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CLIN.CHEM.21/1, 119-124 (1975) Nature of Materials in Serum That Interfere in the Glucose Oxidase-Peroxidase--o- Dianisidine Method for Glucose, and Their Mode of Action Walter J. Blaedel and James M. Uhi Separation of blood serum on Sephadex G- 100 reveals three fractions that interfere with the glucose oxidaseperoxidase method for serum glucose when o-dianisidine is used as the chromogen. A low-molecular-weight fraction containing primarily uric acid, a fraction containing protein with a molecular weight of about 40 000, and a fraction of even higher molecular weight (-.‘ 500 000) each interfered with glucose recovery when glucose was measured by this procedure. The uric acid fraction and the isolated 40 000 molecular weight fraction interfere by competing with o-dianisidine for hydro- gen peroxide in the peroxidase-catalyzed color-formation step. The high-molecular-weight fraction not only interferes with the peroxidase reaction, but also with the glucose oxidase reaction itself. These agents cause values to be low by as much as 20% in the manual determination of glucose in normal serum if thejr interference is not recognized. AddItional Keyphrases: molecular-weight materials interference by uric acid and high#{149} interfering material in urine Keston (1) first conceived an enzymatic method for determining glucose in biological fluids, in which the glucose oxidase1 reaction is coupled to the peroxidase reaction in the presence of a chromogen. Use of o- dianisidine in a quantitative procedure was reported by Teller (2) shortly thereafter. Since then, many glucose oxidase methods, both manual and au- Department Wis. I of Chemistry, University of Wisconsin, Madison, 53706. Terminology reductase, reductase, 1.7.3.3; and Received used: glucose oxidase, l-D-glucose:oxygen EC 1.1.3.4; peroxidase, donor:hydrogen peroxide EC 1.11.1.7; uricase, urate:oxygen oxidoreductase, o- dianisidine, 3,3’-djmethoxybenzidine. July 25, 1974; accepted Oct. 21, 1974. oxidooxidoEC tomated, have been reported. About 20 references to methods reported for clinical use appear in a paper by Miskiewicz et al. (3). Attempts to apply this method directly to plasma or serum samples have not given highly precise results, owing to interferences that are present in varying amounts. Plasma has been shown to contain inhibitors of glucose oxidase methods (4, 5), which are generally removed by protein precipitation (4, 6) or extreme dilution of serum (5, 7) in manual methods. In automated methods, serum is generally dialyzed against a buffer solution to remove the glucose from the protein serum matrix (8-10), sq that interfering material of high-molecular-weight is excluded. Because automated methods are most frequently used for routine analysis, the interferences most often discussed are those caused by substances of low molecular weight, such as uric acid, ascorbic acid, and bilirubin. A recent study (3) showed that uric acid was the only such low-molecular-weight substance having a significant effect on determinations of glucose in normal or slightly above-normal physiological concentration. Interference by uric acid has been reported by many workers and possible mechanisms for it have been proposed (8, 11, 12). In early work on an electrochemical method for tollowing the glucose oxidase reaction (13), a high-molecular-weight serum fraction separated with a column of Sephadex G-50 was found to inhibit glucose oxidase. Separation of serum by use of Sephade, G100 has revealed two separate high-molecular-weight fractions and a low-molecular-weight fraction, each of which interfered with the glucose oxidase-peroxidase method. Preparations of each of the fractions CLINICAL CHEMISTRY, Vol. 21. No. 1, 1975 119 were tested for their effects on each step of the method. On the basis of the results obtained from these tests, a mode of action of each of the interferences is proposed. Materials and Methods Apparatus Visible absorbances at 460 nm were measured with a Spectronic 88 spectrophotometer (Bausch & Lomb, Rochester, N. Y. 14602). Ultraviolet absorbance studies of uric acid were performed with a Cary 14 double-beam recording spectrophotometer (Applied Physics Corp., Pasadena, Calif.). The oxygen electrode used was a Clark-type oxygen sensor, consisting of a platinum wire (20 am in diameter), heat-sealed into a glass tube. The end of the glass tube was ground off and polished, exposing a 20-am cross-section of the wire, which was covered with a polypropylene membrane (0.001 inch thick). The internal reference electrode was silver-silver chloride. The platinum microelectrode was held at -0.5 V with respect to the reference electrode. Currents were measured with a Model 414 S picoammeter (Keithley Instruments, Inc., Cleveland, Ohio 44139) and recorded on an Omniscribe chart recorder (Houston Instruments, Bellaire, Tex. 77401). Rate measurements were carried out in a 4.5-mi cylindrical cavity in a Plexiglas block equipped with a small magnetic stirrer. Solutions were added and withdrawn by means of hypodermic syringes through ports, which could be sealed to prevent access of air to the reaction cavity. Reagents Glucose oxidase solutions of two different concentrations (70 U/mI and 5 U/mI) were prepared from a powder having a glucose oxidase (EC 1.1.3.4) activity of 110 U/mg (Worthington Biochemical Corp., Freehold, N. J. 07728). A reagent solution containing, per liter, 100 mg of o- dianisidine dihydrochloride and 3000 U of horseradish peroxidase (EC 1.11.1.7; Worthington) was prepared in 0.1 mol/liter phosphate buffer (12.814 g of KH2PO4 and 1.02 g of K2HPO4 in 1 liter of solution, adjusted to pH 6.0 with concentrated KOH). A 50 U/liter solution of uricase (urate oxidase; EC 1.7.3.3) was prepared by dissolving 0.0197 g of crude uricase (17 U/g; Sigma Chemical Co., St. Louis, Mo. 63178) in 5 ml of 0.1 mol/liter borate buffer (0.6184 g. of H3B03 in 100 ml of solution, adjusted to pH 8.5 with concentrated KOH). Glucose standard solutions (100 mg/100 ml) were prepared by dissolving 0.1000 g of anhydrous glucose in 100 ml of water. Two uric acid solutions, 7.0 mg/ 100 ml and 4.2 mg/100 ml, were prepared by dissolving 0.0070 g and 0.0042 g of the solid, respectively, in 100 ml of warmed water. A hydrogen peroxide solution having a concentration of about 0.2 mmol/liter 120 CLINICAL CHEMISTRY. Vol. 21, No. 1, 1975 was prepared by two successive 250-fold dilutions of a 30% solution of hydrogen peroxide. All of the above chemicals were reagent grade. Samples of pooled serum and “Q-Pak Automated Chemistry Control Serum” (Hyland Div. Travenol Laboratories, Inc., Costa Mesa, Calif. 92626) were used in the initial chromatographic separations. The Hyland control serum was used in the preparative separation of the two high-molecular-weight frac- tions, because we found it contained greater concentrations of each interference than did the pooled serum. For interference studies, solutions of the two highmolecular-weight fractions were used directly as collected from the preparative separation outlined below. Procedures Serum was separated by gel-chromatography on a 27.5 cm X 1.7 cm column of Sephadex G-100 (Pharmacia Fine Chemicals, Inc., Piscataway, N. J. 08854). To prove the presence of the various interfering agents, 3-ml samples of serum were separated and eluted at about 1 ml/min with phosphate buffer (0.1 mol/liter, pH 6.0). After the first 20 ml was eluted, 5.0-ml fractions were collected. Each fraction was divided into two 2.0-ml aliquots. To each aliquot, 2.0 ml of peroxidase-odianisidine reagent solution was added. To one of the aliquots, 0.1 ml of standard glucose was added, to the other 0.1 ml of the phosphate buffer. To initiate the reaction, we added 0.1 ml of glucose oxidase solution (5 U/ml). The reaction was allowed to proceed for 5.0 mm at 25 #{176}C, and was then quenched by adding 0.1 ml of KOH (400 g/liter). Absorbances were read within 10 mm at 460 nm vs. a reagent blank. We calculated glucose recovery in each fraction by taking the difference between the absorbances developed in the two aliquots and dividing by the