Appendix: analytical methods 173 



1957) has been largely replaced by perchloric acid (King, 1932; see 

 Lowry et al., 1954). When large amounts of organic material are 

 present, as in extracts containing phospholipids or in strips of 

 paper cut from chromatograms, the sulphuric acid-perchloric acid 

 digestion mixture of Hanes and Isherwood (1949) has proved very 

 suitable. Explosions, occasionally obtained with perchloric acid 

 alone, have not occurred and digestion of the last traces of carbon 

 can be assisted by the cautious addition of hydrogen peroxide 

 without excessive risk. Since the mixture contains sulphuric acid, 

 dilution and hydrolysis of the pyrophosphates formed is necessary 

 (Heald, 1956«). Inorganic phosphate in the solution can then be 

 estimated by the modified Berenblum and Chain procedure. 



Enzymic Methods 



Sensitive methods for the analysis of cerebral extracts for 

 adenosine di- and triphosphates, phosphocreatine, di- and triphos- 

 phopyridine nucleotides, hexose phosphates, fructose- 1 : 6-diphos- 

 phate, pyruvic acid and triose phosphates have been described 

 (Kratzing and Narayanaswami, 1953 ; Thorn et al, 1955 ; Clock and 

 McLean, 1955; Tower, 1958). 



With the exception of methods for the determination of di- and 

 triphosphopyridine nucleotides the other phosphates are deter- 

 mined by reactions involving the production of reduced or oxidized 

 diphosphopyridine nucleotide and measurement of the change in 

 absorption. In the method of Kratzing and Narayanaswami 

 (adapted from that of Slater 1953) adenosine diphosphate and 

 phosphocreatine are determined by difference. Adenosine triphos- 

 phate is determined directly after conversion of the y-phosphorus 

 to glucose-6-phosphate. This process appears to be specific since 

 guanosine triphosphate is not determined by the hexokinase 

 reaction (Sanadi et al, 1954). The methods of Thorn et al. 

 estimate adenosine di- and triphosphates in separate reactions. 

 Since both procedures also include certain intermediates such as 

 phosphopyruvate in the values determined corrections must be 

 applied. Normally, with cerebral tissues such corrections are 

 low. Hexose phosphates have been estimated by conversion of 

 fructose-6-phosphate to glucose-6-phosphate by hexose phosphate 

 isomerase followed by the determination of glucose-6-phosphate 

 by glucose-6-phosphate dehydrogenase (Tower, 1958; cf. Thorn 

 et al., 1955). Glucose-6-phosphate was determined directly by 



