Influence of Isolation Media on the Preservation of Mitochondrial Functions

Abstract

The influence of sucrose and mannitol-based isolation media on the degree of functional preservation of rat liver mitochondria was investigated. Apparently intact mitochondria conventionally prepared with a 0.3 M sucrose medium displayed significantly lower rates of state-3 respiration, pyruvate carboxylation, ATP hydrolysis and thiol group production than mitochondria prepared from the same livers with mannitol. Extracts from the latter showed a significantly higher activity of succinate dehydrogenase activity, whereas no difference in glutamate dehydrogenase activity was demonstrable. The low activities apparent with the sucrose medium could be brought to the level of the mannitol medium by the addition of potassium phosphate (4 mM). A similar effect was exerted by K2SO4, whereas KCl and the respective Na salts were significantly less effective. Sucrose-prepared mitochondria display decreased contents of metabolites such as ATP, glutamate, citrate and malate. Comparative studies with a variety of carbohydrates indicated that isolation media based on disaccharides were inferior to those based on monosaccharides in the preparation of functionally intact mitochondria from rat liver. Sucrose-prepared mitochondria have been employed in the past in a great number of studies and are still widely used at present.