THE DETERMINATION OF SUGAR IN BLOOD AND SPINAL FLUID WITH ANTHRONE REAGENT*

THE DETERMINATION OF SUGAR IN BLOOD AND SPINAL FLUID WITH ANTHRONE REAGENT* BY JOSEPH H. ROE (From the Department of Biochemistry, School of Medicine, George Washington University, Washington, D. C.) (Received for publication, July 16, 1954) The anthrone reagent of Dreywood (1) has been applied to the determination of blood sugar by Durham, Bloom, Lewis, and Mandel (2), Fetz and Petrie (3), and Zipf and Waldo (4). In the procedures developed by these authors, the heat resulting from mixing sulfuric acid with water causes the reaction to take place. Greater precision is obtained by heating the mixture of anthrone, sulfuric acid, and carbohydrate for a definite time in a constant temperature bath. Scott and Melvin (5) reported that the heat of mixing procedure is satisfactory if accuracy no better than ~5 per cent is required. They obtained data showing a coefficient of variation of ko.48 per cent in their method, which involves heating in an ethylene glycol bath at 90 for 16 minutes. In our laboratory a method was developed for the determination of dextran in blood and urine in which a mixture of anthrone reagent and dextran solution is heated in a boiling water bath for a definite time (6). In twelve determinations by this method with dextran solution there was observed a coefficient of variation of ko.36 per cent, and in twelve determinations in which the dextran was precipitated from solution by alcohol the coefficient of variation was f0.56 per cent. Our observations with respect to the precision of the heat of mixing procedure, compared with heating for a definite time in a constant temperature bath, are in agreement with the work of Scott and Melvin (5). We have adapted our procedure for the determination of dextran to the estimation of the sugar in blood and spinal fluid. A stabilized anthrone reagent has been developed, and certain findings of interest are reported. Method Reagentsi. Anthrone reagent. A solution containing 0.05 per cent anthrone, 1 per cent thiourea, and 66 per cent by volume HzS04 is used. For each liter of reagent, place in a suitable flask 340 ml. of distilled water and add cautiously 660 ml. of concentrated sulfuric acid, sp. gr. 1.84, of highest * Supported in part by a grant from the Division of Research Grants and Fellowships, National Institutes of Health, United States Public Health Service. 335

336 ANTHRONE BLOOD SUGAR METHOD purity. Prepare a stock of a number of liters of this acid solution. Place in a flask 500 mg. of recrystallized anthrone, 10 gm. of highest purity thiourea, and 1 liter of the 66 per cent by volume H$SOd. Warm the mixture to SO-go, occasionally shaking the flask to mix the contents. Do not overheat the mixture. After solution is complete, cool and store in a refrigerator. To obtain maximal color production, this reagent should be freshly prepared every 2 weeks. 2. Standard glucose solutions. (a) Stock solution. Obtain highest purity glucose and dry in a vacuum oven at 60-70. Dissolve 100 mg. of glucose in 100 ml. of saturated benzoic acid solution. (b) Working standard. Place 10 ml. of the stock solution in a 100 ml. volumetric flask and make up to volume with saturated benzoic acid solution. 1 ml. of this solution, containing 0.1 mg. of glucose, is used as a standard. 3. Deproteinixing reagents. Prepare 5 per cent trichloroacetic acid, or 10 per cent Na2W04.2Hz0 and 0.66 N HzSO+ according to Folin and Wu (7), or 5 per cent ZnSOr.7HzO and 0.3 N Ba(OH)z, according to Somogyi (8). Procedure for Blood Blood is deproteinized with tungstic acid (7), with Ba(OH)z and ZnSOc (8), or with 5 per cent trichloroacetic acid, the preferable dilution being 1: 10. Place 1 ml. of blood filtrate in a photoelectric calorimeter tube of the test-tube type. Continue until 1 ml. of each filtrate to be analyzed has been placed in a matched calorimeter tube. Pipette 1 ml. of standard glucose solution, containing 0.1 mg. of glucose, into a similar calorimeter tube, and also 1 ml. of distilled water into another tube to be used as a blank for setting the calorimeter. Add 10 ml. of the anthrone reagent to each tube. Twirl the tubes to bring about thorough mixing. Stopper each tube by inserting jirmly a rubber stopper through which has been passed a glass tube about 5 mm. in diameter and 4 inches in length. The purpose of this stopper is to keep water from splashing into the tube when placed in the boiling water bath. Place all tubes in a rack suitable for immersion in a water bath. Set the rack of tubes in a tap water bath for 3 to 5 minutes to bring the contents of each tube to the same temperature, and then place the tubes in a boiling water bath for 15 minutes. A metal water bath, admitting minimal amounts of light, should be used. Remove the rack of tubes to a tap water bath and let stand until cool. When the tubes have come approximately to room temperature, remove them from the cooling water bath, wipe dry with a towel, and let stand at room temperature in a moderately illuminated or dark place. Allow the tubes to stand 20 to 30 minutes after removal from the boiling water bath, and then compare in a photoelectric calorimeter, using a 620 rnp filter.

