AMINO-ACID NITROGEN 103 



of the alcohol and blood without essentially affecting the results. 

 The alcohol and blood are thoroughly mixed, the vessel contain- 

 ing them is closed and twenty-four hours are allowed for precipi- 

 tation of the proteins to become complete. The solution is filtered 

 through a dry folded filter into a measuring cylinder without 

 washing the precipitate. The volume of filtrate is noted and is 

 taken for analysis as an aliquot part of the total blood-alcohol 

 mixture. The filtrate is then concentrated to a volume of 3 to 

 5 c.c. and used for determination of amino nitrogen by the Van 

 Slyke nitrous acid method. The use of a few drops of caprylic 

 alcohol to prevent foaming is advisable. 



AMINO-ACID NITROGEN 



Method of Greenwald and Bock l 



Principle. The protein of. the blood is removed by heat 

 coagulation and precipitation with trichloroacetic acid. The 

 amino-acid nitrogen is determined in the filtrate by the nitrous 

 acid method of Van Slyke. 



Procedure. If the blood is not over twenty -four hours old 

 proceed as follows: Introduce into a flask approximately 0.4 gm. 

 of ground (20 mesh) soy bean, 3 to 5 c.c. of water, and 1 c.c. of 3 

 per cent solution of crystallized NaH^POd. Let stand a few 

 minutes with occasional shaking. Run in 20 to 50 c.c. of blood 

 and allow to stand at room temperature for thirty minutes. Heat 

 0.01 N acetic acid to boiling, using five volumes of acid for one of 

 blood. Run in the blood slowly and boil, stirring for one-half 

 minute. Add the same amount of boiling water and boil with 

 stirring for 1 minute. Filter hot through a folded filter and 

 wash the casserole three times with 30 c.c. portions of water, 

 heating the water in the casserole and using a rubber-tipped 

 stirring rod. Boil down the filtrate rapidly over a free flame in a 

 casserole. Transfer to a small graduated flask or cylinder, choosing 

 the size so as to obtain nearly the original volume of the blood. 

 Wash the casserole three times with the smallest possible amount 

 of water. The volume after washing should not be more than 

 three-fourths of the final volume. Add enough trichloroacetic 

 1 Greenwald and Bock: Jour. Biol. Chem., 1916-17, 28, 357. 