absorbance developed in a reaction mixture containing only 0.1 ml of glucose standard solution and the enzyme reagents. We made preparative separations of the two highmolecular-weight fractions by adding 6.0-mi samples of serum to the Sephadex G-100 column. The highmolecular-weight fraction was collected between 20.5 and 27.5 ml of eluent, which corresponded approximately to a 1.25-fold dilution of the interfering protein as compared to its concentration in the blood serum. The 40 000 molecular-weight fraction was collected between 30.0 and 44.0 ml, which approximately corresponded to a 2.5-fold dilution as compared to its concentration in the blood serum. A clean separation was obtained between the two fractions by discarding some of the eluate between the two fractions. The low-molecular-weight fraction was prepared by dialyzing 3.0 ml of serum vs. 3.0 ml of the phosphate buffer for 2 h, by using a piece of Spectrapor Type 2 dialysis membrane (Spectrum Medical Industries, Los Angeles, Calif. 90054). Presence of uric acid in the dialysate olet absorbance was checked at 290 nm. We determined by measuring its ultravi- the effect of interferences on the Table 1. Glucose Recovery Experiment’ Fraction overall glucose oxidase-peroxidase system by reacting a series of solutions containing a fixed amount of glucose and various amounts of each interfering fraction, with all other variables held constant. Each solution of the series consisted of 1.5 ml of chromogenperoxidase reagent solution, 0.2 ml of standard glucose solution, interfering solution, and sufficient phosphate buffer to make 4.0 ml (3.0 ml for the uric 1 2 3 4 Results and Discussion Isolation of Interferences Table 1 shows the glucose recovery data for the fractions collected in a Sephadex G-100 separation of pooled blood serum. The low recoveries in fractions 1-5 and 10-13 indicate the presence of two groups of interferences. Fractions 1 and 2 were cloudy and uncolored, distinctly different from fractions 3-5, which were yellow. Glucose was eluted in fractions 8-11, overlapping the low-molecular-weight interferences in fractions 10-13. ,, Glucose recovery, % 20-25 25-30 30-35 69 74 67 35-40 65 5 40-45 85 6 45-50 50-55 55-60 60-65 65-70 70-75 75-80 80-85 100 98 104 100 57 40 26 77 7 acid studies). The reactions were carried out and recoveries calculated as described above. The effect of an interference on the rate of the glucose oxidase reaction alone, independent of the coupled peroxidasereaction, wasmeasured by noting the rate of decrease in dissolved oxygen concentration with a Clark-type oxygen electrode. For a rate measurement, 0.5 ml of standard glucose solution and a measured quantity of the interfering fraction to be tested were added to the reaction chamber (see section on Apparatus), which was then filled with the phosphate buffer to give a total volume of 4.5 ml. To start the reaction, we introduced glucose oxidase (0.2 ml of 70 U/ml solution) into the reaction chamber with a syringe, and followed the fall-off in current caused by oxygen consumption. Initial rates were calculated from the slope of the current-time curve between 0 and 3 mm, divided by the initial current, which gave the fraction of the oxygen originally present that was consumed per minute. This procedure corrected for run-to-run variations in electrode sensitivity, and for oxygen originally present. The effect of an interference on the peroxidase step of the method alone, independent of the glucose oxidation step, was evaluated by measuring the absorbance developed at 460 nm by 0.5 ml of hydrogen peroxide solution when combined with peroxidaseo- dianisidine solution in the presence of the interference. Concentration of interfering material and chromogen concentration were independently varied in separate experiments, the total reaction volume being adjusted to 4.0 ml with the phosphate buffer. It was not necessary to quench these reactions with KOH because, for the small quantities of H202 involved, the reaction proceeded to completion in less than a minute. Eluent volume, ml no. 8 9 10 