Direct measurement of the state of the lactate dehydrogenase system in hemoglobin-free perfused rat liver

Abstract

Appropriate correction of tissue contents of lactate and pyruvate shows that the cellular contents of these metabolites are of limited cell physiological use, especially in abnormal metabolic states. However, the ratio of contents of α-glycerophosphate/dihydroxyacetone phosphate and of extracellular concentrations of lactate/pyruvate are useful in this context. The extracellular ratio [lactate]/[pyruvate] provides a means to poise the redox potential of cytosolic NADH. This is achieved by stepwise changes of [lactate]/[pyruvate] in perfusate of rat liver perfused in an open system. Dependent intracellular changes are followed (a) by photometric and (b) by analytical methods. Mathematical treatment of observed increments on the basis of the mass action law allows extrapolation to fully reduced and fully oxidized cytosolic NADH. In addition, the apparent equilibrium constant for bound NADH/NAD and lactate/pyruvate is obtained. The ratio of dissociation constants, D NAD/ D NADH, relevant for the cytosolic sites, is calculated at an assumed pH of 7.0. The midpoint potential of bound NADH is around −260 mV.