272 PHYSIOLOGICAL CHEMISTRY 



alcohol solution (after the removal of the proteins) by means of oxi- 

 dation and Nesslerization. The details of the procedure are carried 

 out in the following manner: 



Method of Drawing Blood. Attach, by means of a short piece of pure gum 

 tubing, an hypodermic needle about i mm. in diameter and 25 mm. in length 

 (previously sterilized and paraffined) to the tip of a 2 or 5 c.c. pipette. Introduce 

 into the upper end of the pipette (which must be perfectly clean and dry) a small 

 pinch of powdered potassium oxalate, and allow it to run down into the tip and the 

 needle. Attach a piece of rubber tubing to the upper end of the pipette, and to this 

 a mouthpiece consisting of a short tapering glass tube. Place a pinchcock over 

 the rubber tube near the top of the pipette. To draw the blood, insert the needle 

 into the vein or artery and regulate the flow by means of the pinchcock and suc- 

 tion. The exact quantity of blood desired is thus obtained without any waste or 

 clotting. 



Method of Isolating Non-protein Nitrogen Constituents. Methyl alcohol and 

 zinc chloride are employed as precipitants for the protein materials of the blood, 

 and the determination of the non-protein nitrogen is then carried out upon a 

 portion of the methyl alcohol extract. The procedure is as follows : Transfer 

 the blood, as soon as drawn, to a measuring flask which is half filled with pure 

 methyl alcohol (must be acetone free). Fill to the mark with methyl alcohol 

 and shake thoroughly. (If 2 c.c. of blood are taken, 25 c.c. flasks are used for the 

 precipitation, while for 5 c.c. of blood 50 c.c. flasks are used.) Allow the flask 

 to stand for at least two hours and at the end of that time, or later, filter the con- 

 tents through dry filter paper. Add 2-3 drops of a saturated alcoholic solution of 

 zinc chloride to the filtrate and filter again through a dry filter paper after a few 

 minutes. The zinc chloride brings down an appreciable precipitate and the last 

 traces of coloring matter, so that the second filtrate obtained is perfectly colorless 

 and clear. This filtrate is used for the determination of non-protein nitrogen. 



Trichlor acetic Acid Modification. Green wald 1 has suggested the 

 use of trichloracetic acid as the precipitant for the proteins of the blood, 

 as being more satisfactory than the methyl alcohol and zinc chloride. 

 The objection to the methyl alcohol is that some of the amino-acids 

 (creatine, asparagine, and tyrosine) are insoluble in it and hence pre- 

 cipitated along with the proteins. These acids are not removed by the 

 trichloracetic acid. Certain nitrogenous lipoid substances are precipi- 

 tated by the trichloracetic acid and not by the methyl alcohol. Green- 

 wald suggests that these substances, even though non-protein in char- 

 acter, should not be included with the non-protein nitrogen of the 

 amino-acids and urea. 



Procedure. Dilute the blood to ten times its original volume with 2.5 per 

 cent trichloracetic acid solution. Let stand 30 minutes and then filter. Shake 

 the filtrate with about 4 grams of kaolin per 100 c.c. and filter again. An aliquot 

 of this final filtrate is taken, digested with sulphuric acid and nitrogen deter- 

 mined in the usual way. 



1 Greenwald: /. Biol. Chem., 21, 61, 1915. 



