RELEASE OF ALCOHOLS FROM ESTERS 39 



The Site of Liberation of the Alcohol Moiety of 

 Phosphate Esters 



Although the evidence from the study of precipitated cal- 

 cium phosphate appears to show quite clearly that techniques 

 involving the precipitation of calcium phosphate indicate reli- 

 ably the site of alkaline phosphatase, it nevertheless seemed 

 desirable to reinforce this evidence by attempts to secure a pre- 

 cipitation of the alcohol moiety of the phosphate esters. This 

 would be very difficult to do with glycerophosphate. But with 

 a number of other esters of phosphoric acid it has been found 

 possible to secure the precipitation of the alcohol concerned. 

 The chief substrates which proved useful in this connection 

 are: 



1. Phenolphthalein phosphate. 



2. /3-Naphthol phosphate. 



3. p-Nitrophenylazo-a-naphthol phosphate. 



When phenolphthalein phosphate is studied, the calcium salt 

 is used in saturated solution in an incubation mixture contain- 

 ing calcium. Under these conditions both moieties of the ester 

 are precipitated, the phosphate moiety as calcium phosphate and 

 the phenolphthalein probably as a calcium salt. After incuba- 

 tion the sections are dried in air until the greater part of the 

 water has evaporated and are then exposed to ammonia vapor. 

 This converts the phenolphthalein into the bright-red alkaline 

 form. If a sufficient amount of water has been removed from 

 the section, phenolphthalein does not diffuse out at a significant 

 rate, and its distribution may be photographed. If, however, too 

 much water is removed by drying, ammonia does not convert 

 the phenolphthalein into the red form. To obtain just the right 

 amount of moisture in the section is a somewhat tricky pro- 

 cedure. When the phenolphthalein distribution has been re- 

 corded photographically, the distribution of calcium phosphate 

 may be ascertained by conversion of the calcium phosphate to 

 cobalt sulfide, as in the standard glycerophosphate technique. 

 The section may then again be photographed. It is found by 

 this method that the distribution of phenolphthalein is closely 

 similar to that of calcium phosphate, with the reservation that, 

 as the colour of phenolphthalein is much less intense than that 

 of cobalt sulfide, it is not possible to observe the presence of 



