172 Appendix: analytical methods 



values for phosphocreatine higher than found by other methods. A 

 third ahernative involves the precipitation of the calcium or barium 

 salts of phosphocreatine w^ith ethanol and estimation as creatinine 

 formed in the Jaffe reaction (Mcllwain, 1951) or as creatine, 

 liberated upon acid hydrolysis of the residue under standard 

 conditions, by means of the colour reaction with a-naphthol 

 and diacetyl (Ennor and Rosenberg, 1952). Estimations based upon 

 determinations of creatine require appreciable quantities of tissue 

 (0-3-1 -5 g) the latter method requiring the least (Heald, 1956«). 



With cerebral tissues metabolizing glucose the above methods have 

 yielded similar values for phosphocreatine indicating that the 

 phosphorus determined actually arises from phosphocreatine and 

 not from labile esters such as phosphopyruvic acid or acetyl 

 phosphate (Weil-Malherbe and Green, 1951). The values also 

 agree with values obtained by ionophoretic and enzymic techniques. 



Adenosine Di- and Triphosphates 



Chemical methods are generally based upon the release of 

 inorganic phosphate when the polyphosphates are hydrolysed in 

 N acid for 10 min at 100°. Since the fraction containing the 

 nucleotides is usually obtained by precipitation as the calcium or 

 barium salts, this involves a correction for the inorganic phosphate 

 present. Provided only small quantities of the diphosphate is 

 present, the method is fairly reliable. To estimate adenosine di- 

 and triphosphate by this method the ratios of the acid hydrolysable 

 phosphate and the ribose content are determined and used to 

 calculate the percentage of each phosphate. This method is not 

 particularly accurate and has been stated to yield errors of 20-30% 

 in the values so obtained (Block and Alberty, 1951). Better methods 

 involve estimation by chromatographic or enzymic techniques (see 

 below). Nucleotides other than those of adenosine have been only 

 recently recognized and the chromatographic methods used for 

 identification also serve as a means of estimation by determining 

 the absorption of fractions at 260 m [x. 



Total Phosphorus 



Various methods have been proposed. All involve the removal 

 of organic material by wet oxidation followed by the determination 

 of the inorganic phosphate formed in solution. Digestion with 

 concentrated sulphuric acid and hydrogen peroxide (Le Page, 



