168 III. OXIDATION AND METABOLISM 



these findings, and suggested that pyruvate and acetate combine, resulting 

 in the formation of a-ketogkitarate. Wieland and RosenthaP'^ confirmed 

 these findings. Buchanan and associates^^^ reported that, when acetate 

 or acetoacetate was incubated with kidney homogenates, C^^ was found 

 in the a-ketoghitarate, succinate, and fumarate. The quantity of C^^ 

 obtained accounted for a large portion of the isotopic C which disappeared 

 from the acetate or acetoacetate. Since C^* is in the a-carbon of keto- 

 glutarate, it is believed that citrate is excluded as an important inter- 

 mediate. Evidence is adduced to indicate that no "extra" carbohydrate 

 is formed during the metabolism of fatty acids. 



Both the carboxyl and the carbonyl carbons of acetoacetate have been 

 shown by Weinhouse, Medes, and Floyd^^^-^*^ to enter the tricarboxylic 

 acid cycle. Acetoacetate, marked with C^^ in either of the above positions, 

 was shown to give rise to citrate containing C^'^ when it was incubated with 

 oxaloacetate along with homogenates of kidney, muscle, or brain. It is 

 therefore certain that acetoacetate breaks down to two C2 fragments 

 before reacting with oxaloacetate. 



Some confusion was due to the fact that the disappearance of acetoace- 

 tate reported in the earlier studies of Krebs and Eggleston^*^ with suspen- 

 sions of sheep heart homogenate under anaerobic and aerobic conditions, 

 on the addition of fumarate, L-malate, oxaloacetate, or a-ketoglutarate, 

 was shown in their later results ^*® to have been due merely to reduction of 

 the acetoacetate to /3-hydroxybutyrate rather than to its incorporation 

 into the tricarboxylic acid cycle. When the tests were carried out under 

 aerobic conditions with minced sheep heart or kidney cortex, and using 

 the same metabolites as above, definite proof was obtained that acetoace- 

 tate contributes to the formation of acids of the tricarboxylic acid cycle. 

 Oxaloacetate and fumarate were both shown to increase the rate of oxida- 

 tion of acetoacetate. 



Although the evidence would seem to be sufficient to prove that oxalo- 

 acetate reacts with acetoacetate or with acetate to form citrate, the nature 

 of the compound which is common to these two substances is not known. 

 One would naturally expect acetate itself to be the compound involved in 

 both cases. However, in the tests of Buchanan et al.,^'" when acetoacetate 



"2 J. M. Buchanan, W. Sakami, S. Gurin, and D. W. Wilson, /. Biol. Chem., 159, 

 695-709(1945). 



6« S. Weinhouse, G. Medes, and N. F. Flovd, /. Biol. Chem., 166, 691-703 (1946). 



544 N. F. Floyd, G. Medes, and S. Weinhouse, J. Biol. Chem., 171, 633-638 (1947). 



5« H. A. Krebs and L. V. Eggleston, Biochem. J., 39, 408-419 (1945). 



*« H. A. Krebs and L. V. Eggleston, Biochem.. J., m, 294-305 (1948). 



5«J. M. Buchanan, W. Sakami, S. Gurin, and D, W. Wilson, /. Biol. Chem., 169, 

 403-410(1947). 



