Determination of 14C-labelled ketone bodies by liquid-scintillation counting

Abstract

A method of assaying 14C in ketone bodies present in blood by using liquid-scintillation counting is described. D([long dash])-[beta]-Hydroxy[14C]butyrate is converted quantitatively into [14C]acetoacetate by means of a coupled oxidoreduction reaction involving NAD+, D([long dash])-[beta]-hydroxybutyrate dehy-drogenase and malic dehydrogenase in the presence of a high concentration of oxaloacetate. [14c]Acetoacetate is decarboxylated to acetone and carbon dioxide which are trapped separately in a double-well flask and counted subsequently. The method permits the determination of 14C activity in the individual ketone bodies and allows the activity in the carboxyl carbon atoms of acetoacetate or of D([long dash])-[beta]-hydroxybutyrate to be assayed separately from the activity in the remainder of the molecule. Recoveries of 14C-labelled ketone bodies added to blood approach 100% with good reproducibility in replicate analyses.