Modification of Blood Sugar Method 255 



This fact led to a comparison of sugar determinations made on 

 pure glucose solutions with and without the addition of oxalate, 

 with and without the use of colloidal iron. The amount of oxa- 

 late required to prevent coagulation was about 2 mg. per c.c. 

 It was found that the presence of the oxalate resulted in too 

 low sugar values, i.e., too much thiosulphate was used in the 

 titration, only in case colloidal iron was used. Experiments in 

 which the amount of colloidal iron was progressively increased 

 from 0 to 3 c.c. showed that increasing amounts of thiosulphate 

 were required for titration. Direct determinations of the iron 

 in the filtrate indicated that the reaction involved was between 

 the thiosulphate and the ferric salts. It seemed, therefore, that 

 the choice of colloidal iron as a protein precipitant was an un- 

 fortunate one; although, under the carefully standardized con- 

 ditions described by MacLean, it led to no inaccuracy of results, 

 providing oxalate was not used as an anticoagulant. 



By using phosphotungstic acid as our protein precipitant we 

 were able to obtain results which agreed whether the blood 

 was oxalated or not, and which were comparable with those 

 obtained by the Folin method. 



The procedure now adopted for the precipitation of the blood 

 proteins is as follows : 



1 c.c. of oxalated blood is added to 26 c.c. of distilled water 

 in an Erlenmeyer flask or 50 c.c. centrifuge tube. Five minutes 

 are allowed for laking the blood. 2 c.c. of a 10 per cent, solu- 

 tion of phosphotungstic acid and 1 c.c. of 1 per cent, acetic acid 

 are added. The flask or tube is then vigorously shaken. In 

 the case of pigeon's blood, where precipitation is difficult, it is 

 necessary to gently heat the mixture after the addition of phos- 

 photungstic acid to complete the action. The protein precipi- 

 tate may then be separated by filtration or preferably centri- 

 fugation. 20 c.c. of the water-clear filtrate are transferred to 

 an Erlenmeyer flask, and the determination continued as rec- 

 ommended by MacLean. 



The elimination of sodium sulphate from the solution re- 

 sults in the reduction taking place at a lower temperature. 

 This necessitates the construction of a new table to take the 

 place of the one published by MacLean for the conversion of 

 c.c. of thiosulphate into mgs. of glucose per 100 c.c. of blood. 

 These equivalents are given in Table II. 



