THE

(From

DETERMINATION
BY STANLEY
of Chemistry,

OF BLOOD
R. BENEDICT.
University

SUGAR.
Medical College, New

the Department

Cornell York city.)

(Received for publication,

March 26, 1925.)

There are at present available several methods for the determination of sugar in blood which yield satisfactory results for following the blood sugar in diabetes. These include the picric acid procedure in one or other of its various modifications and the copper methods of Bang, Shaffer and Hartman, and Folin and Wu. To many it may appear unjustifiable to include the picric acid methods here, but the writer ventures to assert that although many thousands of analyses have been made by the picric acid methods, it is doubtful whether a clinician has ever been misled in his interpretation of a diabetic case by the figures obtained for these analyses when properly carried out. Nevertheless, critical studies of the picric acid methods have shown that these consistently yield figures higher than the actual glucose content of the blood. This does not mean, however, that we should not regard the use of the picric acid methods as a legitimate routine procedure by the clinician. Indeed we have frequently noted a fact which indicates that the picrate method may be preferable to a copper method for following the course of the blood sugar in the diabetic. With diet, the blood sugar of the diabetic tends to return more promptly to the normal level as indicated by a copper method (Folin-Wu) than when the determination is made with the picrate method. This point is worthy of further study and we are accumulating data along this line as opportunity permits. We would point out in this connection that for bloods having a normal non-protein nitrogen content the picrate method yields figures practically identical with those given by the Folin-Wu procedure, if applied to a portion of the same tungstic acid j&ate after concentrating a portion of the filtrate by boiling so that the sugar
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Determination

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concentration is high enough to permit an accurate determination by the picrate procedure. Though Csonka and Taggart* report results somewhat at variance with this statement it must be noted that their figures show a much clozer agreement when both methods were applied to the tungstic acid filtrate, and that Csonka and Taggart apparently took no precautions to insure a proper concentration of sugar for the picrate reaction. We have carried out comparative determinations upon some seventy (70) bloods, ,chiefly human, and have found in nearly every instance a close agreement by the two methods when applied to the tungstic acid filtrate, providing there was no appreciable nitrogen retention in the blood. The point is of interest as showing that the difference by the two methods for normal blood is due to some unknown non-protein constituent, which is precipitated by tungstic acid and not by the acid picrate solution. The point deserves further investigation. With the discovery of insulin it has become a matter of considerable importance for a true understanding of sugar metabolism in the body to be able to measure the actual glucose content of the blood just as closely as may be possible. It seems very probable to the present writer that one or more of the non-glucose-reducing bodies of the blood is directly concerned in sugar metabolism. Obviously this point can be studied adequately only as we may be able more and more sharply to limit at least one method strictly to the glucose in the blood. It is doubtful whether this end is yet in sight, but it is the purpose of the present paper to present a new method for blood sugar determinations, which employs a more specific copper reagent than has hitherto been available, and which yields figures for the blood sugar very appreciably lower than those heretofore obtained. The greater delicacy and specificity of copper reagents containing carbonate in place of hydroxide were pointed out by the present writer many years ago .2 In that paper a reagent was proposed for sugar detection which contained copper sulfate, Rochelle salt, and sodium carbonate. This reagent is more delicate than the citrate reagent subsequently proposed, but was not
1 Csonka, F. A., and Taggart, G. C., J. Biol. Chem., *Benedict, S. R., J. Biol. Chem., 1907, iii, 101.
1922,

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liv, 1.

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209

so stable, nor so satisfactory, because of its great sensitiveness as a general qualitative reagent for sugar in urine. Folin and Wu, in constructing their admirable method for blood sugar determination, adopted the tartrate carbonate copper reagent in modified form. The chief point of modification was in dilution. Folin and Wu thus obtained an exceedingly sensitive reagent. It is, however, a fact, as the writer learned when first studying the carbonate reagents, that dilution of such reagents increases the sensitiveness at the cost of some of the specificity. Apparently the copper complex is more ionized in the greater dilution. Thus the Folin-Wu modified copper solution is readily reduced by creatinine, formaldehyde, creatine, chloroform, etc. We do not believe that creatine, creatinine, or other known non-glucose-reducing bodies occur in the blood in large enough amount to affect appreciably this reagent; yet we have felt for a long time that a forward step in blood sugar analysis would be taken if a copper solution could be found which would be satisfactorily sensitive for blood sugar determination, and at the same time which would not be appreciably reduced by such known non-sugar-reducing compounds as may occur in blood or urine. If the Folin-Wu copper reagent is applied directly to normal urine it will show from two to three times as much sugar as is present. Obviously it is theoretically not wholly satisfactory to apply such a solution directly to blood filtrates and assume that all the reduction obtained is due to glucose. We have no wish in this connection to stress any criticism of the procedures elaborated by Folin and by Shaffer for determination of blood sugar. We admire these methods and have employed them freely for a long time. We merely mention the fact to explain why we have kept in mind developing a solution which should give approximately the sugar content of normal urine directly, so that we could find out whether the application of such a solution to blood filtrates would result in lower figures for the blood sugar. More than 2 years ago we devised a copper solution which could be applied directly to urine with an error of about one-tenth that obtained with the Folin-Wu solution. Indeed in many samples of urine this reagent yielded as low figures for sugar when applied directly as did the Folin-Wu solution after treatment of the urine with Lloyds reagent. There were, however, difficulties in the

