Excretion of Creatine in Carbohydrate Starvation. 213 



cooled. The mixture in the boiling tube is neutralised with 1*5 c.c. of 

 10-per-cent. soda* and then 15 c.c. of saturated picric acid and 5 c.c. of the 

 soda are added. The mixture is allowed to stand seven minutes and then 

 the contents of the boiling tube are washed into a 500 c.c. flask and diluted 

 up to 500 c.c. with water. By heating for three-quarters of an hour, the aceto- 

 acetic acid can be completely removed even if it is present in a concentration 

 of 02 per cent. 



If the concentration of aceto-acetic acid is greater than - 2 per cent., the 

 distillation must be continued for a longer tune, and the complete removal of 

 the aceto-acetic acid must be ascertained by testing a control with Eothera's 

 nitroprusside test. We have continued the distillation for one and a half 

 hours, and find that no error in the creatine estimation occurred. 



We have tested the method in the following manner. The amount of 

 creatinine in a normal urine was determined, and aceto-acetic acid was then 

 added in varying amounts to the urine, and the creatinine again estimated. 

 The aceto-acetic acid was then removed by the distillation method, and the 

 creatinine again estimated. The results obtained show that the distillation 

 did not break up any of the creatinine, and that the aceto-acetic acid was 

 completely removed (Table IV). 



Table IV shows that the Error caused by Varying Concentrations of Aceto- 

 acetic Acid in the Urine is completely removed by the Distillation 

 Method. 



Amount of aceto- 

 acetic acid added to 

 urine. 



Creatinine by Folin's method. 



Creatinine after 

 removal of the aceto- 

 acetic acid by the 

 distillation method. 



Urine alone. 



Urine + aceto-acetic 

 acid. 



grm. per 100 c.c. 

 O '088 approx. 

 -044 

 0-18 

 0-27 



grm. per 100 c.c. 

 0-145 

 0-108 

 0-096 

 0-096 



grm. per 100 c.c. 

 0-128 

 -097 

 Not estimated 

 053 



grm. per 100 c.c. 

 0-145 

 0-105 

 0-096 

 0-094 



One of the most important questions to decide was whether creatine was 

 converted into creatinine in this process of distillation. 



Pure crystalline creatinef was added to normal urine in varying amounts, 



* The phosphoric acid must be adjusted against the 10-per-cent. caustic soda, and the 

 correct amount of caustic soda necessary to neutralise 1 c.c. phosphoric acid must be 

 added. 



t We wish to thank Dr. F. G. Hopkins and Mr. Mackenzie Wallis for kindly 

 supplying us with the pure creatine. 