J. H. ROE Calculation s X 0.1 X dilution of blood X 100 = mg. glucose per 100 ml where DU = optical density of the unknown, DX = optical density of the standard, 0.1 = mg. of glucose in 1 ml. of standard solution. Procedure for Spinal Fluid To 1 part of spinal fluid add 9 parts of distilled water or 9 parts of 5 per cent trichloroacetic acid. Mix thoroughly and filter if trichloroacetic acid is used. Water dilution is satisfactory unless a very high protein content is encountered. Place 1 ml. of diluted fluid or filtrate in a matched 14 c 1.2 5 IO h s 2 0% E 04 8 0.2 2s 50 loo 200 300 400 IIICROCRAHS OF GLUCOSE FIG. 1. Curve showing agreement with Beer s law when 25 to 400 y of glucose were submitted to the anthrone procedure. calorimeter tube, add 10 ml. of anthrone reagent, and proceed as directed for the blood sugar determination. DISCUSSION Concentration of H~JOK---A concentration of H804 of 60 per cent by volume was found to be optimal (6). This concentration of acid should always be produced if other quantities of filtrate are used. This is obtained in the regular procedure by adding 10 ml. of anthrone reagent, which contains 66 per cent by volume of HzS04, to 1 ml. of filtrate. Agreement with Beer s Law-The agreement of the color produced with the anthrone reagent with Beer s law is presented in Fig. 1. This curve shows the excellency of agreement up to 200 y of glucose and the deviation from strict linearity with 300 and 400 y. As in this method 1 ml. of a 1: 10 dilution of blood is regularly used, the microgram values on the abscissa correspond to mg. of sugar per 100 ml. of blood. Therefore, concentrations up to 200 mg. of sugar per 100 ml. of blood are correctly determined; with concentrations of 300 and 400 mg. per 100 ml. the results are underestimated 3 and 8 per cent, respectively, if read against a stand-

338 ANTHRONE BLOOD SUGAR METHOD ard within the range of strict agreement with Beer s law. To avoid the error due to deviation from Beer s law at concentrat,ions above 200 mg. per ml., repeat the determination, using more dilute filtrate, or make corrections according to the curve of Fig. 1. For most work, however, values around 300 to 400 mg. per 100 ml. may be accepted without repeating the determination or making corrections. TABLE Comparative Blood Sugar Values Values obtained by the anthrone method with blood filtrates prepared by deproteinizing with 5 per cent trichloroacetic acid, tungstic acid (Folin-Wu), and ZnSOa- Ba(OH)z (Somogyi) reagents. Blood Mean. No. Tungstic (1) acid 1 73.1 82.2 68.5 13.4 2 85.7 89.4 82.2 7.2 3 117.8 124.4 116.9 7.5 4 135.8 148.8 133.9 14.9 5 84.0 95.2 77.5 17.7 6 92.0 101.3 85.6 15.7 7 82.1 83.9 76.2 7.7 8 79.2 81.5 74.5 7.0 9 63.4 68.1 63.9 4.2 10 88.0 83.9 81.5 2.4 11 115.0 113.8 107.2 6.6 12 95.0 91.6 83.0 8.6 92.6 TCA (2) 97.0 I Mg. sugar per 100 ml. blood (3) 87.6 (2) - (3) _- Ba ppt., TCA filtrate, ph 8.3 6.3 5.6 7.2 5.6 5.8 9.1 9.4 6.6 Results with Diflerent Deproteinixing Reagents-The sugar values obtained with anthrone reagent are related to the method of deproteinization of the blood. In Table I are data obtained upon filtrates prepared by three different procedures. The highest values were obtained with trichloroacetic acid (TCA), the lowest with filtrates prepared with Somogyi reagents, and intermediate values with filtrates prepared with tungstic acid. Statistical analysis shows a highly significant difference (2 <O.Ol) between the results obtained by TCA and Somogyi reagents and those by tungstic acid and Somogyi reagents. A moderately significant difference (P <0.05) exists between the data obtained by TCA and those by tungstic acid. The higher values obtained on filtrates prepared by TCA deproteiniza-

