KONRAD BLOCH 



cubaled in the presence of DoO indicate that the deuteriocholesterol 

 formed amounted to at least 10% of the cholesterol present at the 

 start, an amount of change which, although close to the limits of 

 detection by available analytical methods, nevertheless should be 

 demonstrable. 



The available data on the syntheses in which acetic acid is 

 employed indicate that, in the intact animal, fatty acids are regener- 

 ated at a faster rate than cholesterol (17). On the other hand, the 

 corresponding results obtained with liver slices would lead to the 

 erroneous conclusion that cholesterol synthesis is by far the more rapid 

 process. The lack of correlation between the rates in vitro and in vivo 

 must be taken to mean that, in the experiments in which acetic acid 

 was added to liver slices, the conditions chosen happened to be favor- 

 able for cholesterol synthesis, but that a component necessary for fatty 

 acid formation was lacking. 



The use in tissue slice experiments of enzyme poisons, such as 

 malonate, fluoride, etc., makes possible an accumulation of inter- 

 mediates whose identification is thereby facilitated. If specific in- 

 hibitors for a particular reaction are not available, a suspected inter- 

 mediate may escape detection because it does not accumulate. The 

 isotope dilution method has recently been applied to establish the oc- 

 currence of a reaction which is not detectable by the balance method 

 (14). Incubation of kidney slices with acetic acid does not lead to a 

 demonstrable formation of acetoacetic acid. If acetic acid containing 

 heavy carbon is substituted for ordinary acetic acid and some non- 

 isotopic acetoacetic acid is added, subsequent analysis of the aceto- 

 acetate then isolated reveals the presence of heavy carbon in the keto 

 acid. Thus, clearly, acetoacetic acid is synthesized from acetic acid, 

 but its rapid removal by subsequent reactions prevents its accumulation 

 in quantities sufficient for analysis. In experiments of this type, only 

 the isotope concentration and not the absolute amount of acetoacetate 

 is relevant; the rate of acetoacetate synthesis can be calculated from 

 the amount of acetoacetate added initially and from its isotope concen- 

 tration at the end of the experiment. It is evident that many of the 

 difficulties pertaining to the use of tissue preparations can be circum- 

 vented by the use of pure enzymes. 



In devising schemes for metabolic interconversions, the bio- 

 chemist often rests his case on little more than similarities of chemical 



294 