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Determination

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application of the solution to the blood because the quantity of reduced copper oxide yielded by a given amount of glucose was not great enough, and because of difficulty in finding a suitable reagent for color development after the reduced copper oxide was obtained. About a year ago, however, we solved these difficulties, and have since that time employed the method under widely varying conditions so as to test its accuracy as thoroughly as possible. The new developments in reagents and technique are essentially the following. The copper reagent employed is a modification of the qualitative citrate carbonate solution, in which the concentration of citrate has been increased very materially, while the quantity of copper and carbonate has been reduced. A small amount of sodium bisulfite has been added to the reagent, which results in a remarkable increase in the quantity of cuprous oxide obtained from the small amount of sugar contained in the dilute blood filtrate. The addition of sulfite in this connection is apparently a new departure in sugar analysis. Its action is probably through combining with fragments of the sugar molecule and preventing their destruction by the carbonate present until they have reduced the copper. Certainly the action is not due to the sulfite preventing the reduced copper from being reoxidized, because carrying out the reaction in the complete absence of atmosphere oxygen will not result in the increased yield of cuprous oxide. The sulfite itself has no reducing action upon the copper solution. For the development of color by cuprous oxide we make use of the tungstic-arsenic-phosphoric acid reagent which we proposed sometime ago for uric acid determination3 and to which is added about 5 per cent of commercial formalin. Unless the formalin is added the sulfite present in the copper reagent will produce some color. The color reagent of Folin and Wu cannot be used because the color obtained in the presence of the new copper reagent is very weak and fades almost immediately. Our final solutions have slightly more color from the same amount of sugar than is obtained in the Folin-Wu method, and the color shows little tendency to change on standing.
3 Benedict, S. R., J. Bid. Chem., 1922, li, 187.

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211

In order to make for simplicity and uniformity we have made our reagents and technique so that the new process is nearly identical in quantity of reagents used and general technique with that of Folin and Wu. The exact procedure for the method follows. Reagents.
1. Alkaline Copper Solution.6.5 gm. Pure crystallized copper sulfate.. . . .. ... . .. ... ... .. . 200 Sodium citrate.. ... ... . .. .. ..., . .. .. .. .. ... . .. . L 50 carbonate (anhydrous). . . . .. . . . . . . I 1 bisulfite .. .. ... .. .. ... ... .. .. .. ... .. ... .. ... . . Distilled water to make.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1,000 cc. Dissolve the citrate, carbonate, and bisulfite together in about 850 cc. of water. Dissolve the copper sulfate separately in about 100 cc. of water Dilute and add to the other solution, with constant stirring or shaking. the solution to 1 liter. This solution will keep for a year or longer. d. Complex Tungstic Acid Color Reagent.-This is prepared as follows: 100 gm. of pure sodium tungstate are placed in a liter flask and dissolved in 50 gm. of pure arsenic pentoxide are now added, folabout 600 cc. of water. lowed by 25 cc. of 85 per cent phosphoric acid and 20 cc. of concentrated hydrochloric acid. The mixture is boiled for 20 minutes. After cooling, 50 cc. of commercial formalin are added and the solution is diluted to 1 liter.* The technique of the method is as follows: 2 cc. of the 1: 10 tungstic acid filtrate are measured into a Folin-Wu sugar tube,5 followed by 2 cc. of the copper reagent. The contents are mixed by side to side shaking for a moment and placed in boiling water for 4 to 5 minutes. The tubes are then cooled by immersion in cold water and 2 cc. of the tungstic-arsenic acid reagent are added. At the end of 6 to 10 minutes the contents of the tubes 4 Except for the formalin, this reagent is identical with that proposed by the writer for determination of uric acid in blood. If desired, 5 cc. of formalin can be added to 100 cc. of the uric acid reagent previously prepared, and this solution used for color development in the sugar method. 6 In place of the special Folin-Wu sugar tube an ordinary test-tube graduated at 12.5 and 25 cc. may be used with the following simple precaution to After the solutions are placed prevent reoxidation of the cuprous oxide. in the tube 2 or 3 drops of benzene are added and the tube is plugged with cotton. During the heating the benzene is completely vaporized. The heavy vapor remainsin the tube and excludes the air. In the presence of the benzene apparently much more cuprous oxide is precipitated. This is due The final results are the same to the oxide being in a finer state of division. as where the Folin-Wu tube is used.

