Hepatic metabolism during acute ethanol administration: A phosphorus-31 nuclear magnetic resonance study on the perfused rat liver under normoxic or hypoxic conditions

Abstract

The effect of ethanol metabolism on the energetic parameters and intracellular pH of the isolated perfused rat liver from fed rats was studied by phosphorus-31 nuclear magnetic resonance spectroscopy. This technique allowed us to analyze nondestructively and in real time the role of low oxygen tension on the possible injurious effect of ethanol on the liver cells. A quantitative analysis of nuclear magnetic resonance data recorded on a perfused rat liver within a 30 mm diameter probe has been performed at 80.9 MHz. Under normoxic and normothermic conditions, the levels of phosphorylated metabolites detected by nuclear magnetic resonance were 2.8, 0.3 and 2 μmoles per gm liver wet weight for ATP, ADP and inorganic orthophosphate, respectively. The cytosolic pH was 7.25 ± 0.05. During a period of 4 min of hypoxia induced by reducing the perfusion flow rate to 25% of its initial value (i.e., from 12 ml to 3 ml per min per 100 gm body weight), the level of ATP dropped to 2.2 μmoles per gm liver wet weight. Concomitantly, ADP and inorganic orthophosphate increased to 0.6 and 3.3 μmolès per gm liver wet weight. Cytosolic pH fell to 7.02 ± 0.05. Perfusion of the liver with a Krebs medium containing 70 mM (0.4%) ethanol induced a sharp decrease in intracellular inorganic orthophosphate to reach 1.3 μmole per gm liver wet weight and after a lag time of 4 to 6 min, a decrease in ATP level (2.15 μmoles per gm liver wet weight). A large increase in phosphomonoesters (mainly sn-glycerol 3-phosphate) up to 6 μmoles per gm liver wet weight was also observed. No significant cytosolic pH variation was noted after 20 min of ethanol perfusion (pH 7.20 ± 0.05). In the presence of 70 mM ethanol, a 4-min period of hypoxia caused pronounced changes; in particular, the level of ATP dropped to 1.15 μmole per gm liver wet weight, and a large intracellular acidosis occurred (pH 6.80 ± 0.05). Ethanol oxidation during hypoxia is also correlated to a dramatic increase in the cytosolic content of sn-glycerol 3-phosphate, as compared to the level found under normoxic ethanol perfusion. Data presented here indicate that ethanol hepatotoxicity might be enhanced under hypoxia due to accumulation of protons in the cytosol. This severe metabolic acidosis is likely to contribute to metabolic disturbances occurring during ethanol exidation. Ethanol metabolism in fed rats gives rise to a synthesis of sn-glycerol 3-phosphate correlated with a decrease in cytosolic inorganic orthophosphate which in turn becomes limiting for ATP production. Finally, the observed increase in sn-glycerol 3-phosphate under hypoxia indicates that the sn-glycerol 3-phosphate/dihydroxyacetone phosphate shuttle could play a very important role as a compensatory mechanism in hypoxic regions of the liver in order to compensate for the decreased activity of the malate/aspartate shuttle.