Flavin and Pyridine Nucleotide Oxidation-Reduction Changes in Perfused Rat Liver

Abstract

Flavin and pyridine nucleotide fluorescence was continuously monitored from the surface of the hemoglobin-free, perfused rat liver with the use of a new double fluorometer.

In response to anoxia or after addition of inhibitors of the respiratory chain, the fluorescence intensity excited at 436 mµ decreased, while that excited at 366 mµ increased, representing reduction of flavin and pyridine nucleotides, respectively. Subsequent reoxidation of pyridine nucleotides in the cytosol by the addition of pyruvate was not accompanied by a change in flavin fluorescence. Maximal flavin reduction could be achieved by complete inhibition of the respiratory chain in the presence of oxygen. Titration with acetoacetate showed that mitochondrial pyridine nucleotides were in equilibrium with the fluorescent flavoproteins. Microsomal oxidation-reduction reactions appeared to be reflected by small changes in pyridine nucleotide fluorescence, but not by changes of flavin fluorescence. It is concluded, therefore, that the flavin signal is predominantly due to mitochondrial flavoproteins with approximately equal contributions from flavin pools located on both the substrate and the oxygen side of the rotenone block. The continuous monitoring of flavin and pyridine nucleotide fluorescence as an experimental approach for studies of compartmentation of oxidation-reduction systems is discussed.