Appendix: analytical methods 171 



reduction. With solutions containing protein the /^obutanol can 

 be separated by centrifugation and then removed to a separate tube 

 or the protein may first be precipitated by sihco-tungstate as 

 described by Martin and Doty (1949). The latter authors use a 

 mixture of wobutanol/benzene as extractant. Under the conditions 

 of extraction phosphocreatine is not broken down (Ernster et al., 

 1950) and the method may be used to estimate inorganic phosphate 

 in the presence of this ester. The stability of other phosphates 

 under these conditions has been described by Weil-Malherbe and 

 Green (1951). With extracts made in perchloric acid, wobutanol is 

 apparently not so effective in removing the phosphomolybdate 

 complex and can be replaced by xVopropanol (Wahler and WoUen- 

 berger, 1958). If phosphocreatine is also to be estimated, inorganic 

 phosphate is first separated by means of the barium fractionation 

 procedure, the residue being dissolved in OT A^ HCl before the 

 final estimation. The method is extremely sensitive and has been 

 used with samples of brain weighing 50-100 mg (Heald, 1954; 

 Bollard and Mcllwain, 1959). 



Phosphocreatine 



Phosphocreatine is usually determined by methods depending 

 upon the lability of the phosphate group in acid solution. In the 

 methods described by Le Page (1957) and Stone (1948) it is 

 calculated as the difference between the total inorganic phosphate 

 determined by incubating extracts for 20-30 min under acid 

 conditions and the inorganic phosphate determined by the calcium 

 precipitation technique. A procedure based upon the Lowry- 

 Lopez technique has been described using the same principle 

 (Furchgott and GubarefT, 1956). The method is sensitive and has 

 been used with 50 mg of tissue. An alternative procedure for use 

 with small quantities of cerebral tissues involves a preliminary- 

 removal of inorganic phosphate as the barium salt followed by 

 acidification of the extracts and incubation at 25° with molybdate 

 solution. Under these conditions, phosphocreatine is quantitatively 

 hydrolysed in 30 min (Ernster et al., 1950) and the inorganic 

 phosphate released is estimated by the modified Berenblum and 

 Chain procedure (Heald, 1954). Separation of inorganic phosphate 

 is considered to be a necessary preliminary since use of the direct 

 extraction method for inorganic phosphate followed by hydrolysis 

 of phosphocreatine at 30° in the same solution tends to yield 



