APPENDIX 



ANALYSIS OF CEREBRAL TISSUES 

 FOR PHOSPHATE DERIVATIVES 



The determination of phosphorus derivatives in cerebral tissues 

 presents special problems. The quantities of many of the phos- 

 phates are small in comparison with those of other tissues and the 

 amounts of tissue available particularly from experiments in vitro 

 is usually limited to some 100-300 mg wet wt. The presence of 

 large quantities of phospholipids can complicate the preparation of 

 suitable extracts. The following is an account, based on the 

 writer's experience, of the various methods employed in the 

 analysis of cerebral phosphates. No attempt has been made to 

 describe analytical methods in detail, for which the original papers 

 should be consulted. 



Surveys of some analytical methods have been given by Le Page 

 (1948, 1957), Stone (1948), Lindberg and Ernster (1956), and 

 Mommaerts (1958). 



Preparation of Tissues 



The rapid changes in cerebral phosphates consequent upon 

 death can be avoided if the brain in vivo is rapidly frozen and 

 removed while in this state. Small animals such as the mouse, rat 

 or guinea pig can be frozen by dropping into liquid nitrogen 

 contained in a w^ide mouthed vacuum flask (Le Page, 1957; 

 Dawson and Richter, 1950). The body weight should not be in 

 excess of 100 g unless dictated by special circumstances. In the 

 latter case volumes of liquid nitrogen of 1-2 1. are required for each 

 animal. The low temperature of the coolant ( — 180°) provides an 

 excellent temperature gradient and the coolant does not contami- 

 nate the preparation. Most use has been made of this method 

 though where liquid air or nitrogen are not readily available, 

 drowning in a mixture of acetone and solid carbon dioxide (Le 

 Page, 1946; Lindberg and Ernster, 1950; Abood, 1956) would 

 appear to be a satisfactory alternative. Le Page recommends light 



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