Appendix: analytical methods 163 



involved in preventing change the method yields high vakies for 

 the quantities of phosphocreatine, approaching those found in 

 anaesthetized animals, and low values for the quantities of cerebral 

 lactic acid, both of which change markedly within a few seconds 

 when the brain is stimulated. After freezing, the brain can be 

 chiselled out with precooled chisels before further extraction. 

 During removal it is desirable that it be cooled at frequent intervals 

 by returning the animal to the liquid air. With coolants such as 

 acetone-carbon dioxide such a procedure is likely to contaminate 

 the tissue. 



Extraction of the Tissues 



Breakdown of labile phosphates is avoided if the denaturation of 

 the proteins and extraction of the phosphates is rapid. This is 

 assisted if the lumps of tissue are powdered while frozen. An 

 apparatus designed for this purpose has been described by Le Page 

 (1948). Alternatively an ordinary pestle and mortar precooled in 

 liquid nitrogen have proved successful. The sample is cooled in the 

 mortar by covering with liquid nitrogen. When this has boiled off 

 the tissue can be powdered by striking the pestle with a hammer. 

 The powdered residue is transferred rapidly to a weighed, cooled 

 homogenizing tube containing extractant at 0-2° and immediately 

 ground with the homogenizing pestle. Speed is essential for it has 

 been shown that the rate of penetration of the extractant into the 

 frozen tissue is insufficient to prevent extensive breakdown of 

 phosphocreatine and adenosine triphosphate if the lumps of brain 

 are simply allowed to thaw in the extractant before grinding 

 (Table 25). These problems do not arise with tissue slices which 

 appear to be fixed, as judged by overall change of colour, within 

 1-2 sec of immersion into trichloracetic acid. Presumably the thin- 

 ness of the slice permits rapid penetration. 



The extractants most widely,, used are trichloracetic acid 

 (10% w/v), perchloric acid (19% w/v) and, when very labile 

 phosphate esters, such as acetyl phosphate, are to be studied, a 

 saturated solution of ammonium sulphate, buffered at pH 4-0 

 with sodium acetate-acetic acid (Lowry and Lopez, 1946). The 

 removal of glycogen from extracts made in trichloracetic acid 

 (Le Page, 1957; Sacks, 1949; Kaplan and Greenberg, 1944) before 

 fractionating the phosphates is only rarely practised with extracts 

 of cerebral tissues where the glycogen level is about 4-0 /><,moles/g 



