methodforsimultaneousdirectestimationof ... · verysimilaroridentical. o-toluidine alsoreacts...

CLINICAL CHEMISTRY, Vol. 16, No. 2, 1970 85

Methodfor SimultaneousDirect EstimationofGlucoseand Xylosein Serum

Jesse F. Goodwin

A direct procedure for measuring serum monosaccharides is presented.Borate intensifies the colors produced when these substances react witho-toluidine, which, in the case of glucose and xylose, have absorption maximaat 480 nm and 630 nm, respectively. Thus, these sugars may be determinedsingly or simultaneously in a single sample by differential spectrophotom-etry. An alternative technique in which glucose oxidase and o-toluidineare used is also presented for the estimation of xylose and other pentosesugars in the presence of glucose. These two techniques permit the mea-surement of a pentose and a hexose other than glucose in protein solu-tions containing glucose. The techniques have a high degree of correla-tion and reproducibility. The effects of such variables as reagent concen-tration, reaction time, and borate concentration on the o-toluidine reactionare presented, along with an evaluation of bilirubin and hemoglobininterference.

A NUMBER OF PROCEDURES are available for theestimation of serum and plasma glucose. Most

of these techniques are based on the reducing prop-erties of glucose, which are shared to some extentby other reducing compounds present in serum andplasma. In the presence of strong acids, glucose isdehydrated to form hydroxymethylfurfural. Thealdehyde group of this derivative subsequentlyreacts with phenolic compounds to give a colorwhich serves as a means of quantification (1-4).

Glucose oxidase can be used for the specificmeasurement of glucose. However, this enzyme isinhibited by, e.g., uric acid, catechols, glutathione,cysteine, ascorbic acid, bilirubin, and hemoglobin(5-7).

Xylose, a pentose sugar, is used clinically in atest for carbohydrate absorption (8). Ordinarily,

From the Dept. of Biochemistry and the General ClinicalResearch Center for Children, Wayne State University Schoolof Medicine, and the Children’s Hospital of Michigan, Detroit,Mich. 48207.

Presented at the 20th National Meeting of the AmericanAssociation of Clinical Chemists and 12th Annual Meeting ofthe Canadian Society of Clinical Chemists, Washington, D. C.,Aug. 18-23, 1968.

Received April 25, 1969; accepted July 17, 1969.

adults excrete 2 to 5 mg of pentose sugars per kg ofbody wt per 24 h and none is normally detectable inserum (9). Xylose is a reducing sugar and inter-feres in methods for estimating glucose that dependon reduction. Roe and Rice (10) introduced aquantitative procedure for xylose based on thereaction of xylose with furfural in hot acetic acid;this product was then reacted with p-bromoanilineacetate to form a pink complex. Although specificfor aldopentose sugars, the method must be per-formed on blood or serum filtrates and requireslong incubation in darkness for maximum colorformation.

Hultman (11) introduced the use of o-toluicline asa reagent for the measurement of aldosaccharides.He suggested that glucose reacts with o-toluidineand other aromatic amines in hot acetic acid toform an equilibrium mixture of glycosylamines andthe corresponding Schiff base. The resulting greencolor of the reaction product has a maximum atapproximately 630 nrn. Zender (12) demonstratedthat the color formed by furfural and o-toluidinehad a different spectrum from that formed byreaction of o-toluidine with glucose or xylose. Thiswould appear to rule out a dehydration step forproduction of a furfural intermediate prior to

86 CLINICAL CHEMISTRY, Vol. 16, No. 2, 1970

reaction with o-toluidine. Mink and Habets (13)reported a method for the estimation of xylose inblood and urine filtrates with o-toluidine.

Considering that o-toluidine reacts with bothpentose and hexose sugars to yield reaction prod-ucts that differ in the maxima of their absorptionspectra, I have developed a method for the simul-taneous spectrophotometric quantification of glu-cose and xylose. The observation of Shibata (14)prompted the incorporation of borate ion in theo-toluidine reagents. The purposes of this paper are(a) to describe the o-toluidine reaction for hexoseand pentose sugars as applied directly to serum orprotein solutions, (b) to present methods forestimating xylose or glucose separately, (c) topresent a method for measuring glucose and xylosesimultaneously in serum, and (d) to present ameans for measuring xylose in the presence ofglucose in serum.

