J. Biol. Chem.-1925-Benedict-207-13.pdf

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THE DETERMINATION OF BLOOD SUGAR.BY STANLEY R. BENEDICT.

(From the Department of Chem istry, Corne ll U niversity Med ical Colle ge, NewYork city.)

(Received for publication, March 26, 1925.)There are at present available several methods for the deter-mination of sugar in blood which yield satisfactory results forfollowing the blood sugar in diabetes. These include the picric

acid procedure in one or other of its various modifications andthe copper methods of Bang, Shaffer and Hartman, and Folin andWu. To many it may appear unjustifiable to include the picricacid methods here, but the writer ventures to assert that althoughmany thousands of analyses have been made by the picric acidmethods, it is doubtful whether a clinician has ever been misledin his interpretation of a diabetic case by the figures obtained forthese analyses when properly carried out. Nevertheless, criticalstudies of the picric acid methods have shown that these con-sistently yield figures higher than the actual glucose content of theblood. This does not mean, however, that we should not regardthe use of the picric acid methods as a legitimate routine pro-cedure by the clinician. Indeed we have frequently noted a factwhich indicates that the picrate method may be preferable toa copper method for following the course of the blood sugar inthe diabetic. With diet, the blood sugar of the diabetic tends toreturn more promptly to the normal level as ndicated by a coppermethod (Folin-Wu) than when the determination is made with thepicrate method. This point is worthy of further study and weare accumulating data along this line as opportunity permits. Wewould point out in this connection that for bloods having a normalnon-protein nitrogen content the picrate method yields figurespractically identical with those given by the Folin-Wu procedure,if applied to a portion of the same tungstic acid j&ate after con-centrating a portion of the filtrate by boiling so that the sugar

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208 Determination of Blood Sugarconcentration is high enough to permit an accurate determinationby the picrate procedure. Though Csonka and Taggart* reportresults somewhat at variance with this statement it must be notedthat their figures show a much clozer agreement when both meth-ods were applied to the tungstic acid filtrate, and that Csonka andTaggart apparently took no precautions to insure a proper con-centration of sugar for the picrate reaction. We have carried outcomparative determinations upon some seventy (70) bloods,,chiefly human, and have found in nearly every instance a closeagreement by the two methods when applied to the tungstic acidfiltrate, providing there was no appreciable nitrogen retention inthe blood. The point is of interest as showing that the differenceby the two methods for normal blood is due to some unknownnon-protein constituent, which is precipitated by tungstic acidand not by the acid picrate solution. The point deserves furtherinvestigation.

With the discovery of insulin it has become a matter of con-siderable importance for a true understanding of sugar metabolismin the body to be able to measure the actual glucose content of theblood just as closely as may be possible. It seems very probable tothe present writer that one or more of the non-glucose-reducingbodies of the blood is directly concerned in sugar metabolism.Obviously this point can be studied adequately only as we may beable more and more sharply to lim it at least one method strictlyto the glucose in the blood. It is doubtful whether this end is yetin sight, but it is the purpose of the present paper to present a newmethod for blood sugar determinations, which employs a morespecific copper reagent than has hitherto been available, andwhich yields figures for the blood sugar very appreciably lowerthan those heretofore obtained.

The greater delicacy and specificity of copper reagents con-taining carbonate in place of hydroxide were pointed out by thepresent writer many years ago .2 In that paper a reagent wasproposed for sugar detection which contained copper sulfate,Rochelle salt, and sodium carbonate. This reagent is more deli-cate than the citrate reagent subsequently proposed, but was not

1 Csonka, F. A., and Taggart, G. C., J. Biol. Chem., 1922, liv, 1.*Benedict, S. R., J. Biol. Chem., 1907, iii, 101.

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S. R. Benedict 209so stable, nor so satisfactory, because of its great sensitiveness as ageneral qualitative reagent for sugar in urine. Folin and Wu, inconstructing their admirable method for blood sugar determina-tion, adopted the tartrate carbonate copper reagent in modifiedform. The chief point of modif ication was in dilution. Folin andWu thus obtained an exceedingly sensitive reagent. It is, however,a fact, as the writer learned when first studying the carbonatereagents, that dilution of such reagents increases the sensitivenessat the cost of some of the specificity. Apparently the copper com-plex is more ionized in the greater dilution. Thus the Folin-Wumodified copper solution is readily reduced by creatinine, for-maldehyde, creatine, chloroform, etc. We do not believe thatcreatine, creatinine, or other known non-glucose-reducing bodiesoccur in the blood in large enough amount to affect appreciablythis reagent; yet we have felt for a long time that a forward stepin blood sugar analysis would be taken if a copper solution couldbe found which would be satisfactorily sensitive for blood sugardetermination, and at the same time which would not be appre-ciably reduced by such known non-sugar-reducing compounds asmay occur in blood or urine.

