State of oxidation-reduction and state of binding in the cytosolic NADH-system as disclosed by equilibration with extracellular lactate-pyruvate in hemoglobin-free perfused rat liver.

1 The extracellular ratio [lactate]/[pyruvate] is used as a means to poise the cytosolic NADH/NAD+ system. This is achieved by stepwise changes of [lactate]/[pyruvate] in the perfusate in an open system. Fluorescence and absorbance at NADH-specific wavelengths is recorded simultaneously with single or dual wavelength methods. Fluorescence excitation and absorbance difference spectra are obtained with the help of an instrument computer. The readout is calibrated by infusion of internal standards, permitting an estimation of the enhancement of quantum yield of NADH-specific fluorescence. 2 Mathematical treatment on the basis of the mass action law permits extrapolation to fully reduced and fully oxidized cytosolic bound NADH. In addition, the apparent equilibrium constant for bound NADH/NAD+ and lactate/pyruvate is obtained (0.022 to 0.071), corresponding to a midpoint potential of bound NADH of – 250 to – 265 mV. Accordingly, the ratio of dissociation constants, KNAD+/KNADH, relevant for the cytosolic binding sites, is 200–700. Cytosolic NADH-binding capacity is estimated to be around 80 nmol/g liver wet weight. Under normoxic conditions at [lactate]/[pyruvate] of 10 in the perfusate, the ratio of contents of cytosolic bound NADH/free NADH is 100. 3 The existence of near-equilibrium conditions in the system is suggested by measurement of net H+ transport coupled to lactate import, as well as by determination of an intracellular dependent indicator metabolite couple, α-glycerophosphate/dihydroxyacetone phosphate. Problems regarding the validity of tissue contents of lactate/pyruvate as indicator for the cytosolic NADH/NAD+ potential are discussed.