226 CUVETTE COLORIMETRY 



PHOSPHATE AND PHOSPHATASE 



Siwe (1935) has described the use of the Pulfrich step photometer 

 with filter No. 72 (red) for the colorimetric measurement of in- 

 organic phosphorus in small amounts of blood by conversion to 

 phosphomolybdic acid and reduction by aminonaphtholsulfonic acid. 

 The same reaction was employed by Weil and Russell (1940) in 

 their phosphatase method ( page 209) . At about the same time Lund- 

 steen and Vermehren (1936) developed a micro procedure for the 

 determination of inorganic phosphate and alkaline phosphatase in 

 blood plasma based on Miiller's (1935) amidol reduction of phos- 

 phomolydic acid. The Pulfrich instrument with filter No. 72 was also 

 used in this case, and for most measurements the 10 mm. cells were 

 employed, but the 20 mm. cells were required for weaker colors. 50 

 fA. of blood are needed for a duplicate determination, and since the 

 procedure might be adapted to tissue extracts as well, it will be 

 described. 



Conditions for the determination of inorganic phosphate in the 

 presence of labile phosphate esters, such as phosphocreatine, acetyl 

 phosphate, and ribose-1-phosphate, were established by Lowry and 

 Lopez (1946). As these authors pointed out, the usual procedures 

 for measurement of inorganic phosphate in tissue extracts represent 

 the sum of the inorganic phosphate and the phosphate of the labile 

 esters hydrolyzed by the reagents employed in the determination. 

 The procedure of Lowry and Lopez is based on the reduction of 

 phosphomolybdate by ascorbic acid at pH 4.0. 



Bessey, Lowry, and Brock (1946) utilized as the substrate p-nitro- 

 phenyl phosphate, which had been studied by King and Delory 

 ( 1939) , and applied to phosphatase determinations by Ohmori 

 (1937) and Fujita (1939). Bessey et al. were able to determine the 

 phosphatase in as little as 5 ju.1. serum using 0.5 ml. of solution for 

 the colorimetry. The advantage of this substrate is that it is color- 

 less and yields the yellow salt of p-nitrophenol when the phosphate 

 group is split off. Thus the color develops in proportion to the degree 

 of the hydrolysis and no additional reagents are required for the 

 color development. This advantage is also to be found in the use 

 of phenolphthalein phosphate, which was employed by Huggins and 

 Talalay (1945). However, alkaline phosphatase splits the p-nitro- 

 phenyl phosphate 25-30 times faster than the phenolphthalein com- 



