Appeftdix: analytical methods 175 



Generally phospholipids are removed either by heating with 

 mixtures of ether and ethanol, or preferably by the chloroform- 

 methanol mixture of Folch et al. (1951) which appears to be the 

 most effective as regards cerebral tissues both for completeness 

 and extreme rapidity. The tissue remaining is then analysed for 

 nucleic acids, phosphoproteins and residual organic phosphorus 

 by differing methods. These have been critically assessed by 

 Logan et al. (1952). When cerebral tissues are analysed for nucleic 

 acids by the methods of Schneider (1946) or Schmidt and Than- 

 hauser (1945) ribose nucleic acid is heavily contaminated with 

 phosphorus from other fractions, though a reliable value is 

 obtained for desoxyribonucleic acid. The difficulty can be over- 

 come if the urea-saline method of Hammarsten (1947) is used 

 (Strickland, 1952) when only nucleic acids are extracted. These 

 are fractionated by copper precipitation methods. An alternative 

 method for estimating the total nucleic acids involves the deter- 

 mination of the ultra-violet absorption of extracts prepared by the 

 Schneider method (Logan et al., 1952). The residue remaining 

 after removal of the nucleic acids is complex and is either re- 

 extracted with trichloracetic acid according to the Schneider 

 procedure (Strickland, 1952), to remove the last traces of nucleic 

 acids or else digested with normal NaOH at 37° for 18 hr according 

 to the Schmidt-Thanhauser method. On acidification of the digest. 

 a precipitate of an unidentified phosphate is obtained (probably 

 containing a little DNA). The supernatant contains inorganic 

 phosphate from the phosphoproteins and the remaining phosphorus 

 compounds collectively known as '* residual organic phosphorus ". 

 Considerable care is needed when studying the metabolism of 

 such groups of phosphates by means of radioactive phosphorus 

 since traces of inorganic phosphorus adhering to the tissue 

 residues appear in the various fractions. After precipitation 

 with trichloracetic acid the residue must be washed five or six 

 times with fresh trichloracetic acid, sometimes containing added 

 inorganic phosphate as an inactive carrier (Strickland, 1952; 

 Heald, \9Sla). If this procedure does not remove all traces of 

 added radioactivity the samples of radioactive phosphate are 

 suspect. In such cases treatment as described by Ennor and 

 Rosenberg (1954) has usually proved effective in reducing the 

 source of error. The phosphoprotein fraction, estimated as 

 inorganic phosphate, is particularly prone to contamination of this 



