Abstract
Cytoplasmic free NAD(+)/NADH ratios have been calculated from lactate to pyruvate ratios, and mitochondrial NAD(+)/NADH ratios, have been calculated from beta-hydroxybutyrate to acetoacetate ratios in isolated rabbit alveolar macrophages. In freshly harvested cells, assuming a pH of 7 for the two compartments, cytoplasmic NAD(+)/NADH averaged 709 +/-293 (SD), and mitochondrial NAD(+)/NADH averaged 33.2 +/-30.2, values which are significantly different. 30 min of air incubation in a relatively poorly buffered medium showed a significant reduction in calculated mitochondrial NAD(+)/NADH to 10.1 +/-4.8. 30 min of exposure of cells to a hypoxic environment (equivalent to a nonventilated, perfused alveolus) caused significant reductions of NAD(+)/NADH in both compartments. Re-exposure of hypoxic cells to air produced a change toward normal in cytoplasmic NAD(+)/NADH but did not reverse mitochondrial abnormality. Uncertainties concerning the value of cytoplasmic and mitochondrial pH under control conditions and during experimental pertubations, limit absolute interpretation of NAD(+)/NADH ratios calculated from redox pairs, but the data suggest the following: (a) separate cytoplasmic and mitochondrial compartments for NAD(+) and NADH exist in the alveolar macrophage; (b) brief periods of exposure to moderate hypoxia of the degree seen in clinical lung disease produce decreases in both cytoplasmic and mitochondrial NAD(+)/NADH; (c) the mitochondrial changes are less easily reversed than the cytoplasmic changes; (d) measurements of NAD(+)/NADH provide an early sensitive indication of biochemical abnormality; and (e) careful control of extracellular pH is required in studies involving experimental modifications of alveolar macrophage function.