Evaluation of the kinetics of the intracellular reduction of 2,6-dichlorophenolindophenol in normal and transformed hepatocytes measured by amperometric methods

Amperometric methods were used to study the kinetics of intracellular reduction of 2,6-dichlorophenolindophenol (DCIP) in normal and transformed hepatocytes with glucose and succinate as substrates. The curves showing the formation of DCIPred as a function of time were biphasic, the first part obeying the equation of a pseudo-first-order reaction, the final part corresponding to Michaelis-Menten kinetics. A statistical method was used to estimate pseudo-first-order rate constants k as well as Km and Vmax values. At saturating glucose concentrations k, Km and Vmax values were higher in normal compared to transformed cells. Decreasing glucose concentrations revealed lowered saturation concentrations in tumour cells compared to normal cells. With succinate as substrate for hepatocytes, k values were higher than with glucose, while Km and Vmax were about the same. Hepatoma cells did not metabolize succinate. K values could be attributed to intracellular dehydrogenase activities including cytosolic and mitochondrial processes. Differences in pseudo-first-order rate constants between normal and tumour cells may therefore represent characteristic alterations associated with transformation.