Pyridine nucleotide distributions and enzyme mass action ratios in hepatocytes from fed and starved rats

Hepatocytes isolated from fed or starved rats were rapidly lysed using the recently described technique of turbulent flow (M. E. Tischler, P. Hecht, and J. R. Williamson, 1977, Arch. Biochem. Biophys., 181, 278-292). Pyridine nucleotide and metabolite contents were measured in the particulate fraction of both whole and disrupted cells after centrifugation through silicone oil. Lactate/pyruvate, β-hydroxybutyrate/acetoacetate, isocitrate/α-ketoglutarate, and malate/pyruvate ratios were determined for calculation of the free NADH NAD⁺ and NADPH NADP⁺ ratios in the cytosol and mitochondria. Lactate/pyruvate ratios measured in the extracellular and cytosolic compartments were in good agreement. Ratios of β-hydroxybutyrate/acetoacetate measured in the extracellular, cytosolic, and mitochondrial compartments also agreed well. Addition of ammonia to fed or starved rat liver cells incubated with lactate, pyruvate, β-hydroxybutyrate, and acetoacetate caused an oxidation of both the NAD and NADP redox states in the mitochondria and cytosol, although the NADP system was oxidized to a greater extent. Calculation of the free NADH and NAD concentrations in the cytosol provided values of about 1 and 400 to 500 μm, respectively, under control conditions. The concentrations of free NADH and NAD in the mitochondria were considerably higher, being 300 to 400 μm and 4 to 6 mm, respectively. The free andm bound NAD systems in both the cytosol and mitochondria were more oxidized in the presence of ammonia. NAD and NADP redox potential differences across the mitochondrial membrane (ΔE_h) were not significantly affected by ammonia addition and were generally similar in cells from both fed and starved rats: -52 and -56 mV for the NAD system and -19 to -29 mV for the NADP system. For the NAD system the cytosolic potential was -260 mV in the absence of ammonia and -250 mV in its presence, the mitochondrial values being -315 and -303 mV, respectively. The average cytosolic NADP potential, on the other hand, was -400 mV in the absence and -384 mV in the presence of ammonia. The mitochondrial fractions yielded NADP potentials of -420 mV in the absence of ammonia with both fed and starved rats. Ammonia decreased the mitochondrial NADP potential to -404 mV in fed rats and to -415 mV in starved rats. The calculated free NADH NAD⁺ and NADPH NADP⁺ ratios as well as metabolite concentrations were used to evaluate the mass action ratios of both cytosolic and mitochondrial enzymes. Cytosolic alanine aminotransferase remained near equilibrium in the absence and presence of ammonia, while cytosolic and mitochondrial aspartate aminotransferase reactions deviated up to fivefold. The glutamate dehydrogenase reaction was in near equilibrium with the NAD system, but deviated by three to four orders of magnitude from equilibrium with the NADP system in the direction favoring glutamate synthesis rather than deaminatión. Cytosolic malate dehydrogenase deviated from equilibrium by about one order of magnitude, while mitochondrial malate dehydrogenase and citrate synthase deviated by two to six orders of magnitude. These data emphasize the importance of regulation of the citric acid cycle at the citrate synthase step.