540 PHYSIOLOGICAL CHEMISTRY 



With this method the urine should be diluted when the sugar content ex- 

 ceeds 2.5 per cent. Under 0.5 per cent of sugar the figures are likewise inac- 

 curate, and here Benedict's colorimetric method of estimating the sugar content 

 of normal urine should be used. 



Interpretation. See Benedict's Method above. 



3. Folin-McEllroy-Peck Method. 1 Principle. The method is a 

 titration procedure depending upon the use of an alkaline copper 

 solution in which the cupric hydroxide is held in solution by means of 

 phosphate instead of the customary tartrates, citrates, or glycerol. 



The method is applicable to the determination of lactose in milk. 



Procedure. Place 5 c.c. of an acidified 5.9 per cent copper sulphate solution 2 

 in a large, hard glass test tube and add approximately i c.c. of 20 per cent sodium 

 carbonate solution. Shake for a moment and add 4 to 5 gms. of phosphate - 

 carbonate -thiocyanate mixture 3 and a small pebble. Heat gently with shaking 

 until all the salts have dissolved except for a few isolated particles of sodium 

 carbonate. A clear solution is usually obtained in less than i minute at tem- 

 peratures which need not exceed 6oC. From a burette 4 add undiluted urine 

 (0.4 c.c. to i.o c.c.) heat fairly rapidly to boiling and boil very gently for 2 minutes. 

 With the full required amount of sugar present at the beginning (the 5 c.c. of 

 copper solution are reduced by 25 mg. of glucose), the boiling solution becomes 

 suddenly turbid within 5 seconds after the boiling point has been reached. If, 

 within the first 15 seconds of boiling, the contents of the test tube do not thus 

 suddenly become filled with the cuprous thiocyanate precipitate, then less than 

 half the required amount of sugar has been added and more urine should be 

 added without further delay and the gentle boiling be continued. On the other 

 hand, when an excess of sugar has been inadvertantly added at the beginning of 

 the process it is advisable to note the time required for complete decolorization 

 of the copper, for this time (see table) can serve as a guide to the quantity of 

 solution to be introduced at the next titration. 



The only restriction called for hi the final titration is that complete reduction 

 must not occur in less than 4 minutes of boiling. It makes practically no diff er- 



1 Folin and McEllroy: Jour. Biol. Chem., 33 513, 1918; Folin and Peck: Jour. Biol. 

 Chem., 38, 287, 1919. 



2 Prepare this acidified copper sulphate solution by dissolving 59 gm. of CuSO4-i2H2O in 

 water together with 2 c.c. of concentrated sulphuric acid and making up to i liter. Five 

 c.c. of this solution correspond to 25 mg. of glucose or fructose, 45 mg. of anhydrous 

 maltose, or 40.4 mg. of anhydrous lactose. 



3 To prepare the phosphate-carbonate-thiocyanate mixture powder in a large mortar 

 200 gm. of crystallized disodium phosphate (HNa2PO 4 -i2H2O) and sprinkle over it about 

 50 gm. of sodium thiocyanate (or 60 gm. of potassium thiocyanate). Mix for 10 minutes 

 with pestle and spoon, giving a uniform semi-liquid paste. Add about 120 gm. of mono- 

 hydrated sodium carbonate (or 100 to no-gm. of anhydrous carbonate) and mix with pestle 

 and spoon until a rather fluffy, granular powder is obtained. To test the completeness 

 of the mixing add 5 gm. of the powder to 5 c.c. of the copper solution; if any black specks 

 are formed, even temporarily, the mixing is incomplete. A certain amount of green color 

 is, however, practically unavoidable when this test is applied. If no black coloration is 

 obtained allow the mixture to stand in the mortar for a few hours or over night (covered 

 with paper) mix once more and transfer to bottles. In stoppered bottles the mixture keeps 

 indefinitely. 



4 Special 5 c.c. sugar burettes graduated in 0.02 c.c. together with accessory capillary 

 tips for delivering very small drops are made by the Emil Greiner Company, New York. 



