284 PHYSIOLOGICAL CHEMISTRY 



molybdate phosphate solution, a blue color being obtained which is 

 compared with that of a standard. 



Procedure. Transfer 2 c.c. of the tungstic acid blood filtrate to a blood 

 sugar test tube of the type illustrated in Fig. 89 1 and to two other similar test 

 tubes (graduated at 25 c.c.) add 2 c.c. of standard sugar solution containing 

 respectively 0.2 and 0.4 mg. of glucose. 2 To each tube add 2 c.c. of the alkaline 

 copper solution. 3 



The surface of the mixtures must now have reached the constricted part of 

 the tube. If the bulb of the tube is too large for the volume (4 c.c.) a little, but 

 not more than 0.5 c.c. of a diluted (i :i) alkaline copper solution may be added. 

 If this does not suffice to bring the contents to the narrow part, the tube should 

 be discarded. Test tubes having so small a capacity that 4 c.c. fills them above 

 the neck should also be discarded. Transfer the tubes to a boiling water bath 

 and heat for 6 minutes. Then transfer them to a cold water bath and let cool 

 without shaking for 2 or 3 minutes. Add to each test tube 2 c.c. of the molyb- 

 date phosphate solution. 4 The cuprous oxide dissolves rather slowly if the 

 amount is large but the whole, up to the amount given by 0.8 mg. of glucose, dis- 

 solves usually within 2 minutes. When the cuprous oxide is dissolved, dilute 

 the resulting blue solutions to the 25 c.c. mark, insert a rubber stopper, and mix. 

 It is essential that adequate attention be given to this mixing because the greater 

 part of the blue color is formed in the bulb of the tube. Compare hi a colori- 

 meter using the standard which most nearly matches the unknown. 



The two standards given representing 0.2 and 0.4 mg. of glucose are adequate 

 for practically all cases. They cover the range from about 70 to nearly 400 mg. 

 of glucose per 100 c.c. of blood. )/ 



It will be noted that in the process described cooling of the alkaline cuprous 

 oxide suspension before adding the phosphate molybdate solution is suggested. 



1 These test tubes, with or without graduation, may be obtained from Emil Greiner, 

 New York. 



2 Standard Sugar Solutions. Three standard sugar solutions should be on hand: (i) 

 a stock solution, i per cent glucose or invert sugar, preserved with xylene or toluene; 

 (2) a solution containing i mg. of sugar per 10 c.c. ( 5 c.c. of the stock solution diluted 

 to '500 c.c.); (3) a solution containing 2 mg. of sugar per 10 c.c. ( 5 c.c. of the stock solution 

 diluted to 250 c.c.). The invert sugar solution has the advantage that it can be easily 

 prepared from cane sugar, which is pure. When good quality glucose is available, it is, 

 of course, the one to use. The diluted solutions should be preserved with a little added 

 toluene or xylene; it is probably better not to depend on such diluted solutions to keep for 

 more than a month, but the stock solution should keep indefinitely. 



3 Alkaline Copper Solution. Dissolve 40 gm. of pure anhydrous sodium carbonate 

 in about 400 c.c. of water and transfer to a liter flask. Add 7.5 gm. of tartaric acid, and 

 when the latter has dissolved add 4.5 gm. of crystallized copper sulfate. Mix and make 

 up to a volume of i liter. If the chemicals used are not pure a sediment of cuprous oxide 

 may form in the course of i or 2 weeks. If this should happen, remove the clear super- 

 natant reagent with a siphon, or filter through a good quality filter paper. The reagent 

 seems to keep indefinitely. To test for the absence of cuprous copper in the solution, 

 transfer 2 c.c. to a test tube and add 2 c.c. of the molybdate phosphate solution; the deep 

 blue color of the copper should almost completely vanish. In order to forestall improper 

 use of this reagent attention should be called to the fact that it contains extremely little 

 alkali, 2 c.c. by titration (using the fading of the blue copper tartrate color as indicator), 



aequ ;i .n v bout 1.4 c.c. of normal acid. 



4 Transfer -lo a liter beaker 35 gm. of molybdic acid and 5 gm. of sodium tungstate. 

 Add 200 c.c. of 10 per cent sodium hydroxide and 200 c.c. of water. Boil vigorously for 

 20 to 40 minutes so as to remove nearly the whole of the ammonia present in the molybdic 

 acid. (The molybdic acid which may be obtained from the Primos Company, Primos, 

 Pa., contains considerable ammonia.) Cool, dilute to about 350 c.c., and add 125 c.c. 

 of concentrated (85 per cent) phosphoric acid. Dilute to 500 c.c. 



