The Determination of Inorganic Phosphate and Creatine Phosphate in Tissue Extracts

A number of procedures have been developed for the determination of inorganic orthophosphate (IP) and creatine phosphate (CP) in extracts of muscle or nervous tissue (l-6).Probably the simplest procedure in current use is that in which IP is determined in the presence of CP by the method of Lowry and Lopez (7) on one aliquot of extract, and IP plus acidmolybdate-labile organic phosphate is determined by the method of Fiske and Subbarow (8) on a second aliquot.The difference in phosphate concentrations given by the two methods is taken as a measure of the CP.Sometimes an accurate determination of IP by the Lowry-Lopez method is difficult because of the presence of substances in tissue extracts which retard the development of the reduced phosphomolybdate color.'Also, in extracts in which the concentration of IP is much greater than that of CP, a small error in the determination of IP or in the determination of IP plus CP can lead to a large error in the estimation of CP.In the procedure to be reported here the last mentioned disadvantage of the Lowry and Lopez procedure is overcome, since IP and CP are determined successively on the same aliquot of extract during a continuous run in a spectrophotometer.The procedure is essentially a modification of that of Fiske and Subbarow.The IP of an aliquot is first determined in weak acid (pH 2.3) with the aid of added Cu++ to increase the rate of development of the reduced phosphomolybdate color.2Strong acid is then added to the aliquot and the extra IP formed during the subsequent rapid hydrolysis of CP is determined by the further increase in color.