Some Applications and Limitations of the

Enzymic, Reducing (Somogyi), and Anthrone

Methods for Estimating Sugars

Frank W. Fales, Jane A. Russell, and John N. Fain*

The colorimetric glucose-oxidase and the Somogyi-Nelson reducing-sugar methodsyield the same blood-glucose values when applied to carefully prepared zinchydroxide filtrates of blood obtained from fasting individuals. The anthrone meth-od yields values only slightly higher than those obtained by the enzymic and thereducing-sugar methods. Use of the enzymic method in conjunction with thereducing-sugar or the carbohydrate method allows the simultaneous accuratedetermination of other carbohydrates added to the blood, with only microquantitiesof blood being required for analysis. Possible applications include the galactoseand fructose tolerance tests, the inulin clearance test, and the determination ofthe body water “space” of various carbohydrates.

To allow valid glucose determinations by the enzymic method, glutathione,cysteine, and amylase substrates such as glycogen must be absent.

The anthrone reaction with glucose is enhanced by chloride, bromide, and iodide.To eliminate interference of chloride with estimation of blood glucose, the samplesmust be diluted appropriately before analysis.

�HE QUANTITATIVE ENZYMIC METHOD for glucose, the Somogyi meth-

od for reducing sugar, and the anthrone method for carbohydrate,used either singly or in combination, offer a variety of applicationsbecause of the differences in specificity. Because of the specificity ofthe enzymic method for glucose (1), one possible application is itsuse for determining blood-glucose levels in the presence of othercarbohydrates. The Somogyi (2) or other of the more specific re-

From the Department of Biochemistry, Emory University, Atlanta, Ga.

A portion of the material presented in this article was presented before the Annual

Meeting of the American Association of Clinical Chemists, Cleveland, Ohio, Aug. 28, 1959.Received for publication Aug. 12, 1960.‘Public Health Service Predoetoral Feliow, National Institute of Arthritis and Metabolic

Diseases.

289

290 FALES ET AL. Clinical Chemistry

ducing-sugar methods, used in conjunction with the glucose-oxidasemethod, offer an alternative for the determination of reducing sugarsother than glucose added to the blood, as in the galactose or fructosetolerance tests. Likewise, the anthrone method used in conjunctionwith the enzymic method offers a means for determining carbohy-

drate added to blood, but in this instance, the carbohydrate is notrestricted to the reducing sugars. These proposed methods offer

advantages over previous methods that involve the removal of glu-cose by yeast (3) or by glucose oxidase (4) prior to the analysisin that a precise measure of the glucose level is also obtained and

that the methods are more readily amenable to microanalytic pro-cedures.

Of equal relevance to possible applications of the methods are thelimitations that were uncovered during the study reported in this

article. It appears that too much emphasis may have been placedupon the specificity of glucose oxidase in the colorimetric enzymicmethods, but too little attention paid to the many substances thatmay interfere with the method. This has tended to engender the false

supposition that the enzymic methods yield true glucose values re-gardless of the experimental conditions. These methods all employ areagent containing a crude glucose oxidase preparation, peroxidase,

and a chromogenic hydrogen donor for the peroxidase reaction. Thiscombined reagent is sensitive to a number of substances other thanglucose, and some assurance as to the absence of these interfering

materials must be at hand before the “true” glucose values can beconsidered as valid. Apart from the obvious requirement for theexclusion of activators and inhibitors of the two enzymes, other less

obvious materials can cause serious errors. The peroxides that may

be released in acid filtrates of biologic materials cause a positiveerror, whereas materials that compete with the chromogen as hydro-

gen donors, e.g., uric acid, ascorbic acid, bilirubin, and the catechols,

cause a negative error. It has been found that glutathione and

cysteine also strongly inhibit color formation, and it is presumed

that these substances also compete as hydrogen donors. Also, in aninvestigation of the applicability of the enzymic method for the

determination of glucose in tissue extracts, it was found that there

was a large positive error in the presence of appreciable concentra-tions of glycogen, because the glucose oxidase preparations used in

