The observation by others of decreased bile flow and death following sulfobromophthalein (BSP) infusion in rats and other laboratory animals suggested that BSP was a possible metabolic toxin and led to studies of its effect on mitochondria respiration. Measurements of oxygen consumption showed that state 3 oxidation of pyruvate, glutamate, succinate, β-hydroxybutyrate, palmitoyl-L-carnitine, and hexanoate by rat liver mitochondria was inhibited at 3 µg of BSP per milligram of mitochondrial protein, inhibition increasing with increased BSP concentration up to 24 µg/mg. Rat heart mitochondrial oxidation of pyruvate was similarly inhibited by BSP. BSP also inhibited incorporation of 32P into ATP during state 3 respiration. Analysis of polarograph tracings indicated that BSP inhibited state 3 respiration without any consistent effect on state 4 or the ADP to oxygen ratio. Synthetic BSP-GSH conjugate did not inhibit mitochondrial oxygen consumption or prevent inhibition by unconjugated BSP. Adding defatted bovine albumin to the incubation medium decreased BSP inhibition of mitochondrial oxygen consumption. In addition, the introduction of albumin to the medium following inhibition by BSP caused a return to normal rates of oxygen consumption. The mechanism of the reversible inhibition of state 3 oxidation was investigated by studying the effect of BSP on some of the isolated steps of oxidative phosphorylation. BSP did not inhibit the succinate, glutamate, or β-hydroxybutyrate dehydrogenases, or the succinate-cytochrome c reductase or NADH oxidase reactions. Succinate oxidase was inhibited in whole mitochondria but not in sonicated submitochondrial particles. This suggested that BSP may act at the mitochondrial inner membrane, possibly by interfering with mitochondrial uptake of the substrates necessary for coupled oxidative phosphorylation. The effect of BSP was therefore measured at various concentrations of the substrates of coupled oxidative phosphorylation. There was no change in inhibition over a 50-fold range of ADP concentration. However, BSP inhibition varied inversely with concentration of Pi in the medium. Similarly, inhibition by BSP varied inversely with the concentration of succinate but was not affected by changing the concentration of the other oxidizable substrates. Since the mitochondrial uptake of succinate has been shown to require Pi, the data are consistent with an effect of BSP on Pi transport across the mitochondrial inner membrane. These studies suggest that BSP is a potent inhibitor of oxidative phosphorylation in vitro. There is no evidence at present, however, that BSP exerts a similar effect in vivo.
- Copyright ©, 1974, by Academic Press, Inc.