METABOLIC CYCLES AND DECARBOXYLATION 207 



of strong acid. Part of this non-a-ketoglutarate radioactivity can be 

 released as carbon dioxide on treatment with ninhydrin at 100° C, 

 and with chloramine-T (Table 4). This suggests that part of the 

 assimilated carbon dioxide has been converted into an amino acid. 

 Beyond this, however, we know nothing of the chemical nature of 

 the compounds concerned. 



Table 4.— Effect of ninhydrin and chloramine T on non-a- 

 ketoglutarate radioactivity* 



Total 

 a-Keto- Total activity Activity of Activity of 



Experiment glutarate activity of medium CO2 released CO2 released 



synthesized of a-keto- after CO2 by ninhydrin by chloramine-T 

 glutarate removal 



mg. 



1 35.0 5.18 31.8 7.8 — 



2 39.0 6.2 31.2 — 10.39 



3 53.0 1.2 4.2 — 0.78 



4 31.6 0.22 0.929 0.209 — 



* Experimental conditions are similar to those described in Table 3. The experiments 

 with chloramine-T and with ninhydrin were carried out on aliquots of the metaphos- 

 phoric deproteinized reaction mixture under the conditions described, respectively, 

 by P. P. Cohen in Biochem. J., 33, 551 (1939) and by D. D. Van Slyke and R. T. Dillon 

 in Comp. rend, du Lab. Carlsberg, Ser. Chim., 22, 480 (1937). 



In all experiments involving the use of radioactive isotopes or 

 tracer elements it is a matter of primary concern whether the ap- 

 pearance of the tagged atom in a product represents a true meta- 

 bolic reaction or has been introduced by a nonspecific exchange 

 reaction. Oxalacetic acid, for example, breaks down to pyruvic acid 

 and carbon dioxide in tissues, and it might be argued that any 

 reaction involving this compound would yield a radioactive end 

 product if the reaction were carried out in the presence of radio- 

 active carbon dioxide. Such a possibility is difficult to control. How- 

 ever, the very large amounts of carbon dioxide assimilated argue 

 against this explanation of the presence of radioactivity in the 

 alpha-ketoglutaric acid. Also suggestive are experiments in which 

 the possibility of an exchange between the carboxyl group of oxal- 

 acetic acid and carbon dioxide of the medium has been examined. 

 In muscle, alpha-ketoglutaric acid is synthesized by the condensation 

 of oxalacetate with pyruvate to form citrate and the subsequent 

 oxidation of this compound to alpha-ketoglutarate; i.e., in terms of 

 the citric acid cycle this is what occurs. Now, a formation of oxal- 