11 12 13 3.0 ml of pooled G-100, and fractions Additional serum then was chromatographed used in glucose chromatographic recovery studies on Sephadex experiments. with Sepha- dex G-100 and Sephadex G-200 showed that fractions 1-5 (Table 1) contain at least two different interfering substances. The molecular weight of interference in fractions 3-5 was 40 000 to 45 000, as determined by the ratio of elution volume to void volume in several chromatographic experiments. The molecular weight of the cloudy fractions is much higher, greater than 500 000. We made no further at- tempts to identify these two materials more specifically, although they presumably are proteins. The ultraviolet absorption spectrum of a dialyzed serum sample containing the low-molecular-weight interfering material was very similar to that of a 0.1 mmol/liter solution of uric acid in the phosphate buffer. In addition, treatment of another serum sample with uricase before dialysis removed ultraviolet absorbance in the dialysate entirely. Also, after uncase treatment of this dialysate, interference in the glucose determination was decreased to about a tenth of that observed for serum dialysate not treated with uricase. Because uric acid evidently accounts for almost all of the low-molecular-weight interference observed, we studied the nature of its interference by using solutions of uric acid itself rather than dialysates. Urine, when subjected to the same chromatographic procedure, gave early fractions (corresponding to high-molecular-weight substances) that did not interfere at all with glucose recovery. On the other hand, fractions corresponding to 10-13 in Table 1 interfered severely and caused glucose recovery values to be only about 10% of the true figure, an effect we ascribed to high concentrations of uric acid in the urine. Effects of Interferences Figure creasing on the Overall Method 1 shows the effect of the presence of inamounts of each interfering substance on CLINICAL CHEMISTRY, Vol. 21, No. 1, 1975 121 GLUCOSC+ 02 GLUCOSE OXOASE C GlUCONATE + .!i PEROXIDASE U 0 H202 +0-DIANISIDINE t #{174} uJ 0 2 I-lO+OXlDlZED CHROMOPHORE #{174} Fig. 2. Possible sites of interference in the glucose oxidaseperoxidase-o-dianisidine u-i u-i SItes of interference system are Indicated by arabic numerals 0 Lu Lu U.) 0 Nature of Uric Acid Interference 0 2 ID INTERFERENCE ADDE ML Fig. 1. Relation between glucose recovery and increasing amounts of each interfering fraction present in a reaction mixture containing 0.1 ml of glucose solution (1 g/liter) A, UricacId (4.2 mg/100 ml). 8, 40000 molecular weight chromatographic traction. C. Higher molecular weight chromatographic fraction the overall glucose oxidase-peroxidase reaction. Uric acid interferes quite strongly: 0.1 ml on the x- axis of Figure l#{192} is typical of the amounts of uric acid and glucose found in 0.1 ml of normal serum, and this amount causes recovery to be less than 80%, corresponding to a decrease in the apparent value of more than 20%. The effect of the fraction of 40 000 molecular weight is shown in Figure 1B; 0.25 ml of added in- terfering solution, typical of the amount that would be found in 0.1 ml of normal serum, causes a 77% recovery, again corresponding to a decrease in the apparent value of more than 20%. The 500000 molecular weight fraction gives a small but measurable effect (Figure 1C), causing a relative error of about 3% in normal serum. Possible Modes of Interference Five possible ways or modes in which interferences could affect the glucose oxidase-peroxidase-o -dianisidirie system are listed below and summarized in Figure 2. 1. Inhibition of the glucose oxidase enzyme. 2. Nonenzymatic destruction of the H202 produced in the glucose oxidase reaction. 3. Peroxidase-catalyzed chemical reaction with H2O2, in competition with o- dianisidine. 4. Inhibition of the peroxidase enzyme. 5. Destruction (bleaching) of the oxidized form of o- dianisidine. In the following sections, experimental evidence is presented to elucidate and to test the nature of each of the three previously described interferences. 