* Slightly modified.
* Slightly modified.
color, a variation made necessary by the use of sodium sulfite. The uric acid reagent must invariably be added after, and not before, the addition of the sodium carbonate, because in acid solution the sulfite will itself give a blue color with the phosphotung-stic acid.
It may also be worth while to mention that the peculiar increase in blue color obtained by the use of cyanide is not obtained in the presence of sulfite. Opinions will doubtless differ as to whether this is an advantage or disadvantage. The amount of color obtained from 2 cc. of blood is rather weak, and if we could conveniently have retained the intensifying effects of the cyanide we probably should have done so, though the fainter solutions can be read just as readily and accurately as the stronger ones obtained by means of the cyanide. The antifading effects of the cyanide
It was originally our intention to incorporate some adaptation of Benedict's picrate method for the determination of sugar to our tungstic acid blood filtrates. But a few exploratory experiments showed that an intense and stable color reaction can be obtained by the application of the phenol reagent of Folin and Denis to cuprous oxide. The color obtained from a given quantity of sugar is far more intense than that obtained by the alkaline picrate reaction; so that a small fraction of a mg. of dextrose (1 or 2 cc. of blood filtrate) is all that is required for a determination of the blood sugar. Some difficulties were encountered in trying to find the conditions under which the extent of reduction is strictly proportionate to the quantities of sugar used; but, by a systematic study of the various factors involved, these difficulties were overcome and a rapid and convenient process was obtained.
The copper solution used for reduction is a weakly alkaline copper tartrate solution. Qualitatively this solution is an extremely sensitive reagent for traces of sugar, yet is not affected by creatinine or uric acid in quantities corresponding to 50 mg. of each per 100 cc. of blood. We are therefore inclined to regard our method as more accurate than any method as yet proposed for the deter-
The picrate methods,7 whether we use Benedict's last modification or Myers' modification of Benedict's original method, in our hands give almost invariably results that are materially higher than the figures given by our new method. We are under the impression that the picrate methods are subject to sources of error similar to those encountered in Folin's original picrate method for blood creatine. The development of color in blood filtrates seems
7 Lewis, R. C., and Benedict, S. R., A Method for the estimation of sugar in small quantities of blood, J. Biol. Chem1915, xx, 61. Myers, V. C., and Bailey, C. V., The Lewis and Benedict method for the estimation of blood sugar, with some observations obtained in disease, ibid., 1916, xxiv, 147. Benedict, S. R., A modification of the Lewis-Benedict method for the determination of sugar in the blood, ibid., 1918, xxxiv, 203.
not to proceed at the same rate of speed as the color derived from a corresponding amount of dextrose. If the heating is interrupted at the end of 2 to 3 minutes the value obtained for the blood sugar will be nearly 50 per cent higher than when the heating is continued for 10 minutes or more. Such quantitative variations are not encountered in our process when equal amounts of dextrose in the form of pure sugar and of blood filtrate are heated, except that the reduced copper is, of course, more extensively precipitated and visible in the pure sugar solution. It need scarcely be stated that added sugar is quantitatively recovered by our method.
Solutions Needed for Determination of Sugar in Blood.
1. Standard Sugar Solution.—Dissol e 1 gm. of pure anhydrous dextrose in water and dilute to a volume of 100 cc. Mix, add a few drops of xylene or toluene, and bottle. If pure dextrose is not available, a standard solution of invert sugar made from cane sugar is equally useful. Transfer exactly 1 gm. of cane sugar to a 100 cc. volumetric flask; add 20 cc. of normal hydrochloric acid and let the mixture stand over night at room temperature (or rotate the flask and contents continuously for 10 minutes in a water bath kept at 70°C.). Add 1.68 gm. of sodium bicarbonate and about 0.2 gm. of sodium acetate, to neutralize the hydrochloric acid. Shake a few minutes to remove most of the carbonic acid and fill to the 100 cc. mark with water. Then add 5 cc. more of water (1 gm. of cane sugar yields 1.05 gm. of invert sugar) and mix. Transfer to a bottle; add a few drops of xylene or toluene, shake well, and stopper tightly. The stock solution made in either way keeps indefinitely. Dilute 5 cc. to 500 cc., giving a solution 10 cc. of which contain 1 mg. of dextrose or invert sugar. Add some xylene.
2. Alkaline Copper Solution.—Dissolve 40 gm. of anhydrous sodium carbonate in about 400 cc. 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 1 liter. If the carbonate used is impure, a sediment may be formed in the course of a week or so. If this happens, decant the clear solution into another bottle.
3. Phosphotungstic-phosphomolybdic Acid.—Transfer to a large flask 25 gm. of molybdenum trioxide (Mo03) or 34 gm. of ammonium molybdate (NH4MM0O4); add 140 cc. of 10 per cent sodium hydroxide and about 150 cc. of water. Boil for 20 minutes to drive off the ammonia (molybdic acid sometimes contains large amounts of ammonia as impurity). Add to the solution 100 gm. of sodium tungstate, 50 cc. of 85 per cent phosphoric acid, and 100 cc. of concentrated hydrochloric acid. Dilute to a volume of 700 to 800 cc.; close the mouth of the flask with a funnel and watch-glass. Boil gently for not less than 4 hours, adding hot water from time to time to replace that lost during the boiling. Cool and dilute to 1 liter. This solution is identical with the phenol reagent of Folin and Denis. For use in connection with the determination of blood sugar dilute 1 volume (100 cc.) of the reagent with one-half volume (50 cc.) of water and one-half volume (50 cc.) of concentrated hydrochloric acid.
The determination of blood sugar is carried out as follows: Heat a beaker of water to vigorous boiling. Transfer 2 cc. of the tung-stic acid blood filtrate to a test-tube (20 m. X 200 mm.) graduated at 25 cc. The graduated test-tubes used as receivers when distilling off the ammonia in urea determinations (p. 95) are suitable for this work. Transfer 2 cc. of the dilute standard sugar solution to another similar test-tube. Add to each tube 2 cc. of the alkaline copper tartrate solution. Heat in the boiling water for 6 minutes. Remove the test-tube and add at once (without cooling), preferably from a graduated pipette, 1 cc, of the strongly acidified and diluted phenol reagent. This should be done as nearly simultaneously as possible; it is not advisable to use one standard for a set of more than four determinations. The purpose of the added hydrochloric acid in the reagent is to dissolve the cuprous oxide. Mix, cool, and add 5 cc. of saturated sodium carbonate solution. An intense blue color is gradually developed which will remain unaltered for several days. Dilute the contents of both test-tubes to the 25 cc. mark, and after at least 5 minutes make the color comparison in the usual manner.
The depth of the standard (in mm.) multiplied by 100 and divided by the reading of the unknown gives the sugar content, in
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