Characteristics of ethanol and acetaldehyde oxidation on flavin and pyridine nucleotide fluorescence changes in perfused rat liver.

Data presented in this paper describe how the percentage decomposition of the catalase-H202 intermediate by methanol and ethanol as determined from absorbance changes (660 to 640 nm) through a lobe of the perfused rat liver can be used to calculate the intracellular ethanol concentration. Values obtained were similar to those measured in the eftluent fluid from the liver. Using this technique, a value of 0.09 mM for the apparent K,,, of alcohol dehydrogenase in situ for ethanol and a Vrnax value of 1.25 pmol per min per g, wet weight of liver were calculated. The hepatic ethanol concentration giving half-maximal reduction of the pyridine nucleotides as determined from pyridine nucleotide fluorescence changes (366-nm excitation, 460-nm emission) was 0.07 mM. When arterial fluid ethanol concentrations were used, the apparent K, for ethanol was about 0.4 mM. The apparent K, values for ethanol measured in sifu are 5to ZO-fold lower than values measured with isolated rat liver alcohol dehydrogenase, indicating that ethanol metabolism by the liver is not directly limited by alcohol dehydrogenase activity but rather by the rate of reoxidation of cytosolic NADH. At the low rates of hydrogen peroxide production observed in substrate-free perfused livers, rates of ethanol oxidation via alcohol dehydrogenase calculated from the different effectiveness of ethanol and methanol in decomposing the catalase-H202 intermediate were similar to rates of ethanol uptake measured directly from arteriovenous concentration differences. Changes of flavin and pyridine nucleotide fluorescence as measured by surface fluorometry were compared with changes in the ratios of lactate to pyruvate and P-hydroxy butyrate to acetoacetate in the etlluent fluid perfusing the liver. Titrations with octanoate to reduce the pyridine nucleotides in the mitochondrial compartment showed that the oxidation-reduction state of the fluorescent flavoproteins was in rapid equilibrium with the ratio of /3-hydroxybutyrate