The experiments reported here were designed to test the hypothesis that the two-electron quinone reductase DT-diaphorase

The saturation kinetics of NADH and succinate oxidation for Cocnzyme Q (CoQ) has been rc-invest&ted in pcntanccatmckd lyophilizcd beef heart mitochondria rcconstitutcd with cxo~cnous CoQ,,. The apparent '&,,:,' for CoQ,, was one order of mawitude lower in succinatc cytochrome c reductnse than in NADH cytocbromc L' rcductase. The K,,, value in NADH oxidation approaches the natural CoQ content of beef heart mitochondria, whereas that in succinatc oxidation is close to the content of respiratory chain enzymes.

Apoptosis and aging share common mechanisms in oxidative stress and mitochondrial involvement. Treatment of cultured neuroblastoma cells with a radical initiator induced apoptosis; raise in hydrogen peroxide and release of cytochrome c from mitochondria preceded collapse of mitochondrial potential and cell death. In rat hepatocytes treated with adriamycin incubation with exogenous Coenzyme Q10 counteracted the drug-induced increase of hydrogen peroxide and the fall of the mitochondrial potential, thus demonstrating the quinone antioxidant effect. Complex I activity and its rotenone sensitivity decreased in brain cortex non-synaptic mitochondria from old rats; a 5 kb mitochondrial DNA deletion was found only in the old rats. A similar behavior was found in human platelets from old individuals. The postulated energy decline was confirmed by the inhibitor sensitivities of platelet aggregation and lactate production. The lack of the 5 kb deletion in platelets throws doubts on mitochondrial DNA lesions as the only causes of mitochondrial dysfunction in aging.

Rhodamine 123 (Rh123) is widely used as a flow cytometric probe for mitochondrial membrane potential (MMP) in metabolic, pharmacologic, and toxicological studies. However, the use of relatively high concentrations of Rh123 (up to 10 pg/ml) and prolonged incubation times (up to 1 h), including washing steps, may be inconvenient for certain applications in which labile cells are used or which demand rapid or repeated analysis. In this paper we describe a rapid kinetic assay of MMP in isolated rat hepatocytes, based upon the quantitation of the initial rate of Rh123 uptake by living cells, selected by their scattering properties. The results indicate that at an appropriate dye-to-cell ratio (in our experiments, 50 ng Rh123/ml for 250,000300,000 cells/ml), the initial rate of Rh123 uptake is a highly reproducible and sensitive parameter for estimation of MMP, as demonstrated by the effects of substrates and inhibitors of the glycolytic pathway and mitochondrial respiration. Because of its simplicity, rapidity (about 5 min) and metabolic implications, this assay would be also suitable for the routine evaluation of metabolic state of cell suspensions, as a complementary test to the standard dualstaining tests of viability. Other possible applications in screening pharmacologic and toxicological analysis are discussed. o 1994 Wiley-Liss, Ine.Key terms: Mitochondria, mitochondrial membrane potential, rhodamine 123, flow cytometry, hepatocyte, glucose metabolism Mitochondrial membrane potential (MMP) is a key parameter to assess cellular energy metabolism under physiological and pathological conditions (5). However, the conventional approach of studying isolated mitochondria in vitro does not allow integration of the mitochondrial function into the whole cell.Flow cytometry provides convenient methods for the analysis of MMP in situ. Recently, most flow cytometric studies on mitochondrial function have been performed with rhodamine 123 (Rh123), a lipophilic cationic fluorochrome which is incorporated by mitochondria according to transmembrane potential (6,9,11,14,20,22). Rh123 has been applied extensively in flow cytometric studies of mitochondrial metabolism (2,30,33) and metabolic heterogeneity (341, cell activation (81, differentiation (71, oncogenic transformation (32), stem cell characterization and purification (37,381, aging (16), and apoptotic death (lo), as well as for assessment of cytotoxicity (20,22,23,24) and drug resistance (15,17). Some of the multiple applications of Rh123 have been reviewed by Ronot et al. (26,271 and Chen (5,6).The most usual techniques to study MMP by flow cytometry in whole cells follow the one described by Johnson et al. (13,14), with slight modifications. Following a 30-60 min equilibration of cells with Rh123