the reagent invariably have high amylytic activity. This imposes the

Vol. 7, No. 4, 1961 ESTIMATION OF SUGARS 291

added requirement that amylase substrates be absent. Thus, thereis an imposing list of materials that may interfere with the enzymicmethod, and hence, despite the specificity of glucose oxidase, rigid

exclusion of these materials is required.In conjunction with these studies, the anthrone method was ex-

amined for the determination of blood sugar. Since anthrone doesnot react with non-sugar-reducing material (“saceharoids”), one

might have expected the reaction to give true values for total sugars

in blood. However, Roe (5) found that with tungstic acid filtrates,which are rich in saccharoids, about equal values were obtained bythe anthrone and the reducing-sugar methods. For the most part,this positive error in the anthrone reaction appears to be owing tothe enhancement of the anthrone color by chloride ions (6). Wefound that the chloride enhancement can be overcome by dilution,

the positive error of the anthrone reaction being reduced (by theuse of dilute filtrates) from about 20 to 5 mg./100 ml., the residualerror apparently being due to the presence of small amounts ofpolysaccharide. Thus, under properly controlled conditions, the totalsugar in the blood can be determined with reasonable precision.

By the judicious combined use of the Somogyi and the enzymic,

and of the anthrone and enzymic methods, we have obtained excellentrecoveries of galactose and/or sucrose added to the blood. It is sug-gested that this approach may be of value for the galactose andfructose tolerance tests as well as for the determination of the

clearance and the body-water “space” of various carbohydrates.

Methods and Materials

Preparation of Filtrates

Either the Somogyi (7) 0.5N sodium hydroxide, 10% zinc sulfateor the Somogyi (8) 0.3N barium hydroxide, 5% zinc sulfate reagents

may be used with whole blood, but only the latter are suitable withserum or plasma (8, 9). We prefer the former reagents with wholeblood because a larger volume of filtrate is obtained. The concentra-tion of the base and zinc sulfate must be carefully adjusted as di-

rected. The importance of a careful preparation of the ifitrates can-not be overemphasized, since this process accomplishes the removalof materials that interfere with the enzymic as well as with the re-

ducing-sugar procedure. Incomplete removal of these materials cancause large discrepancies between the values obtained by the meth-

292 FALES ET AL. Clinical Chemistry

ods, since the materials cause low values with the enzymic and highvalues with the reducing-sugar method.

We prepared the ifitrates as follows: The blood was laked with 17

or 22 volumes of water. One volume of sodium hydroxide solutionwas added, and then, after thorough mixing but without sufficient

delay to cause a significant enolization of the sugar, 1 volume of zincsulfate solution was added drop by drop, with mixing. The solutionwas then very thoroughly mixed to avoid entrapment of liquid within

the precipitate. This mixing may be accomplished by shaking in aglass�stoppered* Erlenmeyer flask or centrifuge tube by vigorous

swirling in an Erlenmeyer flask, or by vigorous and prolonged stir-ring with a large glass rod in a centrifuge tube. In any event, thefinal mixture should be uniformly opaque, with no discernible layers

or streaks of liquid. After 5 mm., the mixture was filtered or cen-

trifuged. We prefer the above sequence for the addition of the re-agents because the base insures the complete hemolysis of the redblood cells, which is essential for a valid estimate of the whole bloodglucose concentration.

Reducing-Sugar Determination

The improved copper reagent and the method of Somogyi (2) wereused in conjunction with the Nelson (10) arsenomolybdic acid re-

agent. The procedure is readily adaptable for microquantities andso is well suited for the proposed microprocedure for the galactose

tolerance test. The following procedure, which closely follows therecommendations of Somogyi (2), was found satisfactory. To 1.8ml. of water in a colorimeter tube was added 0.2 ml. of 1:20 dilutionfiltrate and 2 ml. of copper reagent. Duplicate tests, standards, and

a blank were capped with glass marbles to prevent evaporation andwere simultaneously placed in a boiling-water bath. After 20 mm.,the tubes were simultaneously removed to a cold-water bath. Aftera few minutes, 2 ml. of arsenomolybdic acid reagent was added toeach tube, the contents of the tubes were thoroughly mixed, and afterseveral minutes the colorimetric readings were made.