Materials and Methods

Reagents

o-Toluidine reagent. Transfer 3 g of thiourea to a3-liter Erlenmeyer flask. Add 1500 ml of glacialacetic acid. Heat slightly to bring into solution.Cool. Add 125 ml of o-toluidine. Mix and dilute to2000 ml with glacial acetic acid. Store in an amber-colored bottle at room temperature (stable in-definitely).

o-Toluidine-borate reagent. Dissolve thiourea inglacial acetic acid as outlined under the prepara-tion of o-toluidine reagent. Add 7.0 g of sodiumborate (Na2B4O7. 10H20), dissolve, and add 125 mlof o-toluidine. Dilute to 2000 ml with glacial aceticacid. (This reagent retains its sensitivity forat least three weeks when stored at room tempera-ture in an amber-colored container.)

Stock standard glucose. One gram of glucose is dis-solved and diluted to 100 ml with benzoic acidsolution (0.1 g/100 ml) in a volumetric flask.

Working glucose standards. Pipet 0.0, 0.5, 1.0,1.5, and 2.0 ml of stock standard glucose intorespective 10-in! volumetric flasks. Add 7.0 ml ofsalt-poor human albumin (10 g/100 ml) to allflasks and dilute to 10 ml with physiologic salinesolution. These samples represent 0, 50, 100, 150,and 200 mg of glucose, respectively, per 100 ml ofsample (the first is the reagent blank).

Stock standard xylose. One gram of xylose is dis-solved and diluted to 100 ml with benzoic acidsolution (0.1 g/100 ml) in a volumetric flask.

Working xylose standards. Prepare in the sameway as working glucose standards, except that thestock standard xylose is substituted for the stockstandard glucose.

“Glucostat” enzyme mixture. Dilute the con-tents of a glucose oxidase 4X vial (“Glucostat,”

Worthington Biochemical Corp., Freehold, N. J.)to 400 ml with water. Use on the day of analysis.(Refrigerate when not in use.)

Procedures

Separate estimation of glucose or xylose. Pipet0.1 ml of serum and standards into a test tube.Add 5.0 ml of o-toluidine reagent or o-toluidine-borate reagent. Mix and heat at 100#{176}Cfor 5 to 10mm. Cool in cold water for 2 to 3 mm. Set the spec-trophotometer with the reagent blank. Read theabsorbances at 630 nm for glucose estimation and480 nm for xylose estimation. Construct a concen-tration vs. absorbance graph and determine theconcentration of either glucose or xylose in theunknown from this graph.

Simultaneous spectro photometric estimation ofglucose and xylose. Pipet 0.1 ml of sample into atube and proceed as outlined under estimation ofglucose and xylose using the appropriate o-toluidinereagent. Read the absorbance of the mixture at 630inn and 480 nm. The calculation of glucose andxylose content follows.

CALIBRATION. Assay 0.1 ml of each of the work-ing standards of both glucose and xylose with o.-toluidine. Read the absorbance of the two sets ofstandards at 480 and 630 nm, respectively, againstthe reagent blank. For each set, divide the con-Scentration of the individual standard in mg per 100ml into its absorbance at each of the wavelengthsspecified to obtain the absorptivity (a). Averagethe absorptivity values of the glucose and xylosestandards at 630 nm (X1) and 480 nm (X2), re-spectively, to obtain the mean absorptivity valuefor glucose (a9) and xylose (as). Substitute into theformula for estimation of glucose concentration inmg/100 ml (C9):

- (a X2) (A X1) - (a X1) (AX2)- (aXj) (aX2) - (a9X2) (aXi)

where Ax1 is the absorbance of glucose and xylosemixture at 630 nm, and Ax2 is the absorbance ofglucose and xylose mixture at 480 nm.

likewise, xylose concentration may be calcu-lated using the following formula (C = xyloseconcentration in mg per 100 ml):

(a9 X1) (A X2) - (a9 X2) (A X1)Cx=

(a9 x1) (aX2) - (a9X2) (aXi)

Estimation of xylose in the presence of glucose.Pipet 0.1 ml of serum and of xylose standards intoseparate test tubes. Add 0.5 ml of the Glucostatenzyme mixture to the tubes. Incubate at 45#{176}Cfor45 mm. Mix occasionally during the incubationperiod. Cool to room temperature. Proceed withthe estimation of xylose as listed under the firstprocedure.

