ANALYSIS OF TISSUES 577 



C. NITROGENOUS EXTRACTIVES. 



The nitrogenous extractives existing in the tissues consist of amino acids, 

 urea, ammonia, creatine, creatinine, uric acid, etc. The amount of amino 

 acids can be determined by Van Slyke's method of estimating amino nitrogen, 

 the amount of the other constituents by Folin's micrt) methods (p. 556). 



I. Amino Acids (Amino Nitrogen). 



(a} Blood. 



30-50 c.c. of freshly drawn blood are mixed with 9-10 volumes of 

 95 per cent, alcohol in a graduated cylinder to precipitate the proteins, or 

 known volumes are mixed. The total volume must be known. The blood 

 and alcohol are thoroughly mixed and allowed to stand for 24 hours. The 

 solution is filtered through a dry pleated filter paper and collected in a measur- 

 ing cylinder. The volume of filtrate is noted and it is concentrated, preferably 

 in vacuo, to a volume of 3-5 c.c. The nitrogen in this volume is determined 

 by the action of nitrous acid as on p. 149. This volume may be diluted to 10 c.c. 

 and 9*5-9 '8 c.c. used, or it may be put directly into the burette together 

 with the washings. To prevent frothing a few drops of caprylic alcohol may 

 be added. 



The amount of nitrogen evolved from the ammonia present in the blood 

 is negligible, but a correction is required for that evolved from the urea ; it 

 amounts to about 3 per cent. This correction is determined by shaking for a 

 period of 4 minutes at below 20, for 3-5 minutes at 20-25, or f r 2 '3 minutes 

 above 25, measuring the gas and again shaking for a similar period and 

 measuring the gas. The amino acids react rapidly, urea reacts slowly. Urea 

 only is decomposed in the second period of shaking ; as it reacts at a 

 uniform rate the amount of gas evolved in the second period is a measure of 

 that evolved in the first period. By deducting this amount the gas evolved 

 from amino acids only is ascertained. The correction may also be determined 

 by noting the amount of gas evolved per minute, if the first measurement is 

 not finished in 2-4 minutes, and calculating for the time of the reaction for 

 the amino acids. 



About 4 mgm. of amino nitrogen are found for 100 c.c. of blood. 