Total Carbohydrate Determination

Total carbohydrate was determined by the anthrone method ofFales (11). Versene or potassium oxalate (no fluoride) was used as

‘A rubber stopper should not be used because derivatives from the rubber inhibit colorformation in the enzymic procedure. This information was provided by the WorthingtonBiochemical Corporation with their reagent.

Vol. 7, No. 4. 1961 ESTIMATION OF SUGARS 293

the anticoagulant. The use of wood applicator sticks, ifiter paper,and cloth or paper pipet swipes was avoided throughout the proce-dure, as they can cause a serious positive error. Enhancement of theanthrone color by chloride (see below) was avoided by dilution.

Glucose Determination

The prepared reagent Glucostat* was used. The reagent was pre-pared as directed for the microprocedure except that 0.05M phos-phate buffer, pH 7, was used as the diluent rather than water. Two

milliliters of reagent were added to 0.2 ml. of blood filtrate. The re-action was stopped after 40 mm. by the addition of 1 drop of 4Nhydrochloric acid. The readings were made after 5 mm. at a wave-

length of 400 m�. It was found desirable to determine frequently therange of glucose concentrations over which a linear response was

obtained, since there seemed to be a variability of response withdifferent batches of reagent. Also, the pH of the final reagent waschecked and adjusted to pH 7.0 if necessary. The pH given by the

buffer incorporated into the reagent by the manufacturer was at con-

siderable variance from pH 7.0 in many batches. The more con-venient reagent and method described by Washko (12) were used in

some of the later experiments.

ResultsLimitations of the Enzymic Method

It was found that the low blood-glucose values obtained by theenzymic method with tungstic acid filtrates result almost entirelyfrom the glutathione contained in the filtrates. The inhibition of

color formation is not brought about by residual tungstic acid in the

filtrates, as suggested by Beach and Turner (13), since equivalent

amounts of tungstic acid added to glucose standards have no meas-urable effect. Also, the concentrations of uric acid and ascorbic acid

in the filtrates were insufficient to cause a measurable effect. How-ever, glutathione strongly inhibited color formation, and the gluta-

thione content of tungstic acid filtrates is sufficient to account for thelow values.

Glutathione evidently competes with the chromogen as a hydrogendonor for the peroxidase reaction. When excess glutathione is addedduring the course of the reaction, color formation is arrested but no

‘Worthington Biochemical Corp.

294 FALES ET AL. Clinical Chemistry

significant bleaching is observed. Therefore, the action of the gluta-thione is not the reduction of the colored product. The reagent is

affected in a similar manner by cysteine. Oxidized glutathione orcystine have no effect. A study of the course of the color formationwith a tungstic acid filtrate indicated that the inhibition was exertedearly in the course of the reaction. After all the glutathione wasoxidized (10 mm., in this case), the color formation proceeded at arate identical to that observed with a zinc hydroxide filtrate of the

same blood. These data are shown in Table 1. Thus, a simple test

Table 1. COTJRSE or Cot.oa DEVELOPMENT WITH TUNOSTIC ACID AND ZINC HYDROXIDE

FILTRATES

Incubation time Apparent glucose concentrations (mg./IOO ml.)

Tungatic acid Zinc hydroxide(tam.)

5 34 6410 40 67

20 47 69

30 53 66

30 minus 10’ 69 69

‘Values based on the changes in absorbancy from the 10. to the 30-mm. readings of the

blood filtrates and of the standard.

for the presence of glutathione or other interfering hydrogen donorsis suggested. If higher values are obtained with increasing incuba-tion times, the presence of a competing hydrogen donor is suggested.Consistently low or decreasing values would be indicative of enzymeinhibition.

A comparison of the blood glucose values obtained with the use of

tungstic acid and zinc hydroxide filtrates was carried out with nineblood samples. Under the conditions used, the average underestima-tion with tungstic acid ifitrates was 13 mg., with a range of 2-24

mg./100 ml. There seemed to be no correlation between the under-estimation and the glucose concentration. The use of tungstic acidfiltrates in the determination of serum or plasma glucose levels by

the enzymic method may be admissible because the blood glutathioneresides almost wholly within the red blood cells.

