Method for Determining Incorporation of Acetate into Citrate

Abstract

Summary

A simple radiometric method is described to determine the incorporation of acetate or pyruvate into citrate. Following in vitro incorporation of these substrates by the tissues, they are converted to ‘active acetate' which then condenses with oxaloacetate to form citrate. When 1-¹⁴C acetate or 2-¹⁴C pyruvate were added as tracers, the citrate was labeled at 1-¹⁴C carboxyl position which on oxidation was released as ¹⁴CO₂. Labeled acetate in the medium did not interfere with the quantitative recovery of small quantities of citrate formed. When monofluoroacetate (MFA) instead of acetate was added with labeled acetate as the substrate, monofluorocitrate was formed which accumulated in the tissues, perhaps due to block in further oxidation. There was an eight-fold increase in the labeled CO₂ evolution by liver tissue when MFA was used as the substrate.

Of the three temperatures at which the reaction was studied, the maximum amount of citrate was formed at 27°. The ability of the different tissues of rats to synthesize citrate was varied. Kidney, liver and heart synthesized far more citrate than the skeletal muscle, pancreas and lungs. In vivo incorporation of acetate into citrate was studied by injecting 1-¹⁴C acetate into rats who were poisoned with MFA before hand, and then sacrificing the animals 30 and 60 min after the labeled injection. Spleen, kidney and lungs contained much greater radioactivity than that present in the whole blood. Heart and liver also exhibited marked elevation when compared with corresponding tissues from saline-injected control rats treated similarly.