326 Amino-Acid Nitrogen in Blood 
but is somewhat troublesome in certain stages of the manipulation. Fil- 
tration is very slow, and the alternative process of centrifuging such large 
volumes (800 to 500 cc.) is not always convenient. To avoid this diffi- 
culty, he recommended heat coagulation followed by trichloroacetic acid 
precipitation. 
From the standpoint of accuracy the procedures of Greenwald 
and Bock are both satisfactory, but some difficulties still remain, 
chiefly the removal of the trichloroacetic acid after precipitation 
of protein. As suggested by Bock, if the trichloroacetic acid is 
not entirely removed it enters into some combination with the 
amino-acids which prevents their quantitative reaction in the 
Van Slyke method. Vacuum evaporation does not suffice for 
this purpose and Bock’s procedure of direct evaporation until the 
color change of the alizarin indicator is not convenient, for, as the 
solution reaches dryness, frothing and bumping often occur; 
further, the operation requires from 30 to 50 minutes. After 
making alkaline to remove ammonia and acidifying again, it is 
difficult to get clear solutions. To avoid this difficulty, I sought 
to obtain a method in which it is not necessary to remove the 
protein precipitant or in which it could be removed withoyt much 
difficulty. Kaolin in large quantities was found appropriate for 
this purpose. The preliminary procedure was carried out ac- 
cording to Bock. The. details of the whole procedure are 4s 
follows. 
A measured volume of blood (80 to 50 ce.) is treated with 0.4 
em. of ground soy bean dissolved in 2 to 3 cc. of water and 1 cc. 
of a 3 per cent solution of NaH:POu,, and allowed to stand at room — 
temperature for 30 minutes. The urea contained in the blood is 
converted into ammonia which is removed afterwards. One 
volume of this blood is slowly added to five volumes of boiling 
0.01 n acetic acid in a casserole and boiled with stirring for half 
a minute. The same amount of boiling water is then added and 
the mixture is boiled with stirring for a moment, giving a clear and 
easily filterable precipitate. This is filtered hot through a folded 
filter and the casserole washed three times with 30 to 50 cc. por- 
tions of boiling water. The filtrate is rapidly concentrated over 
a free flame in a casserole. The residue is transferred quantita- 
tively to a graduated cylinder or flask, making the volume nearly 
that of the original volume of the blood. After adding 0.1 N 