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Determination

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are diluted to 25 cc. with water and compared with a standard in a colorimeter. As a standard solution, pure glucose solution containing 0.1 and 0.2 mg. of glucose per cc. is employed just as in the Folin-Wu process. These can be conveniently prepared by proper dilution of a 0.1 per cent aqueous solution of glucose, to every liter of which a few drops of toluene have been added. Such a solutionwill keep unchanged for years. The diluted standards will also keep for long periods in the presence of a few drops of toluene.

In Table I the results of comparative analyses by the new method and by the Folin-Wu procedure are recorded for fourteen
TABLE I.

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Showing

Comparalive Figures for Blood Sugar by the Folin-Wu Procedure.

and by the New

method Results by Folin-Wu method Kesults by ew method

Blood

No.

SOWX

New _m,*. per

ioocc

- :. m0.

per ioocc.

1 2 3 4 6 ,6 7 8 9 10 11 12 13 14

Human. I I Dog.* Human (mild diabetes). (diabetes). I 1. ; ;: ( Dog (in insulin convulsions).

* 20 hours after ligation

of both ureters.

73 89 100 95 100 73 88 128 152 320 296 516 333 27

65 73 84 76 79 61 73 107 120 277 246 448 304 16

1.12 1.21 1.19 1.25 1.26 1.19 1.20 1.19 1.26 1.15 1.20 1.12 1.11 1.68

blood samples. With a few obvious exceptions, these figures are taken at random from the large number of comparative analyses which we have made. It will be noted that the new method yields figures averaging some 20 per cent lower than those by the Folin-Wu procedure. The discrepancy between some of the figures is so great as to lead to the suspicion that the new method fails to record all the glucose present in the blood. In this connection, we would point out that glucose added to blood filtrates is recovered satisfactorily by the new procedure. The different results

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213

by the new method become more comprehensible when we bear in mind that if the two methods be applied directly to diluted normal uririe the new procedure consistently yields figures about one-third as great as those given by the Folin-Wu modified tartrate reagent. It is hardly surprising that the blood should show differences of the same kind, though of very much less magnitude. The figures reported for the sugar of Sample 14 merit a word of comment. The dog from which this sample of blood was obtained was in convulsions after an enormous dose of insulin. Figures by both the Folin-Wu and the Shaffer-Hartman methods showed a sugar cont,ent for the blood of 27 mg. per 100 cc. The new procedure reduces this figure to 16, a difference of 68 per cent. As a matter of fact, we are inclined to regard this blood as free from glucose, for we believe that the new method still yields results averaging perhaps 15 mg. per 100 cc. too high. Our chief reason for this view is that all the copper methods which we have so far tested Will give a definite increase in the figure for sugar if the heating of both standard and unknown is continued beyond the period required for complete oxidation of the sugar in the standard tube. In the Folin-Wu method the increase in sugar is as much as. 15 to 20 mg, per 100 cc. of blood if the heating is continued 12 to 18 minutes instead of the 6 minutes directed. The new method shows the same curve of increase though quantitatively less. Unless we assume an inhibiting substance in the blood (which cannot be demonstrated and which would invalidate all copper methods as applied to blood) we must assume the presence of a slowly reacting interfering compound which causes plus errors of something over 10 mg. per 100 cc. of blood. Figures by the new method indicate that the normal blood sugar content averages about 75 mg. per 100 cc. We are, however, of the opinion that the actual glucose content of normal human blood does not usually exceed 60 mg. per 100 cc. except for a period after food ingestion. The figure 60 may still be too high. It is obvious that the new method can be readily substituted in the various modifications of the Folin-Wu procedure which have appeared, providing for the use of less blood, and which rep-resent essentially a division though by 10 of the quantity of blood and reagents employed. In a later paper we shall discuss the application of the new copper reagent to the determination of sugar in normal urine.

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