If the Folin-Wu copper reagent is applied directly to normalurine it will show from two to three times as much sugar as ispresent. Obviously it is theoretically not wholly satisfactory toapply such a solution directly to blood filtrates and assume thatal l the reduction obtained is due to glucose. We have no wish inthis connection to stress any criticism of the procedures elaboratedby Folin and by Shaffer for determination of blood sugar. Weadmire these methods and have employed them freely for a longtime. We merely mention the fact to explain why we have keptin mind developing a solution which should give approximatelythe sugar content of normal urine directly, so that we could findout whether the application of such a solution to blood filtrateswould result in lower figures for the blood sugar.

More than 2 years ago we devised a copper solution which couldbe applied directly to urine with an error of about one-tenth thatobtained with the Folin-Wu solution. Indeed in many samples ofurine this reagent yielded as low figures for sugar when applieddirectly as did the Folin-Wu solution after treatment of the urinewith Lloyds reagent. There were, however, difficulties in the

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210 Determination of Blood Sugarapplication of the solution to the blood because the quantity ofreduced copper oxide yielded by a given amount of glucose wasnot great enough, and because of difficulty in finding a suitablereagent for color development after the reduced copper oxide wasobtained. About a year ago, however, we solved these difficulties,and have since that time employed the method under widelyvarying conditions so as to test its accuracy as thoroughly aspossible.

The new developments in reagents and technique are essentiallythe following. The copper reagent employed is a modification ofthe qualitative citrate carbonate solution, in which the concen-tration of citrate has been increased very materially, while thequantity of copper and carbonate has been reduced. A smallamount of sodium bisulfite has been added to the reagent, whichresults in a remarkable increase in the quantity of cuprous oxideobtained from the small amount of sugar contained in the diluteblood filtrate. The addition of sulfite in this connection is ap-parently a new departure in sugar analysis. Its action is probablythrough combining with fragments of the sugar molecule andpreventing their destruction by the carbonate present until theyhave reduced the copper. Certainly the action is not due to thesulfite preventing the reduced copper from being reoxidized,because carrying out the reaction in the complete absence ofatmosphere oxygen will not result in the increased yield of cuprousoxide. The sulfite itself has no reducing action upon the coppersolution.

For the development of color by cuprous oxide we make useof the tungstic-arsenic-phosphoric acid reagent which we pro-posed sometime ago for uric acid determination3 and towhich is added about 5 per cent of commercial formalin.Unless the formalin is added the sulfite present in the copperreagent will produce some color. The color reagent of Folin andWu cannot be used because the color obtained in the presenceof the new copper reagent is very weak and fades almostimmediately.

Our final solutions have slightly more color from the sameamount of sugar than is obtained in the Folin-Wu method, andthe color shows little tendency to change on standing.

3 Bene dict, S. R., J. Bid. Chem., 1922, li, 187.

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S. R. Benedict 211In order to make for simplicity and uniformity we have made

our reagents and technique so that the new process is nearlyidentical in quantity of reagents used and general technique withthat of Folin and Wu. The exact procedure for the methodfollows.

Reagents.1. Alkaline Copper Solution.-

Pure crystallized copper sulfate .. . . . . . . . . . . . . . . . . . . . 6.5 gm.Sod ium citrate.. . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . 200 L carbonate (anhydrous). . . . . . . . . . . . 50 bisulfite 1 I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Di still ed 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 waterand add to the other solution, with con stant stirring or shaking. Dilutethe solution to 1 liter. Th is solution will keep for a year or longer.

d. Complex Tun gstic Acid Color Reagen t.-This is prepared as follows:100 gm. of pure sodium tungstate are placed in a liter flask and dissolved inabout 600 cc. of water. 50 gm. of pure arsenic pentoxide are now added, fol-lowed by 25 cc. of 85 per c ent phospho ric acid and 20 cc. of concentratedhydrochloric acid. The mixture is boiled for 20 minutes. After cooling ,50 cc. of comm ercial formalin are added and the solution is diluted to1 liter.*

The technique of the method is as follows: 2 cc. of the 1: 10 tungstic acidfiltrate are measured into a Folin-Wu sugar tube,5 followed by 2 cc. of thecopper reagent. The contents are mixed by side to side shaking for amoment and placed in boiling water for 4 to 5 minutes. The tubes are thencooled by immersion in cold water and 2 cc. of the tungstic-arsenic acidreagent are added. At the end of 6 to 10 minutes the contents of the tubes

4 Except for the formalin, this reagent is identic al with that proposedby 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 previouslyprepared, and this solution used for color development in the sugarmethod.

