APPENDIX 



455 



Nonprotein Nitrogen of Blood. Normally we have from 25 to 35 mg. of non- 

 protein N in 100 c.c. of blood. This is greatly increased in chronic nephritis and 

 especially in uraemia. The nonprotein N is more easily and accurately determined 

 than the urea N. Of course, the urea N makes up about one-half of the normal 

 nonprotein N and in uraemic conditions it may constitute from 80 to 90% of the 

 nonprotein N. Hawk gives urea N as 12 to 23 mg. per 100 c.c. of blood. In 

 uraemia it may amount to 70 to 300 mg. with a nonprotein N increase of from 90 

 to 350 mg. per 100 c.c. of blood. 



To Estimate Nonprotein N. Take 5 c.c. blood in a graduated centrifuge tube 

 with powdered potassium oxalate as for blood sugar. Be sure not to use ammonium 

 oxalate. Now gradually add, with constant stirring, 75 c.c. of absolute alcohol, 

 mix thoroughly, filter through dry filter, wash precipitate with several small portions 

 of alcohol, evaporate filtrate and washings to dryness on water-bath, using a small 

 porcelain dish for the purpose, and with smallest possible quantity of water take up 

 and transfer residue to a small flask. Add i to 2 grams potassium sulphate and 2 

 c.c. of concentrated sulphuric acid, place over a small flame and heat until all water 

 is driven off and the contents of flask, after having become thoroughly charred, 

 have again become a pale yellow or colorless. Cool, add about 20 c.c. of water, 

 again cool, add 4 or 5 drops of phenolphthalein, gradually add 10% solution of sod- 

 ium hydrate, keeping contents of flask cool by frequent immersion in cold water, 

 until exact point of neutrality is reached. Now add 5 grams of neutral potassium 

 oxalate, stir until dissolved and then add about 10 c.c. of neutral 20% formaldehyde 

 (50% formalin) and then from burette add N/50 NaOH until permanent pink 

 color is obtained. 



The number of cubic centimeters N/50 NaOH required times 0.28 equals number 

 milligrams nonprotein nitrogen in 5 c.c. of blood. 



Urea N of Blood. The following method suggested by Dunning has been 

 modified in our laboratory. For urea estimation take 5 c.c. blood as for nonprotein 

 N and dilute with 25 c.c. distilled water and add 30 grams of crystallized sodium 

 sulphate or 13 grams of anhydrous sodium sulphate. The mixture is then warmed 

 to 35C. when the sodium sulphate is dissolved and the blood proteins precipitated. 

 The mixture is then made up to 50 c.c. with a saturated solution of sodium sulphate, 

 thoroughly shaken and allowed to stand a few minutes. It is then filtered and 

 25 c.c. of the filtrate used for the test and 10 c.c. for a control. Each ic.c.c. repre- 

 sents i c.c. of the oxalated blood. 



To 25 c.c. of the nitrate add distilled water to 100 c.c. and one finely powdered 

 Urease-Dunning tablet and digest for i hour at 50 C. or over night at room tem- 

 perature. 



After digestion filter through double dry filter and to 40 c.c. of this nitrate, which 

 represents i c.c. of the blood, add 2 drops of methyl orange. 



Now add 2 drops of methyl orange to the control and titrate with N/2O HC1 

 to a faint pink color. Then titrate the urease-treated solution to exactly the same 

 shade of pink as the control. 



The amount of N/20 HC1 required to neutralize the urease-treated solution, less 

 the amount required for the control, multiplied by 0.0015 will give the urea in i c.c. 

 of the blood: or multiplied by 0.0007 will give the urea nitrogen in the i c.c. of blood. 



The end reaction is not at all sharp and requires considerable experience to deter- 



