520 PHYSIOLOGICAL CHEMISTRY 



substances. The benzole acid is extracted with chloroform and ti- 

 trated with sodium ethylate. 



Procedure. Measure 100 c.c. of urine into a porcelain evaporating dish by 

 means of a pipette. Add 10 c.c. of 5 per cent NaOH and evaporate to dryness 

 on the steam-bath. Transfer the residue to a 500 c.c. Kjeldahl flask by means of 

 25 c.c. of water and 25 c.c. of concentrated HNO 3 . Add 0.2 gram of copper 

 nitrate, a couple of pebbles or glass pearls and boil very gently for four and one- 

 half hours over a micro-burner. Fit the necks of the flasks with condensers of 

 the Hopkins type made from large test-tubes fitted with two-hole rubber stoppers, 

 the inlet tubes extending near the bottom of the test-tubes while the outlet tube 

 is shorter. These condensers should fit rather loosely. A good current of water 

 flowing through the condensers prevents loss of benzoic acid or change in con- 

 centration of the nitric acid. 



After cooling, rinse the condensers down with 25 c.c. of water and transfer the 

 contents of the flask to a 500 c.c. separatory funnel, with the aid of 25 c.c. more 

 of water. The total volume of the solution is now 100 c.c. Add to the solution 

 sufficient ammonium sulphate to just saturate it (about 55 grams). Make four 

 extractions with freely washed chloroform, using 50, 35, 25, and 25 c.c. portions. 

 The first two portions may be used to further rinse out the Kjeldahl flask. 



Collect the successive portions of chloroform in another separatory funnel. 

 Add to the combined extracts 100 c.c. of a saturated solution of pure sodium chlo- 

 ride, to each liter of which has been added 0.5 c.c. of concentrated HC1. Shake 

 well, draw the chloroform into a dry 500 c.c. Erlenmeyer flask and titrate with N/io 

 sodium alcoholate, 1 using 4 or 5 drops of phenolphthalein as an indicator. The 

 first distinct end point should be taken, although it may fade on standing a short 

 time. 



Calculation. Multiply the number of cubic centimeters of alcoholate used by 

 the factor for hippuric acid as determined by standardization to obtain the 

 amount of hippuric acid hi the 100 c.c. of urine used. One c.c. of exactly N/io 

 sodium alcoholate is equivalent to 0.0179 gram of hippuric acid. Calculate the 

 daily output of hippuric acid from the 24-hour volume. 



Interpretation. The average excretion of hippuric acid by a normal 

 adult man is about 0.7 gram per day. The amount is increased by 

 the ingestion of benzoic acid or fruits containing it (plums, prunes, 

 cranberries). It arises in part apparently from putrefaction products 

 formed in the intestine. In herbivora it is often the most abundant 

 nitrogenous constituent of the urine. 



2. Dakin's Methods. 2 Preliminary Procedure. Place 150 c.c. (or more) of the 

 urine under examination in a porcelain evaporating dish and evaporate almost to 

 dryness upon a water-bath. Add about i gram of sodium dihydrogen phosphate, 



1 The sodium alcoholate is made by dissolving 2.3 grams of cleaned metallic sodium in i 

 liter of absolute alcohol. It is advisable that it be slightly weaker rather than stronger 

 than tenth-normal. It may be standardized against pure benzoic acid in washed chloro- 

 form. It may also be standardized against N/io HC1 provided the alcoholate solution 

 contains not more than traces of carbonate. 



2 Private communication to the author from Dr. H. D. Dakin. 



