CHAPTER I 



DESTRUCTIVE AND NON-DESTRUCTIVE 

 METHODS IN MODERN BIOCHEMISTRY 



I. FROM THE WHOLE ORGANISM TO THE PURE ENZYME 



Many types of biochemical research are carried out on the whole organism, 

 with a minimum of disturbance. This is the case, for example, when a given 

 compound is added to the food and the excreta is investigated for this 

 substance or its transformation products. 



An example of important information obtained by this non-destructive 

 procedure is the demonstration of nitrogen equilibrium in the adult 

 animal, as in the classic experiments of Bischoff and Voit on the dog. If 

 meat is given to a dog continuously over a period of several days, and the 

 nitrogen excretion is measured over this time, it is found that no nitrogen 

 is retained. The nitrogen excreted corresponds to the nitrogen ingested; 

 the animal is said to be in nitrogen equilibrium. 



The organism can also be subjected to experimental intervention such as, 

 for example, the removal of an organ. The normal mammal converts the 

 nitrogen of amino acids into urea, but this is no longer the case when the 

 organism is deprived of the liver. The amino acids and ammonia accumu- 

 late in the blood. Alternatively, the urea formed when the liver is present is 

 eliminated by the kidneys and it is the urea which would accumulate in the 

 blood if the kidneys were removed. 



A more destructive process is experimentation using isolated or perfused 

 organs. Thus it is possible to introduce a substance to an organ by way of 

 the perfusion fluid and to observe the changes it undergoes in the organ by 

 studying the efferent fluid. For example, introduction of ammonia into 

 the liquid perfusing the isolated liver of a dog results in the appearance of 

 urea in the efferent liquid. Alternatively, the same experiment using isolated 

 goose liver would show the appearance of uric acid. It is evident that an 

 isolated perfused organ, whether perfused with "physiological saline" or 

 with blood, is in fact a highly abnormal experimental material. It is ab- 

 normal when we consider the supply of oxygen to the cells, the supply of 

 blood, the inervation of the material, etc. Even more destructive is experi- 

 mentation using thin tissue slices. Such slices of tissue, provided that they 

 have been prepared under suitable conditions, will survive for several 

 hours, the cells appearing to lead a normal life. The pressure of oxygen 

 must be sufiicient to meet the oxygen requirements of the tissue. If the 



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