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0PERCENT BORIC ACID

Results and Discussion


The concentration of o-toluidine in the o-tolui-dine reagent affects the color intensity of thereaction. o-Toluidine reagent contains less than4 ml/100 ml, giving progressively much less coloron reaction with glucose and xylose. The amountof color formed reaches constancy when the o-toluidine reagent contains 5 to 10 ml of o-toluidineper 100 ml. The concentration of the reagent chosenfor the procedure is 6.25 ml/100 ml of glacialacetic acid.

Shibata (14) reported a sensitivity increase of1.56 times for glucose estimation after adding asaturated solution of boric acid (12 g/100 ml) to ano-amninobiphenyl-acetic acid reagent. My pre-liminary experiments with o-toluidine and aqueousboric acid showed that the color could be increased1.60 times. The presence of water in the o-toluidinereagent decreased the color yield of the o-toluidinereaction with glucose and xylose. For this reason,solid boric acid was added directly to the reagent.The effect of borate ion on the o-toluidine reactionis shown in Fig. 1. Freshly prepared o-toluidinesolution plus borate gives a color yield two to threetimes that of o-toluidine reagent without borate inthe reaction with glucose. A 10-mm heating timewith o-toluidine-borate reagent and glucose pro-duces more color than a 5-mm heating. The o-toluidine-borate reagent proposed here increasesthe sensitivity of the glucose reaction by a factor of

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2.5. The o-toluidine reaction with xylose is onlyslightly affected by the addition of borate. When a10-mn heating time is used, the addition of borateto the o-toluidine reagent has very little effect onthe color intensity of the o-toluidine-xylose reac-tion products, except for a possible slight initialdepression in sensitivity when the reagent con-tains less than 0.35 g of boric acid per 100 ml. Onthe other hand, the presence of borate enhancescolor formation by not more than 1.19 times whenthe heating time is 5 mm. The o-toluidine-boratereagent retains a stable and high sensitivity forat least 21 days. Addition of water tends to reducethe sensitivity. Boric acid forms a complex withhydroxyl groups, thus increasing ionization of thealpha glucose moiety (15). This complexing ofhydroxyl groups may tend to produce greaterreactivity of the free-aldehyde form or other reac-tive forms as opposed to the usual ring formation ofthese compounds in solution. Perhaps this in-creased aldehyde or keto activity in the case offructose enhances the production of an aldimine orketimine that reacts with o-toluidine. The sug-gested result of this reaction is the formation of aproduct or products which yield characteristicspectral absorption curves for hexose and pentosesugars.

Maximum spectral absorbance is obtained byheating glucose with o-toluidine reagent for 10 mmand a decrease in color formation is noted after 20mm of heating. However, a 5-mm heating time

Fig. 1. Effect of borate on the o-toluidine reaction with glucose and xyloseo.Toluidine reagent was reacted with albumin standards containing either glucose or xylose in the amountsspecified

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Fig. 2. Comparison of glucose reaction ratio with that for other sugars

Mannose I Gtucurono- i Ribose i Lyxose

Galoctose Frutose XyIise Arasnose

The glucose ratio was calculated by dividing the absorbance reading for the reaction product of 100 pgof glucose reacted with o-toluidine into that similarly obtained with 100 pg of the specified carbohy-drates. The modified o-toluidine reagent contained 0.1 g borate per 100 ml


may be used without a decrease in precision. Theglucose-o-toluidine reaction occurs when the ratioof reagent volume to sample volume is 20:100.However, greatest sensitivity is obtained when theratio of reagent volume to sample volume is 20:60.The ratio of sample volume to reagent volume inthe proposed technique is 50.

