110 1. MALONATE 



difficult then with incomplete knowledge of the cycle and related pathways 

 to understand how such conversions could take place; it still is. That is, 

 if there is no significant impairment of the utilization of citrate by mal- 

 onate, it is difficult to conceive of a pathway for the formation of citrate from 

 malonate that would be so rapid as to lead to a large rise in the citrate level. 

 A possibility that seems not to have been considered is that the substance 

 determined as citrate may not have been citrate but a related tricarboxylic 

 acid or some other compound giving a positive test. Although Hallman 

 (1940) examined the specificity of the determination, there are many 

 substances that have not been tested. For example, it is easy to formulate 

 reactions in which malonyl-CoA could react with various carbonyl sub- 

 stances, such as glyoxylate or pyruvate, to form tricarboxylate anions which 

 might be oxidized to pentabromacetone in the citrate test and be mistaken 

 for citrate. Certain dicarboxylates. such as itaconate, also are determined in 

 this test. Such substances may not be readily metabolized and hence would 

 accumulate much more readily than citrate. Although this posibility may 

 seem far-fetched, it would be well to make certain that it actually is citrate 

 that is accumulating during the action of malonate. It would be necessary 

 to convert only a small fraction of the administered malonate to such a 

 compound, since a dose of 26 millimoles/kg (see column 3 of Table 1-22) 

 would theoretically give rise to almost 1 g of a substance with a molecular 

 weight near that of citrate, whereas actually only around one-twentieth 

 of this was determined as citrate. Of course, such estimations depend on the 

 degree of sensitivity of the test to the compound. If there is any validity in 

 this suggestion, it may be that depressions of citrate levels may occur in 

 those preparations or tissues where such reactions of malonate or its meta- 

 bolic products do not occur, i.e., where the response to malonate is the one 

 expected on the basis of its inhibition of the functioning of the cycle. 



Accumulation of a-Ketoglutarate 



Very large increases in urinary a-ketoglutarate following the administra- 

 tion of malonate to rabbits and rats were reported by Krebs et al. (1938) 

 (Table 1-17) and this has been confirmed by El Hawary (1955), who found 

 a 3.7-fold increase in serum a-ketogluterate 30 min after the intraperitoneal 

 injection of 20 millimoles/kg malonate. As in the case of citrate, Krusius 

 (1940) found that a-ketoglutarate excretion is increased not only by mal- 

 onate but by many organic anions: malonate (46.4), maleate (42.0), malate 

 (17.7), succinate (17.7), /5-hydroxybutyrate (15.7), acetate (14.3), pyruvate 

 (12.1), fumarate (9.3), and sodium bicarbonate (0.6-7.8) (the figures give 

 urinary excretion in milligrams/day). He concluded that essentially all the 

 substances that increase citrate also raise the a-ketoglutarate excretion. 

 However, glutarate is a notable exception, for it potently augments citrate 

 formation but has no effect on a-ketoglutarate excretion. This would seem 