J. H. ROE 339 tion appeared to be due to carbohydrate in phosphate combination, which would be precipit,ated by barium when blood is deproteinized with Somogyi reagents. To test this assumption, the TCA filtrate was alkalized to phenolphthalein, treated wit.h small amounts of Somogyi reagents, centrifuged, and washed twice with 50 per cent alkaline alcohol; the carbohydrate content of this barium precipitate was determined by the anthrone method. The results (last column of Table I), ranging from 5.6 to 9.1 mg. per cent, account fairly well for the differences in the blood sugar values obtained upon filtrates prepared with TCA and Somogyi reagents. Probably a considerable part of the fraction in. the barium precipitate from blood, which we have determined by the ant,hrone procedure, is hexose diphosphate. This suggestion is based upon the fact that, of the bariumprecipitable carbohydrate compounds, hexoses yield optimal or near optimal color production with anthrone reagent in 15 minutes boiling, while pentoses react very rapidly with this reagent and the color produced fades considerably in the boiling period used. Ribose and arabinose yielded less than 10 per cent of the optical density produced by an equivalent amount of glucose when the anthrone method, as outlined above, was used. The barium-precipitable carbohydrate fraction in blood is of considerable interest because it probably consists of intermediates in the carbohydrate metabolism of the blood cells. This assumption is supported by our observation, discussed later, of no difference in the values obtained with TCA and Somogyi reagents upon spinal fluid, a biological fluid that is essentially cell-free. Comparison of Anthrone Procedure with Copper Reduclion-The anthrone method compares most closely with the Nelson-Somogyi (9, 8) copper reduction procedure when tungstic acid blood filtrate is used. In a series of nineteen comparative blood sugar determinations on tungstic acid filtrates, the mean values per 100 ml. of blood were 108.84 mg. for anthrone and 110.94 mg. for copper reduction. Analysis of variance of these data showed that there is no significant difference between the results by the two procedures. Clinically, the values obtained by the proposed anthrone method may be interpreted as similar to the results obtained by copper reduction methods with tungstic acid filtrates. Eflect of Tungstate-Tungstic acid is precipitated from aqueous solution by excess sulfuric acid. The tungstate ion is also reduced by anthrone reagent, yielding a blue color t.hat absorbs at 620 rnp wave-lengths. Tungstic acid, as a deproteinizing agent, might offer serious interference in anthrone procedures, but, fortunately for clinical usage, the small amount of tungstate left in the Folin-Wu blood filtrates does not interfere. The