The possible use of the enzymic method for the determination ofglucose content of tissue extracts was investigated. Upon testing the

effect of various constituents of the tissue extracts, it became evidentthat glycogen strongly interfered with the determination. When the

20 30 40TIME. MINUTES

Vol. 7, No. 4, 1961 ESTIMATION OF SUGARS 295

reagent was added to glycogen solutions, a considerable color forma-tion was observed. The course of the color development with various

concentrations of glycogen and with a glucose standard is shown in

Fig. 1. The autocatalytic reaction curves with the glycogen solutions

Fig. 1. Increase in absorbancy with

time by the glucose oxidase method

with various amounts of glycogen andwith glucose: A, 25 pg. of glycogen;

B, 100 pg. of glycogen; C, 400 pg. of

glyeogen; D, 5 mg. of glycogen; andE, 50 pg. of glucose.

suggest an a-amylase contaminant in the reagent. The endoamylasecatalyzes the hydrolysis of the polysaccharide into small fragmentsthat are further split into maltose units, but a molecule of glucose isderived from fragments having an odd number of glucose residues.Although the relative yield of glucose from glycogen is not large,very much more glycogen than free glucose is likely to be present in

most tissues, and so the results obtained when crude oxidase is ap-plied to aqueous protein-free extracts are entirely unreliable. Pre-

liminary observations indicate that the amylase can be removedfrom a solution of the crude glucose oxidase by passing it through a

starch column under properly controlled conditions of pH and ionic

strength. However, even with such a preparation, it would still benecessary to remove glutathione and other reducing materials fromthe tissue extracts before the method can be applied to them withconfidence.

Limitations of the Anthrone Method

Enhancement of the reaction by chloride, mentioned above, wasobserved some time ago by one of us (J. A. R.) and was briefly men-

296 Clinical Ch.misfry

I-.ztaJU

wa.

z0

I-

I-zL,J

I,D

FIg. 2. Enhancement of anthrone

absorbancy with glucose (40 pg.) bythe halides. Halide concentrations are

expressed in terms of the final sam-pie-reagent mixture. Fluoride values

are approximate, owing to some etch-ing of the glass.

-6 -5 -4 -3 -2 -ILOG.(M) HALIDE CONCENTRATION

FALES ET AL.

tioned by Scott and Melvin (6). However, no extensive report ofthis potential error has appeared, and Mokrasch (14) erroneouslyreported that chloride at a concentration of 1M did not interfere.The enhancement of the anthrone reaction with glucose by the

halides is shown in Fig. 2. Included with the chloride data are twoexperiments by the method of Morris (15), in which reagent is addedto the sample in a ratio of 10:5, and two experiments by the methodof Fales, in which the ratio is 10:1. It is evident that the enhance-ment is dependent upon the final concentration of halide, regardless

of the ratio of sample to reagent. Investigations at higher concentra-tions were not feasible because of the etching of glass with fluoride,

the fuming of hydrogen halides with chloride and bromide, and theformation of iodine and other colored products with iodide. The dataindicate that the maximal halide concentrations free from interfer-

ence are 0.04, 4 X 10�, 1.8 X 10�, and 4.5 X 10� molar, respective-ly, for fluoride, chloride, bromide, and iodide. With the assumptionof a whole-blood chloride concentration of 85 mEq./L., a 65-fold dilu-

tion of the blood by the Morris method and a 20-fold dilution by theFales method would be required to remove the chloride effect. The

data also point to the danger of interference in specimens frompatients on bromide and iodide therapy. Although fluoride at the

‘Values based on glucose standard. These figures do not necessarily represent the actual

concentration of nonfermentable carbohydrate, because the carbohydrate in question maygive an absorbancy value with anthrone quite different from that given by glucose.

f Same blood as previous sample but with 1:20 dilution of tungstic acid filtrate ratherthan 1:20 of dilution zinc hydroxide filtrate, as was the case in the other samples.

