57$ PRACTICAL ORGANIC AND BIO-CHEMISTRY 



(b) Tissues. 



5-30 gm. of tissue (weighed to -01 gm.) are cut into small pieces and 

 covered with boiling water containing i c,c. of 50 per cent, acetic acid per 1000 

 c.c. and heated on a boiling water-bath until the proteins are coagulated 

 (20-30 minutes). The pieces are lifted out with forceps and minced finely, 

 returned to the same water and heated for another 10 minutes. The super- 

 natant liquid is decanted through glass wool and the residue heated with 

 fresh boiling acidified water for 5-10 minutes, using 5-10 c.c. per gm. 

 of tissue. The solution is decanted through the same filter. This is repeated 

 4-5 times so as to extract the amino acids completely. The extracts are 

 concentrated in vacuo to about 20 c.c. The concentrated solution is trans- 

 ferred with a minimum amount of wash water to a conical flask and mixed 

 with 9-10 volumes of 95 per cent, alcohol, or 5 volumes of absolute or 

 methyl alcohol. The small amount of protein not coagulated by heat is thus 

 precipitated. After standing for 24 hours the solution is filtered, the residue 

 washed with 80 per cent, alcohol and concentrated in vacuo to 10-20 c.c., 

 a few drops of phenolphthalein and 25 per cent, sodium hydrate being added 

 to render the solution alkaline. Ammonia is thus driven off during the con- 

 centration. If the distillate be collected in '02N acid, the amount of am- 

 monia is also determined (about 10 mgm. per 100 gm.). When the solution 

 has been concentrated to about 10 or 20 c.c., the distillation in vacuo is inter- 

 rupted and the solution acidified with 50 per cent, acetic acid. About 50 c.c. 

 of water are added and the concentration continued to a volume of a few c.c. 

 If the amount of tissue used be less than 10 gm., it is advisable to concentrate 

 finally in a smaller flask of 300-500 c.c. capacity. The concentrated solu- 

 tion is transferred to a 10 c.c. or a 25 c.c. measuring flask and the amino 

 nitrogen determined by the action of nitrous acid : 10 c.c. portions are taken 

 if the larger apparatus, 2 c.c. portions if the micro apparatus, be used. The 

 time of reaction is 4-5 minutes at 15-20, 3 minutes at 20-25, and 2-2*5 

 minutes above 25. A correction for urea, etc., is determined in the same 

 way as described under blood and is deducted. 



The correction for urea, etc., may be omitted, also the removal of ammonia. 



The chief error is in the sampling of the tissue ; two portions of the same 

 organ seldom give the same results ; it is almost impossible to obtain absolutely 

 homogeneous samples of tissue. 



From 50-73 mgm. of amino nitrogen are given by 100 gm, of tissue. 