340 ANTHRONE BLOOD SUGAR METHOD data of Table II make this clear. In this study 1 part of 10 per cent sodium tungstate was mixed with 1 part of 0.66 N HsS04 and 8 parts of water, making a 1 per cent H2WO4 solution. Different aliquots of this solution were mixed with anthrone reagent, and the usual procedure for blood sugar determination was carried out. The interference resulting from 0.2, 0.3, 0.5, 0.7, and 1 ml. of 1 per cent HzW04 was equivalent, respectively, to 0, 1, 2, 29, and 216 mg. of glucose per 100 ml. These data show that not until more than one-half of the amount of tungstate used in protein precipitation of blood remains in the filtrate will serious interference occur. Hence, only when bloods containing less than onehalf the usual amount of protein are encountered will error arise due to tungstate left in the filtrate. With spinal fluid, however, tungstic acid deproteinization cannot be used. We observed serious interference from - TABLE Effect of Tungstic Acid on Color Prod~uction by Anthrone Reagent 1 ml. solution containing 1 per cent H2WOn ml. II Optical density Mg. per cent as glucose 0.2 0 0 0.3 0.0044 1.0 0.5 0.0090 2.1 0.7 0.1249 29.0 1.0 0.9210 216.0 tungstate in published procedures for spinal fluid when our anthrone method was applied. Xtabilixed Reagent-In solution, anthrone is an equilibrium mixture of the tautomeric forms of 9-ketoanthracene. The enol modification, anthranol, is the active form in the reaction of anthrone reagent with carbohydrate; it condenses with furfural, or a furfural derivative, to form the blue-colored product. The assumption that anthranol is the active form is supported by the fact that the reaction is prevented by oxidizing agents and is continued or supported by reducing agents. Perhaps the most serious limiting factor in the use of anthrone methods is the instability of anthrone in sulfuric acid. Upon standing, the anthrone reagent develops a brownish color and its chromogenic property decreases. This is due to an oxidative change. We have overcome this limitation by introducing an antioxidant, thiourea, into the reagent. When thiourea in 1 per cent concentration is added to the sulfuric acid solution of anthrone and the mixture is kept in a refrigerator, the reagent does not change color upon standing. The chromogenic capacity of this improved anthrone reagent remains essentially unchanged for 2 weeks, but diminishes there-

J. H. ROE 341 after. After 3 and 4 weeks of standing at 6 its capacity to produce color with glucose decreased 11 and 16 per cent, respectively. This improvement in the keeping quality of the anthrone reagent brings the anthrone method into the realm of practical routine procedures. The quality of the blue color is also improved by thiourea, which prevents the development of greenish tinges. Effect of Light-We have previously shown that the blue color formed by the reaction of anthrone reagent with carbohydrate fades rapidly in sunlight (6). It has been observed further that the results obtained when the tubes containing the reaction mixture are boiled in water in a glass beaker are more variable than when placed in boiling water in a metal container to which minimal amounts of light are accessible. In sunlight the anthrone reaction with carbohydrate takes place faster and fading occurs sooner. Since the tubes in a transparent water bath receive a variable exposure to light, it is obvious that the precision will not be as good under such conditions. Filter Paper-Some filter papers contain appreciable water-soluble carbohydrate. We determined the amount of error possible from using seven different grades of filter paper by passing 25 ml. of 5 per cent trichloroacetic acid through an 11 cm. paper and analyzing the filtrate by the anthrone method. Calculated as a blood filtrate diluted 1: 10, and expressed as glucose, the following values were obtained: 4.8, 3.0, 1.0, 1.0, 5.0, 2.4, and 1.0 mg. per 100 ml. Whatman s acid-washed filter paper No. 42 is free from water-soluble carbohydrate. A blank test should be carried out upon the filter paper, and paper of a grade suitable for the work at hand should be selected. Comparative Study of Spinal Fluid Procedures-The results of a comparative study of anthrone and copper reduction procedures with spinal fluid extracts prepared by water dilution and by deproteinization with trichloroacetic acid and the ZnS04-Ba(OH)z reagents of Somogyi (8) are presented in Table III. Analysis of the variance of the data in Table III shows the following relationships: With the anthrone method there is no significant difference between the results obtained upon filtrates prepared by Hz0 dilution and trichloroacetic acid deproteinization (Columns 1 and 2) and in tjhe results with extracts prepared by Hz0 dilution and by deproteinization with trichloroacetic acid and with the Somogyi reagents (Columns 1 and 2 and 3 in Spinal Fluids 5, 6, 7, 8). The values obtained by the anthrone and copper reduction methods upon spinal fluids diluted with Hz0 also do not vary significantly (Columns 1 and 4). However, the results obtained by the copper reduction method with extracts prepared by He0 dilution and by Somogyi reagents (Columns 4 and 5) vary significantly. The data in Table III show that there is essentially no phosphorylated

