VI. EFFECTS OF DEFICIENCY 235 



in a variety of ways, and the explanation for such inhibition has been that 

 the inhibitino' agent in some way affects an enzymic process, the ultimate 

 aim of which is the liberation of inorganic ph<jsphorus from j^liosphoric 

 acid esters. The source of this inorganic phosphorus has been a stumbling 

 block in any theory of calcification. Shipley ct al.^^ produced in vitro calci- 

 fication with artificial serum ultrafiltrates containing an adequate concen- 

 tration of both calcium and inorganic phosphorus. Robison^* demonstrated 

 the presence of alkaline phosphatase in the hypertrophic cartilage cells and 

 produced calcification in vitro using solution of calcium and hexosephos- 

 phoric acid. The latter was hydrolyzed by alkaline phosphatase, thus 

 liberating inorganic phosphorus. However, Shipley et at. pointed out that 

 plasma and presumably tissue fluid contain only traces of organically bound 

 phosphorus. Furthermore, preparatory cartilage cells contain only miiuite 

 amounts of organic phosphorus although rich in glycogen and phosphoryl- 

 ase. The problem of finding a source of organic phosphorus seemed to 

 constitute an unsurmountable obstacle until Gutman^^ suggested the proc- 

 ess of phosphylative glycogenolysis as a possible source of inorganic phos- 

 phorus and produced confirmatory evidence in the finding that substances 

 which interfered with this process inhibited in vitro calcification. 



Glycogen, like alkaline phosphatase, may be demonstrated in the car- 

 tilage cells of the hypertrophic cartilage, just before and during calcifica- 

 tion, only to disappear as the process of calcification becomes complete. 

 That glycogen may play an important role in endochondral calcification is 

 indicated by the fact that in vitro calcification will not take place if the 

 glycogen is removed with ptyaline. 



The role of phosphatase in calcification has received the most intense 

 study. ^^ It is presumed to play an important role for the following reasons. 



1. It is present in the preliminary stages wherever tissue is about to be 

 calcified, i.e., in cartilage matrix, in osteoid, and at the site of metastatic 

 calcification. 



2. Substances that inhibit phosphatase activity inhibit in vitro calcifica- 

 tion. Some of these substances are potassium cyanide, hydrocyanic acid, 

 and fluoride. 



3. In vitro calcification takes place in living cartilage in the presence of 

 phosphatase when phosphoric acid esters represent the only source of inor- 

 ganic phosphorus. 



On the other hand, calcification in vitro can definitely occur in the ab- 



«•' P. G. Shipley, B. Kramer, and J. Howland, Biochem. J. 20, 379 (1926). 



6' R. Rohison, Biochem. ./. 35, 304 (1924). 



^^ A. B. Gutman and T. F. Yu, Trans. 2Hd Conf. Metabolic Interrelations p. 167, 



1950. 

 6" R. H. Follis, Jr., Bull. Johns Hopkins Hospital 85, 360 (1949). 