Vol. 7, No. 4, 1961 ESTIMATION OF SUGARS 297

concentration used as a preservative does not interfere, it appears to

lower the permissible chloride concentration. Also, troublesomebubbles often adhere to the sides of the tubes when fluoride is pres-ent, and there is danger of etching the colorimeter tubes.

Another difficulty in the use of the anthrone method for the deter-

mination of blood sugar is some lack of specificity. Both zinc hy-droxide and tungstic acid filtrates contain small amounts of non-fermentable carbohydrate. The nonfermentable carbohydrate found

in blood filtrates by the anthrone method is shown in Table 2. Thefermentations were performed by the method of Somogyi (16), ex-cept that double centrifugations were carried out to insure the com-plete removal of the yeast and that two water blanks were included

with each run to correct for the small quantity of carbohydrateexuded from the yeast. The nonfermentable carbohydrate contentof the blood ifitrates appeared to be in no way related to the blood-sugar levels or to the method of protein precipitation. It appearsthat the nonglucose carbohydrate is not accounted for by sugarphosphates, as suggested by Roe (15), since identical results were

obtained when anthrone was used with ifitrates prepared either withzinc sulfate and sodium hydroxide or with zinc sulfate and bariumhydroxide. The barium would induce the precipitation of the sugarphosphates. This suggests that sugar phosphates in the erythrocytes

Table 2. TOTAL AND FERMENTABLE CARBOHYDRATE IN Bwon FILTRATES DETERMINED WITh

ANTHEONE

Carbohydrate (mg./100 ml.)

Nonfsrmentable*Sample No. Total Fermentable

1 92 5 87

2 99 7 92

3 147 7 136

4 78 8 704f 77 6 71

3 81 6 75

Sf 81 6 75

6 90 3 87

Of 97 4 93

7 104 4 100

7f 105 5 100

298 FALES ET AL. Clinical Chemistry

Table 3. CoMPAR�rIvE Bwon GLUCOSE vALuES (Mo.J100 ML.) OF FASTING INDIvIDUALS

BY 3 INDEPENDENT METHODS OF ANALYSIS

Sample No. Glucose Oxidas. Somogyi-Nelson Ant hron,

1 50 48 55

2 56 56

3 63 60

4 65 665 66 70 736 66 71 72

7 67 68 71

8 68 69 77

9 68 70

10 70 72

11 72 67 74

12 73 80

13 75 8214 75 75

15 76 7516 80 75

17 82 87

18 86 83

19 84 86

20 86 86

21 86 85

22 94 98

23 113 112

24 117 123

25 120 123

26 130 132

are in concentrations that are insignificant as compared to the bloodglucose. The identity of the nonfermentable carbohydrate found in

the filtrates is unknown, but the fact that it is nonreducing in charac-ter suggests that it may be polysaccharide. The error in blood-sugardeterminations with anthrone attributable to this factor is not seri-ous, averaging about 5 mg./100 ml., but it must be considered in thecomparison of the anthrone and the enzymic blood-glucose values.

Applications

In Table 3 are shown the blood-sugar values obtained on fasting

individuals* with three independent methods of analysis. These data

‘The use of blood from fasting individuals was indicated for this study because fructose

and galactose transiently appear in the blood following a meal containing sucrose andmilk products.

Vol. 7, No. 4, 1961 ESTIMATION OF SUGARS 299

indicate that glucose was the only reducing substance present in thefiltrates at an appreciable concentration. The values obtained by the

three methods are in quite good agreement, except that the anthronevalues tend to be slightly higher than the enzymic or the reducing-

sugar values. If 5 mg./100 ml., the average overestimation of glucosededuced from the fermentation studies (Table 2), is subtracted from

the anthrone values, good agreement between the three methods is

obtained.The recovery of galactose added to blood, determined by the pro-

posed procedure for the galactose tolerance test, is shown in Table4. The total reducing value was determined by the Somogyi-Nelsonmethod, and the glucose by the enzymic method. Both glucose and

galactose standards were included with the reducing-sugar determi-nations because glucose has a higher reducing power than doesgalactose. Since the same standard glucose solution and the same

blood filtrate were used in both methods, the portion of the totalSomogyi-Nelson absorbancy resulting from the oxidation of theblood glucose was calculated directly from the colorimetric measure-ments as follows:

AG = AR/AE X Au

where A0 is the absorbancy due to glucose; A5, that of the glucose

Table 4. RECOVERY OF SUGARS ADDED I’O BLooD

Milligrams sugar per 100 ml. of whole blood

Glucose* Gahx.ctose Galactossf Sucroce Sucrose�

�lood sample found added found added found

1 131 0 1

1 127 40 421 131 80 811 129 120 1231 131 160 1632 72 0 0 52 73 0 40 422 72 0 80 89

2 72 0 120 1292 71 0 160 164

2 74 80 78 80 82

‘Determined by the glucose oxidase method.

$Determined by the combined use of the somogyi-Nelson reducing-sugar and the glucoseoxidase methods.

tDetermined by the combined use of the anthrone and the glucose oxidase methods. Thelast figure in the column was determined by the combined use of all three methods. If the

average overestimation (5 mg./100 ml.) by the anthrone method is subtracted, improved

recovery values are obtained (see Table 2).

300 FALES fT AL. Clinical Chemistry

0 I 2 3TIME Hours

standard with the reducing sugar method; A5, that of the glucosestandard with the enzymic method; and A�, that of the blood ifitratewith the enzymic method. The absorbancy due to galactose was cal-

culated by difference, and the concentration of galactose was deter-mined in the usual way with the use of the absorbancy of the galac-

tose standard. The results of a recovery study with added sucroseare also shown in Table 4. In this instance the sucrose was deter-mined by the combined use of the anthrone and the enzymic methods.In one case, both sucrose and galactose were added, and the total

carbohydrate was determined by the anthrone method, the total re-ducing sugar by the Somogyi-Nelson method, and the glucose by theenzymic method. Glucose, sucrose, and galactose standards wereincluded with the anthrone determinations since they give differentabsorbancy values per unit concentration, that for sucrose beingsomewhat higher and that for galactose being considerably lowerthan that of glucose. These data point to the general utility of theproposed method of parallel analyses. In addition to its use for thegalactose tolerance test, it offers an alternative method for thefructose tolerance test. Also, it can be applied to the measurementof the distribution of various carbohydrates in the body water andto the measurement of the clearance of various carbohydrates fromthe blood. In Fig. 3 are shown the results of a galactose tolerance

test carried out by the proposed method on a galactosemic infant.The normal infant has a very high tolerance for galactose, the bloodgalactose level declining after 1 hour and falling close to zero after3 hours. In addition to the information gained from the galactose

Fig. 3. Galactose tolerance test on

galactosemic infant carried out bycombined use of enzymic and

Somogyi-Nelson methods.

Vol. 7, No. 4, 1961 ESTIMATION OF SUGARS 301

levels, the precise measurements of the changes in the blood-glucoselevel may be of some value in conjunction with the intravenousgalactose tolerance test carried out as a measure of liver function.

In this instance, both the capacity of the liver to remove galactose

and its capacity to restore the blood-glucose level would be measured.

Discussion

In the present study, we obtained excellent agreement between theSomogyi-Nelson and the enzymic methods for whole blood-glucosefrom fasting individuals. Beach and Turner (13) likewise found nosignificant differences between the values obtained by the two meth-

ods. Also, Middleton and Griffiths (17) found agreement with theenzymic method when they used the Asatoor and King (18) modi-

fication of the Harding and Downs (19) procedure. The copper re-

agents used in these procedures are modifications of the Shaffer-Somogyi Reagent 50 (20), as is the Somogyi-Nelson reagent, andeach is a low-alkaline reagent having a bicarbonate :carbonic acidratio of maximal sensitivity for glucose relative to “saccharoids.”Recently, Kingsley and Getchell (21) obtained excellent agreementbetween the enzymic and Somogyi-Nelson methods for plasma glu-

cose levels. However, in contradiction to these four reports of agree-ment, Saifer and Gerstenfeld (22) found consistently lower plasmaglucose levels with the enzymic than with the Somogyi-Nelson pro-

cedure. The discrepancy was quite closely proportional to the glu-cose level and ranged from several milligrams with plasma of lowglucose content to over 45 mg./100 ml. with plasma having a high

