Appendix: analytical methods 169 



were made upon portions of the total trichloracetic acid extracts of 

 cerebral tissues and since the nucleotides were detected with 

 ultra-violet light, the position of inorganic phosphate and other 

 phosphates is not clear. The method appears to be a good one and 

 has been used to study changes in the nucleotides in brain in vivo 

 following anaesthesia, ischaemia and anoxia. 



The detailed separation of nucleotides and nucleosides and 

 derivatives upon ion exchange columns described by Schmitz 

 et al. (1954) has not yet been applied extensively (see however 

 Mandel and Harth, 1958) probably owing to the length of time 

 involved in a single run. However, the basic procedure is simple 

 and offers considerable scope for the detection and isolation of new 

 nucleotides and derivatives (Koransky, 1958). It is to be noted 

 that the extract containing the acid-soluble nucleotides also contains 

 phosphocreatine, inorganic phosphate and the hexose and triose 

 phosphates, and the position of these relative to the nucleotides in 

 the eluants from such columns have not yet been determined. A 

 separation of phosphocreatine and the hexose phosphates upon a 

 resin column has been described by Abood (1956). Extracts were 

 absorbed on Dowex-1 -formate and eluted with formic acid. 

 Separation of inorganic phosphate, phosphocreatine, adenosine 

 mono-, di- and triphosphates, glucose-6-phosphate and fructose- 

 1 : 6-diphosphate was achieved. The analytical figures for phos- 

 phocreatine in brain (4-25 /xmoles/g) appear to be higher than 

 normally found but the separation yielded this phosphate free 

 from contamination with radioactive inorganic phosphate. A 

 similar separation of adenosine mono-, di- and triphosphates, 

 phosphocreatine and inorganic phosphate from brain extracts has 

 been described by Lin et al. (1958). 



Although many basic methods have been described for the 

 separation of hexose phosphates and derivatives by means of paper 

 chromatography or ionophoresis (Hanes and Isherwood, 1949; 

 Bandurski and Axelrod, 1951; Ganguli, 1953; Lindberg and 

 Ernster, 1956; Wade and Morgan, 1956; Schwimmer et al., 1956), 

 application to the analysis of cerebral extracts has not been exten- 

 sive. Tower (1958) found that the separation of hexose and pentose 

 phosphates from trichloracetic acid extracts of cerebral slices was 

 readily achieved. The phosphates were first precipitated as the 

 barium salts and, after removal of barium by cation exchange 

 resins, were separated by descending chromatography in an acetic 



