This paper presents a theoretical justification for usine 3-hydroxy[3-"Clbutyrate to label the intramitochondrial pool of acetyl CoA and hence for calculating the percentage of citrate which is oxidized by the tricarboxylic acid cycle. Knowledge of this percentage can be used to calculate the rate of oxidation of glucose through the tricarboxylic acid cycle, pyruvate dehydrogenase, and the pentose phosphate pathway. Experiments on adipocytes are described which confirm that no unexpected results are obtained. It is concluded that the method is valid at least for adipose tissue.
Ketone bodies are oxidized by intact mitochondria
(1). Three enzymes are required to convert 3-hydroxybutyrate into acetyl-CoA (2). These are 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30), 3-oxo-acid CoA-transferase (EC 2.8.3.5), and acetoacetyl-CoA thiolase (EC 2.3.1.9). The transferase and most of the thiolase are located in particulate fractions of tissue homogenates (3). This fact and the impermeability of the mitochondrial membrane to derivates of CoA suggest that the intramitochondrial pool of acetyl-CoA can be specifically labeled in the carboxyl carbon by incubating intact cells with 3-hydroxy[3-14C]butyrate. The labeling of this pool in this way should make it possible to measure the fraction of the mitochondrial acetyl CoA which is oxidized through the tricarboxylic acid cycle.
In making the calculation it is assumed that there are two possible fates for mitochondrial citrate formed from acetyl-CoA by citrate synthase (EC 4.1.3.7). It may be oxidized to oxaloacetate by the enzymes of the tricarboxylic acid cycle or it may be carried out of the mitochondria and be broken down by citrate lyase (EC 4.1.3.6) to oxaloacetate and acetyl-CoA. The stereospecificity of the synthase and lyase enzymes ensures that the cytoplasmic acetyl-CoA is formed from the same carbon atoms as were added to the mitochondrial oxaloacetate (4).
Any label in the cytoplasmic acetyl-CoA will be incorporated into newly synthesized fatty acids. When citrate is formed from carboxyllabeled acetyl-CoA and then oxidized by the tricarboxylic acid cycle, the label is equally distributed between the carboxyl groups of the oxaloacetate. Its subsequent fate depends on whether or not gluconeogenesis occurs in the tissue concerned.