The reduction of acetoacetate to β-hydroxybutyrate in animal tissues

Abstract

Acetoacetate, when added aerobically to sheep-heart-muscle homogenates, is partly oxidized to CO2 and water, and partly reduced to D([long dash])-[beta]-hydroxybutyrate. The ratio of oxygen equivalents used to acetoacetate oxidized is near 4. This suggests that acetoacetate is oxidized in preference to endogenous substrates. In sheep heart, but not in the hearts of rats or pigeons and various other tissues tested, more [beta]-hydroxybutyrate. is formed aerobically than anaerobically when acetoacetate is the sole added substrate. [alpha]-Oxoglutarate, fumarate and pyruvate accelerate both the anaerobic or aerobic conversion of acetoacetate into [beta]-hydroxybutyrate. This can be explained by coupled oxidation-reduction mediated by diphosphopyridine nucleotide (DPN). Succinate increases the rate of the aerobic reduction of acetoacetate but not of the anaerobic reduction. This is discussed and it is suggested that succinate, on account of its ready dehydrogenation, blocks the transfer of electrons from reduced DPN to the cytochrome system and thereby increases the supply of reduced DPN available for the reduction of acetoacetate. The action of dinitrophenol on acetoacetate metabolism varied with the presence of other substrates. 0.01 mM-Dinitrophenol caused large increases in the oxygen uptake and acetoacetate oxidation, especially when [alpha]-oxoglutarate and pyruvate were also added. At 0.2 mM, it inhibited the oxygen uptake and acetoacetate oxidation when added alone or with fumarate, but not in the presence of [alpha]-oxoglutarate. The reasons for these differences are discussed. Anaerobically, relatively slight inhibitions of the reduction of acetoacetate were observed. The oxidation of L(+)-[beta]-hydroxy-butyrate is inhibited by dinitrophenol, whereas that of the D([long dash])-form is not. This is related to the fact that only the L(+)-form requires conversion into the coenzyme A derivative. The differences in the behavior of the L- and D-forms towards dinitrophenol were used to examine the configuration of the [beta]-hydroxybutyrate formed in sheep-heart muscle aerobically and anaerobically. In both cases the D-form only was found.