104 1. MALONATE 



concentrations of inhibitor with the metabolic disturbance produced. 

 Second, the inhibition occurs under physiological conditions, rather than 

 in slices or minces or other preparations in which the cell metabolism may 

 be very abnormal. Last, one is able to compare the different tissues with 

 respect to their metabolic patterns and perhaps determine some of the 

 reasons for the selective actions of inhibitors or drugs. These methods of 

 investigation, called "m vivo metabolic blocking techniques" by Busch 

 and Potter, if applied properly, would help to provide a more rational 

 basis for development in chemotherapy and the selective depression of 

 tumor growth. 



ACCUMULATION OF CYCLE SUBSTRATES 

 OTHER THAN SUCCINATE 



Specific inhibition of succinate oxidase would be expected to lead to the 

 accumulation of succinate but of no other cycle intermediates, because 

 the free energy differences between them are of such magnitude that no 

 backing-up from succinate would be anticipated. When other members of 

 the cycle are found to accumulate in the presence of malonate, it is generally 

 considered to be evidence that either the action of malonate is not specific 

 or that secondary reactions are proceeding. Malonate has been shown many 

 times to cause an accumulation of certain cycle intermediates, especially 

 citrate and a-ketoglutarate, in cell suspensions, slices, and whole animals. 

 The nature of these effects will first be summarized and then some possible 

 mechanisms will be considered. 



Accumulation of Citrate 



The administration of malonate to dogs (Orten and Smith, 1937), rabbits 

 (Krebs et al., 1938),- and rats (Busch and Potter, 1952 a) leads to an increased 

 urinary excretion of citrate (Table 1-17 and Fig. 1-13). There is also a rise 

 in plasma citrate following injections of malonate in rabbits (Forssman, 

 1941) and dogs (Stoppani, 1946). Tissue citrate also rises in mice injected 

 with 10 millimoles/kg malonate: kidney (16 to 20), heart (40 to 70), liver 

 (5 to 10), and diaphragm (70 to 225) (values in milligrams per kilogram 

 wet weight) (Chari-Bitron, 1961). Brain, however, shows no increase in cit- 

 rate, perhaps due to the poor penetration of malonate. Some accumula- 

 tion of citrate has also been observed in suspensions of Ashbya gossypii 

 mycelia metabolizing acetate and oxalacetate (Mickelson and Schuler, 

 1953), Schizophyllum commune mycelia metabolizing pyruvate and malate 

 (J. G. H. Wessels, 1959), and Ehrlich ascites tumor cells metabolizing fu- 

 marate (Kvamme, 1958 c) in the presence of malonate. Thus this phenom- 

 enon is widespread, occurring in different types of organism and under a 

 variety of conditions. 