The relative amounts of color produced withvarious hexose and pentose sugars and glucurono-lactone is shown in Fig. 2. The color sensitivity ofthe reaction products of various sugars has beencompared to the reaction products of glucose underthe same conditions of analysis and is expressedrelative to glucose. The color yields at 630 nm ofgalactose, mannose, fructose, arabinose, lyxose,and glucuronolactone relative to that of glucoseare increased in the presence of boric acid; thoseof xylose and ribose are decreased slightly. There aresimilar increases for galactose, mannose, fructose,ribose, arabinose, and lyxose at 480 nm, whereasthe xylose color yield is decreased.

The reaction of o-toluidine with fructose, a keto-hexose, is noteworthy. Hultman (11) indicatedthat the reagent was specific for aldosaccharides.The addition of borate enhances the fructose-o-toluidine reaction considerably. The spectral ab-sorbance curve of the fruetose-o-toluidine reactionproducts appears to be identical to the spectral

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absorbance curve of other hexose sugars. This ob-servation suggests that the reaction products arevery similar or identical. o-Toluidine also reactswith glucosamine, mannose, and lactose to give acolor.

The spectral characteristics of the products ofthe reaction of glucose and xylose mixtures witho-toluidine, with and without added borate, areshown in Fig. 3. The spectral maxima of theglucose and xylose reaction products are suffi-ciently separated for excellent simultaneous spec-trophotometric measurement by differential tech-niques. A crossover, or isobestic point, occurs atapproximately 552 mm in the presence of borate.In the absence of borate, there appears to be abathochromic shift of the isobestic point to 564nm. The rather slight increase in absorbanee at620 mm in the presence of borate is caused by waterin the o-toluidine-borate solution. Once the ab-sorptivity of pure and known concentrations ofxylose and glucose is known, it is a relativelysimple matter to substitute it into the equationnoted and estimate both components simultane-

ously.Recovery of xylose added to pooled serum

specimens containing 50 to 125 mg of glucose per 100ml as estimated by a glucose oxidase method (17),is shown in Fig. 4; the simultaneous spectro-

WITHOUT BORATE

340 380 420 460 500 540 580 620 666 700 380 420 460 500 540 580 620 666 700

nm nm

GlucosefoundXylose recoveredXylose addedGlucoseinpooledsample (mean value)


photometric assay technique was used. Recoveryof glucose ranged from 96 to 105% in the pooledserum specimens examined. Xylose was added tothe pooled specimens in amounts ranging from 25to 108 mg/100 ml of serum. Residual amounts ofpentoses, 0.3 to 2 mg/100 ml, were quantifiedin some of the pooled specimens by the simul-taneous assay technique with o-toluidine-borate.The serum on which simultaneous assay is per-

formed must be free of visible hemolysis, icterus,or gross lipemia.

Amounts of xylose added to pooled serumsamples and amounts recovered by the simul-taneous spectrophotometric techniques are cor-related in the seattergram in Fig. 5. Xylose wasadded to pooled serum samples containing variableamounts of glucose to yield xylose concentrationsranging from 25 to 150 mg/100 ml. Recovery

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Fig. 3. Absorbance of reaction products of glucose and xylose with o-toluidine

Glucose and xylose mixtures were reacted with o-toluidine: (1) 10 mg glucose and 90 mg xylose/100 ml, (2) 30mg glucose and 70 mg xylose/100 ml, (3)50 mg each of glucose and xylose/100 ml, (4) 70 mg glucose and 30 mgxylose/100 ml, (5) 90 mg glucose and 10 mg xylose/100 ml, (6) 100 mg xylose/100 ml, (7) 100 mg glucose/100 ml.In the spectrum with borate an aqueous saturated borate concentration of 0.1 g/100 ml was used; the ratio ofreagent volume to sample volume was 70

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Fig. 4. Recovery of glucose and xylose from pooled serum samples by simultaneous assay witho-toluidineA-F are pooled sera plus known. Various amounts of xylose were added to fractions of each pool. Glucose andxylose are assayed simultaneously

V ‘-143+0997X(odded (recoveredXyIoe) Xylose)

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(X) mg Xylose p-Bromooniline Method

