Pyridine‐nucleotide oxidation, Ca²⁺ cycling and membrane damage during tert‐butyl hydroperoxide metabolism by rat‐liver mitochondria

As tert‐butyl hydroperoxide is metabolized by the glutathione peroxidase–glutathione reductase enzyme system present in liver mitochondria, rapid and extensive oxidation of NADH and slow NADH oxidation are observed. This NAD(P)H oxidation can be prevented, or reversed, more effectively by 2‐hydroxybutyrate than by isocitrate, indicating an important role of mitochondrial NAD(P)⁺ transhydrogenase activity in maintaining a high NADPH/NADP⁺ ratio for glutathione reductase. In Ca²⁺ ‐loaded mitochondria tert‐butyl hydroperoxide‐induced NAD(P)H oxidation is followed by Ca²⁺ release from the mitochondria. If either 2‐hydroxybutyrate or isocitrate is present, no Ca²⁺ release can be induced by the hydroperoxide. Following Ca²⁺ efflux the NAD(P)H oxidation process becomes irreversible and membrane damage occurs. These late effects do not take place if ruthenium red is added to prevent re‐uptake of released Ca²⁺ by the mitochondria. Thus, we conclude that the metabolism of tert‐butyl hydroperoxide leads to a release of mitochondrial Ca²⁺ via oxidation of pyridine nucleotides, and that subsequent membrane damage is not directly associated with this Ca²⁺ efflux but results from continued cycling of released Ca²⁺.