170 Appendix: analytical methods 



acid-amyl acetate solvent. Provided the conditions of separation 

 have been acidic, phosphates on the paper can readily be made 

 visible by means of an acetylsalicylic acid + ferric chloride reagent 

 (Wade and Morgan, 1953). This reagent possesses the advantage 

 of not degrading phosphates such as adenosine triphosphate which 

 can be eluted intact from the paper after detection. 



Analysis of Phosphates 

 Chemical Methods 



Inorgajiic phosphate. — In extracts of tissue inorganic phosphate 

 can be determined directly by the method of Lowry and Lopez 

 (1946). This involves the measurement of the blue colour develop- 

 ing upon the reduction of phosphomolybdate with ascorbic acid at 

 pH 4-0. By careful choice of the strengths of the reagents the 

 method can be kept sensitive to inorganic phosphate without 

 hydrolysing labile phosphates. Many extracts inhibit the reaction. 

 This can be overcome either by diluting some 10-100 fold as 

 recommended by Lowry and Lopez or by the inclusion of Cu^^ 

 ions in the reaction mixture to a final concentration of 5 X 10-*M 

 (Peel, 1955; Furchgott and Gubareff, 1956). With copper ions 

 present, dilution of the extract by a factor of 5 is the maximum 

 needed. Extracts made in trichloracetic acid or perchloric acid 

 require adjustment to neutrality either using an external indicator 

 or by adding a predetermined quantity of alkali before sampling 

 for the final analysis. 



A method most widely used is that of Fiske and Subbarow 

 (1925, see Le Page, 1957) which is fully adequate when applied to 

 extracts of cerebral tissues. Inorganic phosphate must first be 

 separated from phosphocreatine by means of the calcium salt. 

 The chief disadvantage is the relatively large quantity of phosphate 

 required (range 4-40 jitg) necessitating 0-3-0-5 g cerebral tissue. With 

 experiments in vitro this is equivalent to 3-4 slices. A more sensi- 

 tive method is that of Berenblum and Chain (1938) which has been 

 modified so that estimations can be carried out in a single tube 

 (Long, 1943). This method involves the extraction of phospho- 

 molybdate from acidic solution into wobutanol and the reduction 

 of the inorganic complex with stannous chloride. The extraction 

 is extremely rapid and complete at 0° and separates inorganic 

 phosphate from many substances likely to interfere in the final 