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(X) mg Xylose o-ToIu,dine Method

Fig.5. Recoveryof added xylose to pooled serum: a comparison of o.toluidine and p-bromoanilinemethodsKnown concentrations of xylose were added to freshly collected serum samples and assayed by the simul.taneous o.toluidine spectrophotometric assay and p-bromoaniline procedure (10)

Table 1. Quantification with o-Toluidine of Xyloseand Galactose in Serum in Presence of Glucose

Glucose In mixture,mg/100 ml

Concentration (mg/100 ml)Amount Amountadded found


values shown on the left in the scattergram (Fig.5) show an excellent correlation with the amountsof added xylose. The scattergram on the right com-pares xylose values obtained by the simultaneousassay of xylose with o-toluidine and by thep-bromoaniline method of Roe and Rice (10).Both methods show excellent correlation on the 30samples analyzed. Glucose was measured in thesesame samples by a glucose oxidase method (17) andthe values obtained were compared with those ob-tained by the o-toluidine method. The relationshipwas V = 2.467 + 0.948 X, where Y is the o-tolu-idine method and X is the glucose oxidase method.The glucose concentrations of the samples rangedfrom 48 to 130 mg/100 ml, with most above 80

Xylose 100 25 25100 50 50100 75 78100 100 9850 50 4550 100 9650 150 14550 175 170

Galactose 100100505050

5010050

100150

5010251

101152

mg/100 ml. The correlation coefficient (r = 0.99)showed that there is excellent correlation betweenthe two methods of assay.

Acetic acid reacts with bilirubin, converting itto biliverdin, a green pigment. In view of this, theeffect of bilirubin on the glucose-o-toluidine reac-tion was examined. Bilirubin (purified) was addedto albumin solution containing 120 lug of glucoseper 100 ml and analyzed for glucose content bythe o-toluidine method. Interference was not ap-parent at bilirubin concentrations below 10 mg/100ml. Serum bilirubin interferes with the o-toluidinereaction for xylose when present in amounts above5 mg/100 ml. Bilirubin interference in theassay may be eliminated by using a Folin-Wuserum filtrate for the estimation. Added hemo-globin in concentrations above 226 mg/100 mlinterferes with the o-toluidine procedure forxylose. However, hemoglobin in concentrations be-low 570 mg/100 ml did not interfere with glucosemeasurement.

An alternative method for the quantification ofxylose in the presence of glucose consists of glucosedestruction with the aid of glucose oxidase priorto xylose measurement. The enzyme mixture wasincubated with serum samples at 45#{176}or 56#{176}C.Incubation for 30 mm proved to be sufficient formost samples. However, a 60-mn incubation is re-quired for samples containing more than 400 mgof glucose per 100 ml of serum. For the purposesof the procedure, a 45-mm incubation was adopted.Martinek (18) recommended a 90-mm incubationfor complete destruction of glucose prior to esti-mation of xylose by a copper-reduction procedure.This time has been reduced by the use of an in-

References


creased enzyme concentration and an increasedincubation temperature. This procedure avoidsthe application of simultaneous equations forquantifying xylose. The disadvantage of the tech-nique is the necessity of using an alternativemethod for estimating glucose, if data on the con-centrations of both carbohydrates are desired.However, the technique may prove ideal for thelaboratory that seldom assays samples for xylose.In the event that glucose values are above 250mg/100 ml, incubation with glucose oxidase shouldbe prolonged to 60 mm. Results for glucose andxylose by this technique are in excellent agree-ment with those obtained by the simultaneousspectrophotometric assay procedure.

An additional advantage of the technique inwhich glucose is destroyed before estimatingpentoses is its versatility for the measurement ofhexoses such as galactose, fructose, and(or) man-nose when they are present with glucose. By useof the combined glucose oxidase-o-toluidine andsimultaneous spectrophotometric techniques, it ispossible to measure a mixture of three sugars inserum, two hexoses and a pentose, providing oneof the hexose sugars is glucose. Recovery of bothxylose and galactose in the presence of glucosewith this technique is excellent (Table 1). Theglucose oxidase method for removal of glucose be-fore assay of xylose with o-toluidine comparesvery favorably with the o-toluidine technique forthe simultaneous assay of xylose in the presenceof glucose. Twenty-three samples, assayed by bothmethods, yielded means of 56.6 mg/100 ml for thesimultaneous method and 55.3 mg/100 ml for theglucose oxidase method (V = 4.6 + 0.94 X,S = 2.48, and r = 0.99, where V = simul-taneous assay, and X = glucose oxidase-o-tolu-idine assay).

