URINE 525 



second stage of the control. The formaldehyde mixture is now added ; 10 c.c. 

 for each 20 c.c. of the solution, and the mixture again titrated to the second 

 stage with N/5 alkali. 1 



Two drops of the N/5 alkali are now added to the control solution which 

 assumes a deep red color (third stage). Fifth normal alkali is now added to 

 the solution under examination until it assumes a color corresponding to the 

 third stage of the control. This completes the titration. 



Calculation. The calculations are similar to those which pertain to any 

 acidimetry procedure. Each cubic centimeter of an N/5 alkali or acid solution 

 is equivalent to 0.0028 gram of nitrogen. An example will illustrate the pro- 

 cedure: 40 c.c. of solution (16 c.c. of urine) required 5.10 c.c. N/5 NaOH; con- 

 trol, o.io c.c. N/5 NaOH; total required for amino-acids 5.00 c.c. equivalent to 

 0.014 gram of nitrogen. Ammonia nitrogen in 16 c.c. of urine 0.007 gram N. 

 Then 0.0140.007 = 0.007 gram ammo-acid nitrogen in 16 c.c. of urine. 



Interpretation. The excretion of total amino-acid nitrogen by a 

 normal adult averages between 0.4 to i.o gram per day or from 2 to 6 

 per cent of the total nitrogen. Free amines-acid nitrogen (see Van 

 Slyke procedure) " is considerably less than this, ordinarily 0.5 to i.o 

 per cent of the total nitrogen. The amount may be largely increased 

 in disorders associated with tissue waste as typhoid, in pronounced 

 atrophy of the liver, acidosis, etc. 



2. Benedict-Murlin Modification. 2 Principle. In this method the ammonia is 

 removed by means of phosphotungstic acid, and excess acid as well as carbonates 

 and phosphates carried down with barium. 



Procedure. Measure into a 500 c.c. Erlenmeyer flask 200 c.c. of a 24-hour urine 

 which has been diluted to 2000 c.c. (or its equivalent). Add an equal volume 

 of 10 per cent phosphotungstic acid (Merck) 3 in 2 per cent HC1. Let stand at least 

 three hours, better over night. Pour off 250 c.c. of the clear fluid, add i c.c. of a 

 0.5 per cent solution of phenolphthalein and then barium hydroxide in substance 

 until the whole fluid turns decidedly pink. The barium hydroxide should be added 

 a very little at a time. Let stand one hour. Filter off two 100 c.c. samples ( = 50 

 c.c. urine). Neutralize these samples to litmus (using good quality litmus paper) 

 with N/5 HC1. Add at once 10-20 c.c. of neutral formalin 4 and titrate cautiously 

 to a deep red color, i.e., until the drop produces no additional color with N/io 

 NaOH. Deduct from the result thus obtained the amount of N/io NaOH neces- 

 sary to produce the same depth of color in an equal quantity of water, freed from 

 carbon dioxide by boiling and cooling, and to which an equal volume of neutral 

 formalin has been added. 



Calculation. One c.c. of N/io NaOH is equivalent to 1.4 nag. of amino-acid 

 nitrogen. Multiply the number of cubic centimeters of N/io NaOH used (after 

 deducting for control as indicated above) by 1.4 and by 2 (as the equivalent of 50 



1 This is best accomplished by adding alkali until the color is -deeper than that of the 

 control, then acid again until lighter and finally alkali to the desired color. 



2 Benedict and Murlin: Jour. Biol. Chem., 16, 385, 1913. 



3 Kahlbaum's preparation is a very different substance. 



4 To 50 c.c. commercial formalin solution (30-40 per cent) add i c.c. of phenolphthalein 

 solution and then N/5 NaOH to a very faint pink color. The solution should be freshly 

 prepared. 



