620 3. DEHYDROACETATE 



method of synthesis involves heating ethyl acetoacetate with a small amount 

 of NaHCOg at 200o-210o for 7-8 hr with subsequent distillation at 1280-140° 

 in vacuo, the yield being over 50% (Arndt, 1955). It is also formed by the 

 tetramerization of ketene, and by suitable catalytic means may be almost 

 quantitatively obtained from diketene (Steele et al., 1949), the reaction 

 presumably proceeding through the enolized forms of the diketene. The 

 simplest method of purification of dehydroacetic acid is probably recrys- 

 tallization from ethanol. 



Estimation 



Two methods suitable for tissue analyses were developed by Woods et al. 

 (1950). A colorimetric test, based on the reaction of the acetyl group with 

 salicylaldehyde in alkaline solution to give a red-orange color, is sensitive 

 in the range 10-200 //g. The spectrophotometric test, based on absorption 

 at 312 m//, is approximately 10-fold more sensitive. Both tests depend on 

 the proper pre treatment and extraction of the tissue samples, since neither 

 test is particularly specific. The spectrophotometric tests give the best re- 

 coveries and are preferable for most analyses. 



INHIBITION OF ENZYMES 



Dehydroacetate at concentrations between 2.3 and 93 vaM progressively 

 depresses the oxygen uptake of slices and minces of cerebral cortex and 

 kidney respiring endogenously, but the excess oxygen uptake following ad- 

 dition of various substrates is inhibited only in the case of succinate (Seevers 

 et al., 1950). This suggested that dehydroacetate might inhibit succinate 

 oxidase and thus this enzyme was examined in some detail. 



Succinate Oxidase 



Inhibition of succinate oxidase is proportional to the logarithm of the 

 dehydroacetate concentration from 27 to 77% inhibition (Seevers et al., 

 1950). Although no data were given, it was stated that the substrate con- 

 centration has little or no effect on the degree of inhibition, indicating it is 

 not competitive. If this is true (see page 621), if ^ would be around 11.6 xaM 

 indicating much less affinity of the enzyme for dehydroacetate than for mal- 

 onate. The site of inhibition in the succinate oxidase sequence was determin- 

 ed in two types of experiment. In the first, the cytochrome system was block- 

 ed by cyanide and cresyl blue added as a hydrogen carrier; dehydroacetate 

 inhibited this system to the same degree as the normal one, indicating the 

 action not to be on the cytochrome system (see accompanying tabulation). 

 In the second, the cytochrome system was studied directly and no inhibition 



