TRIGLYCERIDES AND FATTY ACIDS 169 



containing C^' in both carboxyl and carbonyl positions, and ordinary 

 acetate, were incubated with kidney extracts containing a-ketoglutarate, 

 the acetate isolated at the conclusion of the tests contained much less C^* 

 than did a-ketoglutarate. This was interpreted to mean that acetoacetate 

 did not pass through the stage of acetate while being incorporated into the 

 tricarboxylic acid cycle. In later studies by Buchanan and co-workers,^''^ 

 it was found that acetoacetate per se behaved differently from the aceto- 

 acetate originating when octanoate was oxidized. Acetoacetate was 

 found to be resistant to splitting into 2-carbon fragments and recombining 

 at random. Since it is known, however, that all 4 carbons of acetoacetate 

 are convertible into citrate,^" this would appear to necessitate the prelimi- 

 nary formation of two 2-carbon fragments. This intermediate is not the 

 ordinary acetate; therefore, it is believed that it may be an activated form 

 of acetate (see page 15). 



The citric acid cycle is operative in effecting the oxidation not only of 

 acetoacetate but also of the longer aliphatic fatty acids. Thus, Lehn- 

 inger^*" found that the 2-carbon fragments formed on oxidation of octanoic 

 acid with liver slices containing ATP are condensed to citric acid (if 

 oxaloacetate is present), or to acetoacetic acid. He states that neither 

 butyric acid, crotonic acid, vinj^lacetic acid, DL-/3-hydroxybutyric acid, 

 aldol, nor acetopyruvate is an intermediate. 



According to Stern and Ochoa,^'*^ the formation of citric acid from oxalo- 

 acetate and acetate involves a 2-stage reaction which is as follows: 



(1) Formation of the active acetate derivative. This results from the 

 action of an acetate-activating enzyme system which includes CoA and 

 ATP. The active acetate is believed to be acetylphosphate or a compound 

 closely related to it. 



HO HO 



„ I II Acetate I 11 



H— C— C— OH + ATP > H— C— C— X 



I Acitivating enzyme I 



H H 



Acetic acid Active acetate 



(2) Condensation of active acetate and oxaloacetate to form citrate 

 through the action of a condensing enzyme. This apparently involves an 

 addition reaction rather than a condensation between the molecules, with 

 the elimination of a small molecule. The reaction proceeds as follows: 



'« J. M. Buchanan, W. Sakami, and S. Gurin, /. Biol. Chem., 169, 411-418 (1947). 

 "» J. R. Stern and S. Ochoa, Federation Proc, 9, 234-235 (1950). 



