In-vivo external sensor for mitochondrial injury: Circulating cytochrome c

Abstract

Low-abundance, low-molecular-weight serum proteins are useful in diagnosing the change in expression of some serum proteins which may indicate an altered physiology and serve as biomarkers of the respective disease. Recent studies suggest the molecular mechanism responsible for the translocation of cytochrome c from mitochondria to the cytosol during apoptosis. We hypothesize that mitochondrial injury causes the final release of cytochrome c from cytosol to the bloodstream, which acts as a mitochondrial injury external sensor. Serum cytochrome c was dissociated from large, abundant proteins by acetonitrile precipitation pretreatment and then measured using reverse-phase high performance liquid chromatography (HPLC). For quantification of serum cytochrome c levels, internal standard curves (0.2 to 20 mug/ml) were prepared using rat heart cytochrome c dissolved in serum obtained from healthy pentobarbital-anesthetized rats. Serum samples taken from severe mitochondrial injury animals (heart attack rats and diabetes mice) were measured and shown a dramatic increase in cytochrome c levels compared to healthy animals. Irreversible mitochondrial injury (death) occurred once serum cytochrome c level exceeded 2.0 mug/ml as the average cytochrome c level for healthy animals is reported less than 0.2 mug/ml. We conclude that serum cytochrome c may represent a novel in-vivo external sensor of mitochondrial injury.