On the mechanism of acetoacetate synthesis by guinea pig liver fractions.

1. Commercially available coenzyme A preparations were found to contain sufficient amounts of glutathione so that in the reaction with diketene significant quanties of acetoacetyl glutathione were formed. Unless allowances are made for this, significant errors in the interpretation of results can be made because liver mitochondria contain a very active acetoacetyl glutathione hydrolase. 2. Purified acetoacetyl coenzyme A was not deacylated by solubilized mitochondrial preparations under conditions of this assay. Since no acetoacetate was synthesized when /?-hydroxy-fl-methylglutaryl coenzyme A synthesis was inhibited, it was concluded that in the guinea pig liver acetoacetate formation proceeds entirely through fi-hydroxy-/3methylglutaryl coenzyme A. 3. The results indicated that during ketosis, significant quantities of acetoacetate may be synthesized by an extramitochondrial pathway. On the basis of experimental evidence, it was considered unlikely that the enzymes were eluted from mitochondria during the isolation procedure. 4. Acetoacetate synthesis was strongly inhibited by free coenzyme A. This inhibition could not be overcome by increasing the concentration of acetyl coenzyme A several-fold. On this basis, the suggestion was made that the degree of ketosis in the intact animal may relate closely to the levels of free coenzyme A in the liver and that the concentration of acetyl coenzyme A may be of lesser importance.