6 In place of the spe cial Folin-Wu sugar tube an ordinary test-tube grad-uated at 12.5 and 25 cc. may be used with the following simp le precaution toprevent reoxidation of the cupro us oxide. After the solution s are placedin the tube 2 or 3 drops of benzene are added and the tube is plugged withcotton. During the heating the benzene is completely vaporized. Theheavy vapor rem ainsin the tube and exclu des the air. In the presen ce of thebenzene apparently much more cuprous oxide is precipitated. Th is is dueto the oxide being in a finer state of division. The final results are the sameas where the Folin-Wu tube is used.

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212 Determination of Blood Sugarare diluted to 25 cc. with water and compared with a standard in a colorim-eter. As a standard solution, pure glucose solution containing 0.1 and 0.2mg. of glucose per cc. is employed just as in the Folin-Wu process. Thesecan be conveniently prepared by proper dilution of a 0.1 per cent aqueoussolution of glucose, to every liter of which a few drops of toluene have beenadded. Such a solutionwill keep unchanged for years. The diluted stand-ards 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 newmethod and by the Folin-Wu procedure are recorded for fourteen

TABLE I .Showing Comparalive Figures for Blood Sugar by the Folin-W u and by the NewProcedure.

Blood No. SOWX

12346,6789

1011121314

Human.II

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

_-m

-

,*. per ioocc7389

100951007388128152

32029651633327

N- -:. m

-

ew method

0. per ioocc.65738476796173107120

27724644830416

-Results byFolin-WumethodKesults byew method

1.121.211.191.251.261.191.201.191.261.151.201.121.111.68

* 20 hours after ligation of both ureters.blood samples. With a few obvious exceptions, these figuresare taken at random from the large number of comparative analy-ses which we have made. It will be noted that the new methodyields figures averaging some 20 per cent lower than those by theFolin-Wu procedure. The discrepancy between some of the figuresis so great as to lead to the suspicion that the new method failsto record all the glucose present in the blood. In this connection,we would point out that glucose added to blood filtrates is re-covered satisfactorily by the new procedure. The different results

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S. R. Benedict 213by the new method become more comprehensible when we bearin mind that if the two methods be applied directly to dilutednormal uririe the new procedure consistently yields figures aboutone-third as great as those given by the Folin-Wu modified tar-trate reagent. It is hardly surprising that the blood should showdifferences of the same kind, though of very much less magnitude.

The figures reported for the sugar of Sample 14 merit a word ofcomment. The dog from which this sample of blood was obtainedwas in convulsions after an enormous dose of insulin. Figures byboth the Folin-Wu and the Shaffer-Hartman methods showeda sugar cont,ent for the blood of 27 mg. per 100 cc. The new pro-cedure reduces this figure to 16, a difference of 68 per cent. As amatter of fact, we are inclined to regard this blood as free fromglucose, for we believe that the new method still yields resultsaveraging perhaps 15 mg. per 100 cc. too high. Our chief reasonfor this view is that all the copper methods which we have so fartested Will give a definite increase in the figure for sugar if theheating of both standard and unknown is continued beyond theperiod required for complete oxidation of the sugar in the standardtube. 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 to18 minutes instead of the 6 minutes directed. The new methodshows the same curve of increase though quantitatively less.Unless we assume an inhibiting substance in the blood (whichcannot be demonstrated and which would invalidate al l coppermethods as applied to blood) we must assume the presence of aslowly reacting interfering compound which causes plus errors ofsomething over 10 mg. per 100 cc. of blood.

Figures by the new method indicate that the normal blood sugarcontent averages about 75 mg. per 100 cc. We are, however, ofthe opinion that the actual glucose content of normal humanblood does not usually exceed 60 mg. per 100 cc. except for aperiod after food ingestion. The figure 60 may still be too high.

It is obvious that the new method can be readily substitutedin the various modifications of the Folin-Wu procedure whichhave appeared, providing for the use of less blood, and which rep--resent essentially a division though by 10 of the quantity ofblood and reagents employed.

In a later paper we shall discuss the application of the newcopper reagent to the determination of sugar in normal urine.

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J. Biol. Chem.-1925-Benedict-207-13.pdf