glucose content of some diabetic patients. Despite the preponder-ance of evidence for agreement between the enzymic and the more

specific copper reduction methods, this single exception has gen-erated some confusion. Because of. the specificity of the glucoseoxidase, ‘Saifer and Gerstenfeld contend that the disagreement must

be due to reducing substances other than glucose (“saccharoids”).This would not necessarily follow since, as we have shown, there is

little to choose between the two methods on the basis of interferingmaterials. On the other hand, there are those who would insist thatif the blood-sugar values obtained by the enzymic method are to beconsidered valid, they must agree with those obtained by theSomogyi methods, because of the large body of evidence supportingthe validity of the latter methods.

302 FALES fT AL. Clinical Chemistry

Because of the confusion concerning this point, this evidence will

be reviewed. Using the Shaffer-Somogyi Reagent 50 and a modifica-tion thereof, Somogyi (7, 9) found that little or no reducing materialremained in the zinc hydroxide filtrates after the removal of the

sugar with yeast. This finding was confirmed by MacKay (23). Wehave repeatedly verified this finding, using the Somogyi reagents

modified for colorimetry by Nelson (10) and by Somogyi (2) in theperformance of galactose tolerance tests by the fermentation method

in the clinical laboratory. Of the materials that have been suggestedby various authors (5, 24-27) to account for the high values obtainedwith tungstic acid filtrates (glutathione, ergothioneine, glucuronic

acid, uric acid, creatinine, creatine, ascorbic acid, and sugar phos-phates), only the sugar phosphates are acted upon by yeast, and

they are fermented at such a slow rate-less than one-twentieth therate of glucose fermentation (28)-that they would remain largelyunfermented after the 10-mm. fermentation period employed bySomogyi (16). Thus, the absence of nonfermentable reducing ma-

terial in the zinc hydroxide filtrates, as measured by the more specificcopper reagents, constitutes strong evidence in support of the spe-cificity of these methods for blood sugar. This conclusion is in noway vitiated by the several reports of the presence of nonferment-

able reducing material in zinc hydroxide filtrates measured by lessspecific reagents. In this category are the reports of Benedict (29)

and of Fujita and Iwatake (26), who used a copper reagent of highalkalinity and an alkaline ferricyanide reagent, respectively. Thelatter authors found non-sugar-reducing material equivalent to 5-10mg. of glucose per 100 ml. in zinc hydroxide ifitrates when measuredwith the ferricyanide reagent, but with these same filtrates they fullyconfirmed the validity of the blood-sugar values obtained with the

Somogyi low-alkali copper reagent.Further support for the validity of the Somogyi-Nelson procedure

is its agreement with chemical methods not of identical specificity.

The relative agreement presented in this paper with the fermentable

sugar determined with anthrone, a reagent specific for carbohy-

drates, may be cited, as well as the excellent agreement obtained byAnthanail and Cabaud (30) with the o-aminodiphenyl reagent. The

latter is a modified Tauber reaction with the requirement of a sugarhaving a potentially free or easily accessible aldehyde group (31, 32),

the green-colored product measured by Anthanail and Cabaud being

Vol. 7, No. 4, 1961 ESTIMATION OF SUGARS 303

quite specific for the aldoses (32). Unlike the anthrone reaction, nocolored product is obtained with polysaccharides because the acetic

acid contained in the reagent is not of sufficient strength to causetheir hydrolysis (33). Because of the wholly different specificitiesof these chemical methods, their agreement strongly supports thevalidity of the Somogyi-Nelson procedure.

References

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18. Asatoor, A. M., and King, B. J., Biooiein. .T. 56, xliv (1954).19. Harding, V. J., and Downs, C. B., J. BiO1. Chem. 101, 487 (1933).20. Shaffer, P. A., and Bomogyi, M., 1. BIO1. Chem. 100, 695 (1933).21. Kingsley, G. B., and Getehell, G., Clin. Chem. 5, 466 (1960).22. Saifer, A., and Gerstenfeld, B., J. Lab. GUn. Med. 51, 448 (1958).

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