CH. X.] BLOOD SUGAR. 251 



NOTES. i. It is instructive to perform similar experiments on blood 

 which has been incubated at 37 C. for 24 hours, toluol being added as an 

 antiseptic. It will be found that the sugar has disappeared, the " glycolysis " 

 being due to the action of special oxidising enzymes. 



2. For the action of colloidal iron in precipitating proteins see page 10. 



311. The estimation of sugar in blood (Benedict's method).* 



Principle. The blood is laked, treated with picric, acid and 

 filtered. An aliquot part of the protein-free filtrate is heat eg! with 

 alkali. The glucose reduces some of the picric acid to picramic acid 

 (Ex. 1 08), the amount of which is estimated in a colorimeter against 

 a suitable standard. 



Solutions and Apparatus required: 



1. Picric-picrate mixture. To 125 cc. of N. soda in a i litre measuring 

 flask add about 700 cc. of hot distilled water, and then 36 grams, of pure, dry 

 picric acid. Wash in with a little water, shake at intervals till dissolved, cool 

 thoroughly, make up to i litre with distilled water, mix and filter if the solution 

 is not crystal clear. 



It is important to use pure dry picric acid. It is advisable to recrystallise 

 the picric acid of commerce from boiling water, filtering the hot saturated 

 solution through a hot water funnel. The cold solution is filtered through 

 a Buchner funnel and the crystals spread out on layers of filter paper in a 

 warm room till quite dry. The mother liquors can be used for the preparation 

 of the stock solution of glucose mentioned below. It must be remembered 

 that picric acid is an explosive, and that it is not safe to grind it in a mortar, 

 or to send it through the post without damping it. 



2. Standard solution of glucose. The stock solution required is a i per 

 cent, solution in saturated picric acid. This can be prepared by dissolving 

 i gram, of pure glucose (previously dried in a vacuum desiccator) in cold 

 saturated picric acid and making the volume up to 100 cc. with the same 

 solution. Or a strong (10 per cent.) solution can be estimated accurately 

 by means of a polarimeter and a volume that contains exactly i gram, diluted 

 to make 100 cc. with the saturated picric acid. 



From this stock (which keeps for a considerable time) is prepared daily a 

 solution which contains 0-64 mg. in i cc., by measuring 3-2 cc. into a 50 cc. 

 flask and making up to the mark with distilled water. 



An alternative standard is prepared from pure picramic acid, should 

 this be available.f The stock solution contains 100 mg. of picramic acid and 

 200 mg. of anhydrous sodium carbonate per litre. 126 cc. of this solution are 

 treated with i cc. of 20 per cent, sodium carbonate solution and 15 cc. of the 

 picric-picrate mixture and diluted to 300 cc. with distilled water. This 

 solution exactly matches the colour from 0-64 mg. of glucose when treated 

 in the way described below and diluted to make 12-5 cc. The picramic acid 

 standard is not heated with the blood sugar. 



* Journ. of Biological Chemistry, xxxiv., p. 203 (1918). 



t For a method of preparation see Egerer, Journ. of Biological 

 Chemistry, xxxv., p. 565. 