Normal values for glucose by the o-toluidinehave been estimated (16, 19, 20). The values ob-tained by the o-toluidine method are comparableto those obtained by the Somogyi-Nelson andglucose oxidase methods.

Supported by Research Grant FR-74 from the National In-stitutes of Health, U. S. Public Health Service.

1. McDonald, H. J., Bermes, E. W., and Spitzer, R.. H., Clinicalsignificance of ionography. Fed. Proc. 14, 733 (1955).

2. Barac, G., and Delvenne, J., A new technique for the de-termination of blood sugar. Bull. Soc. Chim. Biol. 29, 1094(1947).

3. Groger, W. K. L., l)etermination of sugars in biologicalmedia with thymol in sulfuric acid. Clin. Chim. Ac/a 6, 866(1961).

4. Morris, D. L., The quantitative determination of carbo-hydrates with Dreywood’s anthrone reagent. Science 107, 254(1948).

5. Cawley, L. P., Spear, F. E., and Kendall, II., Ultramicrochemical analysis of blood glucose with glucose oxidase. Amer.J. C/in. Pathol. 32, 195 (1959).

6. Saifer, A., and Gerstenfeld, S., The photometric microde-termination of glucose with glucose oxidase. J. Lab. Clin. Med.51, 448 (1958).

7. Fales, F. W., Russell, J. A., and Fain, J. N., Applicationsand limitations of the enzymic reducing (Somogyi) and anthronemethods for estimating sugars. CLIN. CHEM. 7, 389 (1961).

8. Helmer, 0. 1I., and Fout,s, P. J., Gastrointestinal studies;excretion of xylose in pernicious anemia. J. Clin. Invest. 16,343 (1937).

9. Tower, D. B., Peters, E. C., and Pogorelsk.in, M. A., Natureand significance of pentosuria in neuromuscular disease. Neu-rology 6, 125 (1948).

10. Roe, J. H., and Rice, E. W., Photometric method for de-termination of free pentoses in animal tissues. J. Biol. Chem.173, 507 (1948).

11. Hultman, E., Rapid specific method for determination ofaldosaccharides in body fluids. Nature 183, 108 (1959).

12. Zender, R., Automatic microdetermination of aldohexosesin biologic fluids with o-toluidine. Clin. Chim. Ac/a 11, 88 (1965).

13. Mink, L. J. K., and Habets, L., Microdeterrnination of

d-xylose in blood and urine. C/in. Chem. Ada 14, 704 (1966).

14. Shibata, S., Improved technique of o-aminobiphenyl methodfor the determination of glucose in serum. Bull. Yamaguchi Med.School 9, 13 (1962).

15. Boekesen, J., L’in.fluence de quelques glycols sur la conduct-ibiit#{233}electrique de l’acide borique. Rec. Tray. Chim. 34, 96(1915).

16. Dubowski, K. M., An o-toluidine method for body fluid

glucose determination. CLIN. CuxM. 8, 215 (1962).

17. Kingsley, G. R., and Getchell, G., Direct ultramicro glucoseoxidase method for determination of glucose in biologic fluids.OLIN. CHEM. 6, 466 (1964)).

18. Martinek, 11. G., Micromethod for the estimation of xylosein serum and urine. C/in. Chim. Ac/a 12, 176 (1965).

19. Russell, S., Bryant, M., and Morrison, D. B., A rapid bloodglucose method. OLIN. CHEM. 10, 641 (1964) (Abstract).

20. Hyvarinen, A., and Nikkila, E. A., Specific determination ofblood glucose with o-toluidine. Clin. Chim. Ac/a 7, 140 (1962).

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