342 ANTHRONE BLOOD SUGAR METHOD sugar in spinal fluid, as the values obtained with filtrates prepared by Somogyi reagents are, within the limits of error, the same as those obtained with filtrates prepared with trichloroacetic acid. Sugar in the spinal TABLE Comparative Analyses of Spinal Fluids The anthrone and copper reduction (Nelson-Somogyi) methods were used. The filtrates were prepared by H,O dilution and by deproteinization with 5 per cent trichloroacetic acid and with the Somogyi reagents, diluted 1:lO. III Spinal fluid No. Hz0 dilution (1) (2) 1 58.9 61.3 2 58.4 57.5 3 66.2 70.3 4 55.2 55.6 5 59.6 60.6 6 66.2 66.7 7* 89.3 89.3 8 60.1 64.1 * Contained 260 mg. per cent of protein. Mg. sugar per 100 ml. of spinal fluid Anthrone method Copper reduction method I -I- 5 per cent CClsCOOH ZnSOa-Ba(OH)z Hz0 dilution ZnSOeBa(OH)n TABLE (3) (4) 60.6 66.2 88.8 61.1 IV 63.8 57.7 60.0 54.5 66.4 65.4 52.1 55.7 64.5 60.0 67.2 63.0 92.3 88.0 61.4 63.0 Effect of Protein on Color Production by Anthrone Reagent Blood albumin in glucose solution mg. per ml. 0.1 0.2 0.4 1.0 I Per cent increase in optical density 1.4 2.2 3.7 8.4 fluid is, therefore, not undergoing intermediary metabolism, as would be expected in a biological fluid that is almost cell-free. The practical importance of the data of Table III is that they show that the sugar in spinal fluid can be determined by the anthrone method without deproteinization of the fluid. It is true that protein increases the optical density in the anthrone procedure, but the amount of protein in most spinal fluids does not cause appreciable interference. 0.1 mg. of albumin per ml., corresponding to the protein concentration of filtrate

J. H. ROE 343 prepared from a spinal fluid containing 100 mg. per cent, the fluid being diluted 1: 10, yielded an increase in optical density of 1.4 per cent (Table IV). Thus, the increase in sugar content in most analyses, when the procedure is carried out upon spinal fluid diluted with water, would be less than 1 mg. per cent, as all normal and many pathologic spinal fluids have a protein content of less than 100 mg. per cent. Spinal Fluid 7 (Table III) contained 260 mg. per cent of protein, and the values obtained by the anthrone procedure upon the three types of filtrate are about the same. In Table IV a protein concentration of 1 mg. per ml., corresponding to filtrate from a spinal fluid containing 1000 mg. per cent of protein, resulted in an increase in optical density of only 8.4 per cent. This increase would not influence the clinical interpretation; however, when precise values are desired, the spinal fluid may be treated with 5 per cent trichloroacetic acid and filtered before applying the anthrone procedure. SUMMARY 1. A method has been developed for the determination of sugar in blood and spinal fluid with anthrone reagent. A mixture of deproteinixed filtrate and anthrone reagent in a calorimeter tube is heated in a boiling water bath for 15 minutes, cooled, and compared calorimetrically with glucose standard solution treated similarly. 2. The stability and chromogenic property of the anthrone reagent have been greatly improved by introducing thiourea into the reagent. 3. With the anthrone method a considerable amount of barium-precipitable carbohydrate compounds was demonstrated to be present in blood. BIBLIOGRAPHY 1. Dreywood, R., Innd. and E ng. Chem., Anal. Ed., 18, 499 (1946). 2. Durham, W. F., Bloom, W. L., Lewis, G. T., and Mandel, E. E., Pub. Health Rep., U. S. P. H. S., 65, 670 (1950). 3. Fetz, R. H., and Petrie, L. M., Pub. Health Rep., U. S. P. H. S., 65, 1709 (1950). 4. Zipf, R. E., and Waldo, A. L., J. Lab. and Clin. Med., 39, 497 (1952). 5. Scott, T. A., Jr., and Melvin, E. H., Anal. Chem., 25, 1656 (1953). 6. Roe, J. H., J. Biol. Chew, 208, 889 (1954). 7. Folin, O., and Wu, H., J. Biol. Chem., 38, 81 (1919). 8. Somogyi, M., J. Biol. Chem., 160, 69 (1945). 9. Nelson, N., J. Biol. Chem., 153, 375 (1944).

THE DETERMINATION OF SUGAR IN BLOOD AND SPINAL FLUID WITH ANTHRONE REAGENT Joseph H. Roe J. Biol. Chem. 1955, 212:335-343. Access the most updated version of this article at http://www.jbc.org/content/212/1/335.citation Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/212/1/335.citation.full.h tml#ref-list-1