The paper presents a protocol for the evaluation of energy metabolism in hepatocytes with the aid of high-resolution oxygraphy in permeabilized cells. Optimal conditions are suggested for the isolation of hepatocytes from the hepatic tissue of the rat and the proper technique for control of their structural integrity. Our work describes in detail the experimental schemes for oxygraphic evaluation of the individual enzyme complexes of the respiratory chain (Complex I, II, IV) and methods for the assessment of the mitochondrial function of phosphorylation using the respiratory control index and oligomycin inhibitory effect on ADP-activated respiration. On the example of oxidative damage of hepatocytes by tert-butylhydroperoxide, we demonstrate the utility of oxygraphy in the diagnostics of pathologic conditions caused by disturbances in energy metabolism of the cell.

Univerzita Karlova Praha Lékarská fakulta v Hradci Králové Ustav fyziologie