ADVANCED CHEMICAL PHYSIOLOGY 327 



of ordinary (96 per cent.) alcohol are then added to the solution, the 

 mixture shaken and allowed to stand for several hours (preferably 

 overnight). 



The more or less white precipitate of glycogen will by this time have 

 settled down, so that the supernatant reddish fluid can with care be 

 poured off into a beaker, after which it is filtered through a filter paper 

 of suitable size, so as to collect on the filter any particles of glycogen 

 which the decanted fluid may contain. The precipitate of glycogen is 

 now thoroughly shaken with about ten times its volume of 66 per cent, 

 alcohol (about 700 c.c. alcohol and 300 c.c. water) containing 1 c.c. per 

 litre of a saturated solution of NaCl. This washing fluid removes 

 many of the impurities which adhere to the glycogen. 



After settling, the wash fluid is decanted into the same beaker as was 

 employed for receiving the original supernatant fluid, and filtered 

 through the same filter. This process is repeated at least once again, 

 after which the precipitate is shaken with ordinary alcohol (about ten 

 times its volume), and the suspension thrown on to the same filter paper 

 as used above. 



When the alcohol has all drained off, the precipitate is washed on the 

 filter paper with ether. All the washed glycogen has thus been collected 

 on the filter paper and must now be dissolved, for which purpose the 

 filter is filled up with boiling water, and the solution of glycogen allowed 

 to filter through into a clean Erlenmeyer flask. When the first added 

 water has completely drained through the filter, the filter is filled up 

 with boiling water a second and a third time. It is essential to allow 

 the filter to drain completely before adding more water. To be certain 

 that all the glycogen has been dissolved, some of the final filtrate should 

 be tested with alcohol for glycogen. 



The resulting opalescent solution can now be employed either for the 

 preparation of pure glycogen or for its quantitative estimation. For the 

 former purpose the glycogen is precipitated by alcohol ; for the latter 

 purpose the glycogen solution is made up to a litre in volume, and of 

 this 200 c.c. are taken, mixed with 10 c.c. HC1 (cone.) (i.e. 5 c.c. HC1 to 

 100 c.c. of glycogen solution), and heated in a flask on the waterbath 

 for three hours. 1 Complete hydrolysis of the glycogen is certain within 

 this time, although the resulting solution often contains a flocculent 

 precipitate which is probably protein in origin. The solution, after 

 cooling, is neutralised with 20 per cent. KOH and filtered into a 

 250 c.c. measuring flask through a small filter (10 cm.) paper. 



The flask used for inversion is rinsed three times with distilled water, 

 the washings being each time poured on to the filter and added to the 

 contents of the measuring flask. In this way the volume of the dextrose 

 solution is brought exactly to 250 c.c. 



Where only 10 or 20 gms. of liver were originally employed, the above 

 measurements must of course be altered, it being usually best to take 

 all of the glycogen solution for inversion and bring it to a definite 

 volume after neutralising. 



The estimation of the sugar may be carried out either by a volumetric 

 or a gravimetric method. 



Estimation of Cholesterol in Tissues. Based on the discovery of 



1 If the glycogen be reprecipitated and redissolved in a known volume of 

 water the resulting solution can be examined in the polarimeter and its 

 glycogen content calculated according to the formula on p. 214, 

 (a) D = + 196-63. 



