprague-Dawley rats]

A method for gas chromatographic/mass spectrometric analysis of low‐molecular‐weight organic acids in liver tissue is described. The method involves formation of methoximes of oxo acids, preventing exchange of hydrogen by enolization. The acids are separated as methyl esters by gas chromatography on an SP‐1000 capillary column. The origin of fragment ions in the mass spectrometric analysis was studied by analysis of deuterated analogs. Lactate, pyruvate, 3‐hydroxybutyrate, fumarate, succinate, malate, 2‐oxoglutarate and citrate were analyzed in freeze‐clamped livers of rats given [1, 1‐2H]ethanol, (1R)‐[1‐2H]ethanol and (1S)‐[1‐2H]ethanol. No deuterium was incorporated in succinate and the oxo acids analyzed. The incorporation of deuterium from [1, 1‐2H2]ethanol in one position of malate and fumarate was about the same as in lactate (18–23% of the atoms), but the deuterium in lactate originated to a larger extent from the 1‐pro‐R position than in malate and fumarate. This indicates lack of equilibration of the NADH pools utilized by cytosolic malate dehydrogenase and lactate dehydrogenase, and that the former pool is more closely related to cytosolic acetaldehyde oxidation. 3‐Hydroxybutyrate incorporated the 1‐pro‐S hydrogen of ethanol to a larger extent than the 1‐pro‐R hydrogen. This is in agreement with the presence of 3‐hydroxybutyrate dehydrogenase and aldehyde dehydrogenase in the mitochondrial compartment. Hydrogen in citrate appears to be derived from C‐1 of ethanol both via oxaloacetate and via intramitochondrial isocitrate dehydrogenase.