242 CUVETTE COLORIMETRY 



Borsook Method for Creatine and Creatinine 



SPECIAL REAGENTS 



0.1 N Hydrochloric Add. 



0.01 N Hydrochloric Acid. 



Lloyd Reagent {Eli Lilly and Co.). 



Alkaline Picrate Solution. Combine 10 parts saturated picric acid 



and 1 part 10% sodium hydroxide. 

 Creatinine- Zinc Chloride Standard. Dissolve 1.61 g. creatinine-zinc 



chloride (Benedict, 1914) in N/14: hydrochloric acid and make 



up to 1 1. This soln. has 1 mg. creatinine/ml. Make up fresh at 



least once a month. 



PROCEDURE 



1. Convert creatine to creatinine: Place 1-5 ml. of sample in a 

 Pyrex test tube ( 125 X 9 mm. inside) provided with a ground-glass 

 stopper, add one fourth the vol. of 0.1 A?" hydrochloric acid and mix 

 by inversion. Insert a piece of thread into the neck of the tube and 

 stopper. Then autoclave for 20 min. at 30 lb. (130°). Omit this step 

 for preformed creatinine. 



2. After cooling, add 30-40 mg. Lloyd reagent and, with thread 

 removed, stopper tightly and shake continuously for 10 min. 



3. Centrifuge, pour off supernatant, and remove the last drop 

 from the lip of the tube with filter paper. 



4. Resuspend the precipitate in 1 ml. 0.01 A^ hydrochloric acid 

 and use another 1 ml. portion of the acid to wash down the stopper 

 and the sides of the tube. 



5. Again centrifuge and discard the supernatant, removing the 

 final drop from the lip with filter paper. 



6. Add 3 ml. sodium picrate soln. to remove the creatine from 

 the Lloyd reagent, and shake gently for 10 min. to develop the color 

 fully. 



7. Centrifuge, and measure the color of the liquid spectrophoto- 

 metrically at 525 m/x. A linear relationship exists between the 

 absorption and the creatinine concentration from to 2.0 milligram 

 per cent. 



8. Adsorb standards on Lloyd reagent and treat as above from 

 step 2 on. 



