318 PRACTICAL PHYSIOLOGY 



to each of the standards, mix well, let stand ten minutes, fill to the mark 

 with water, mix again and then do colour comparison. 



Calculation. If the weaker standard, for example, be set at 20 mm., 

 then 20 multiplied by 2-5 divided by the reading of the unknown gives 

 mg. uric acid in 100 c.c. blood. Note the blood nitrate taken corre- 

 sponds to 2 c.c. blood and the standard is diluted to twice the volume 

 of the unknown. 



Determination of Sugar in Blood (Folin and Wu). Reagents. 1. To 

 200 c.c. 10 per cent. NaOH and 200 c.c. water add 35 gms. molybdic acid 

 and 5 gms. sodium tungstate. Boil vigorously for twenty to forty 

 minutes so as to remove nearly all the ammonia present in the molybdic 

 acid. Cool, dilute to about 350 c.c. and add 125 c.c. 85 per cent, 

 phosphoric acid. Dilute to 500 c.c. 2. Alkaline copper solution. 

 Dissolve 40 gms. pure anhydrous sodium carbonate in about 400 c.c. of 

 water and transfer to a litre flask. Add 7-5 gms. of tartaric acid and 

 when this has dissolved add 4-5 gms. crystallised copper sulphate. Mix 

 and make up to a volume of 1 litre. (In the event of a precipitate 

 forming filter this off. Reagent should keep indefinitely. ) 3. Standard 

 sugar solutions. Stock solution of 1 per cent, pure dextrose preserved 

 with xylol or toluol. Keeps indefinitely. Prepare from this two 

 solutions, (a) containing 1 mg. dextrose per 10 c.c. (5 c.c. stock diluted 

 to 500 c.c.) and (6) containing 2 mg. dextrose per 10 c.c. (5 c.c. of stock 

 diluted to 250 c.c. ). Preserved with xylol or toluol these solutions keep 

 for about one month. 



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

 test tube (see later), and to two other similar test tubes (all graduated 

 at 25 c.c.), add 2 c.c. of standard sugar solution containing respectively 

 0-2 and 0-4 mg. of dextrose. To each tube add 2 c.c. of the alkaline 

 copper solution. Transfer the tubes to a boiling waterbath and heat for 

 six minutes. Then transfer them to a cold waterbath and let them 

 cool, without shaking, for two to three minutes. Add to each test tube 

 2 c.c. of the molybdate phosphate solution. The cuprous oxide dissolves 

 rather slowly if the amount is large, but the whole, up to the amount 

 given by 0-8 mg. of dextrose, dissolves usually within two minutes. 

 When the cuprous oxide is dissolved, dilute the resulting blue solutions 

 to the 25 c.c. mark, insert a rubber stopper, and mix. Mixing must be 

 thoroughly carried out. Make the usual colour comparison in the 

 colorimeter. The depth of the standard (a) (in mm. ) multiplied by 100 

 and divided by the reading of the unknown gives the sugar content in 

 mg. per 100 c.c. blood. Necessary correction must be used for standard 

 (6) which is double the strength of (a). 



Folin recommends (Jour. Biol. Chem., 41, 1920, 367) that,'in order to 

 prevent reoxidation of the copper, test tubes (200 x 25 mm.) be used 

 in which the bottom part is drawn out to a neck about 4 cm. long and of 

 8 mm. diameter, leaving a bulb at the bottom of approximately 4 c.c. 

 capacity in order to contain almost exactly the blood filtrate and copper 

 solutions. These tubes can be readily made in the laboratory or may 

 be purchased ready for use. The mixture should just fill the bulb. 

 If the bulb is just too large the volume of fluid may be increased by the 

 addition of diluted alkaline copper solution (1 : 1), but not more than 

 0-5 c.c. in all may be added (Fig. 202). 



If only small amounts of blood are available estimation of dextrose 

 by Maclean's method (Biochem. J., 1919, 13, 135) gives very good results. 



Determination of Cholesterol (Myers). 1 c,c, of blood is pipetted 



