ON THE ESTIMATION OF AMINO-ACID NITROGEN 
IN THE BLOOD. 
By SEIZABURO OKADA. 
(From the Medical Clinic, Imperial University of Tokyo.) 
(Received for publication, December 13, 1917.) 
Since the publication of the nitrous acid method for the deter- 
mination of amino-acid nitrogen by Van Slyke! in 1909, the lit- 
erature upon the occurrence of amino-acids in blood. and tissues 
has grown rapidly. Various procedures have been proposed by 
different authors for the preliminary removal of protein necessary 
to the analysis. For the most part the original method of Van 
Slyke and Meyer? has been followed, in which the blood proteins 
are removed by precipitation with nine or ten volumes of 95 
per cent alcohol. 
Recent investigations by Folin and Denis,? Greenwald,‘ and Bock,® 
however, have shown that the use of alcohol is not effective owing to the 
fact that amino-acids do not completely escape precipitation by alcohol. 
Folin and Denis state that certain nitrogenous substances such as creatine, 
asparagine, and tyrosine added to the blood could not be recovered quan- 
titatively after precipitation with methyl alcohol. Greenwald found 
that methyl alcohol filtrates contained only a little over half of the amino- 
acids added to the blood. After a number of experiments with several 
protein precipitants, he found that trichloroacetic acid closely approxi- 
mated the ideal precipitant. Nine volumes of 2.5 per cent solution to 
one volume of blood were adopted and the filtrates treated with a little 
kaolin. It was found, in every case, that the trichloroacetic acid filtrate 
contained all the added amino-acid nitrogen. The liquid remains clear 
after the addition of picric acid or of potassium mercuric iodide, giving 
no biuret reaction. Bock, as a result of his study on this question, con- 
cluded that the trichloroacetic acid procedure gives satisfactory results, 
1Van Slyke, D. D., Proc. Soc. Exp. Biol. and Med., 1909-10, vii, 45; 
J: Biol. Chem., 1911, ix, 185, | 
2 Van Slyke, D. D., and Meyer, G. M., J. Biol. Chem., 1912, xii, 399. 
’ Folin, O., and Denis, W., J. Biol. Chem., 1912, xi, 527. 
* Greenwald, I., J. Biol. Chem., 1915, xxi, 61. 
‘ Bock, J. C., J. Biol. Chem., 1916-17, xxviii, 357. 
325 