122 CLINICAL CHEMISTRY, Vol. 21, No. 1, 1975 The presence of as much as 1.0 ml of uric acid solution (42 mg/liter) in the reaction chamber had no effect on the rate of glucose oxidase reaction as measured with the oxygen electrode. Because half this amount of uric acid elicited only 30% recovery in the full coupled method, Mode 1 (above) is ruled out. On the other hand, uric acid had a marked effect on the peroxidase reaction. Chromogen formation in an H2O2-peroxidase-o- dianisidine system decreased with increasing amounts of added uric acid, the relationship between absorbance and uric acid concentration closely resembling the curve of Figure l#{192}. Interference Mode 2 was ruled out when it was found that the order of adding H202 and peroxidase to a uric acid buffer system made no difference in the final absorbance reached. If HO2 were destroyed nonenzymatically by uric acid, less absorbance would be expected in those reactions in which H202 and uric acid were allowed to mix before addition of per- oxidase. To test Mode 3, we increased the amount of peroxidase-o- dianisidine reagent in a series of solutions, uric acid and H202 being held constant. The resulting absorbance increased correspondingly, as shown in the second column of Table 2, indicating that uric acid interferes by Mode 3. The nonlinear increase in absorbance with increasing chromogen concentration is expected for a system in which two reactants (chromogen and uric acid) compete for a constant amount of H202 substrate. Further evidence for Mode 3 was obtained by noting that the absorbance changes in Table 2 did not occur in the absence of uric acid. We also found that uric acid was consumed during the interfering process, as indicated by a decreased absorbance of the reaction mixture at 290 nm (where o- dianisidine does not absorb) after the reaction. The decrease in uric acid concentration was accompanied by a corresponding decrease in absorbance at 460 nm (where the oxidized chromophore absorbs). This could be explained by reaction of uric acid with H202, which in turn causes decreased reaction of H202 with the odianisidine. Inhibition of peroxidase (Mode 4) could not be observed because the peroxidase reaction was so rapid. For the glucose quantitation procedure used here, the peroxidase activity was made sufficiently high to give creased by increasing Table 2. Relation Between Amount of Chromogen and Final Absorbance in the Presence of Uric Acid or 40 000 Molecular Weight Interfering Material’ o.Dianisidine. peroxidase reagent, ml Absorbance with 0.2 ml Absorbance of uric acid (7 mg/i00 ml) present 0.5 1.0 1.5 2.0 0.175 0.248 0.273 0.342 with 1.0 ml of 40000 mel wt fraction present 0.091 0.136 - 0.191 Absorbance at 460 nm developed by 0.5 ml of H,O, (0.2 mmol/ liter), the noted volumes of interfering material, and dianisidineperoxidase reagent, plus buffer to make a total volume of 4.0 ml. Table 3. Effect of the High-Molecular-Weight Inhibitor on the Rate of the Glucose Oxidase Reaction’ Vol of inhibitor solution, ml 0 0 0 1.0 2.0 Initial slope, pA/mm” 88 98 92 74 58 Initial current, pA’ 486 536 511 507 519 Reaction rate, oxygen used per mm % 18.1 18.3 18.0 14.6 11.2 “Reaction mixture consists of the indicated volume of inhibitor solution, 0.5 ml of glucose (1 mg/mI), 0.2 ml of glucose oxidase (70 U/mI) and buffer to give a total volume of 4.5 ml. From a current-time curve obtained with a Clark-type oxygen electrode immersed in the reaction mixture. Initial currents given are corrected for background currents of 6-11 pA. the concentration of the 40 000 molecular weight protein present in H202-peroxidase-o- dianisidine systems. The third column of Table 2 shows that the absonbance developed in a solution containing a fixed amount of the 40 000 molecular weight protein increases with the amount of peroxidase-odianisidine reagent. No such absorbance increases were observed in the absence of the interfering material. These results indicate that the way in which this fraction interferes is the same as that in which uric acid interferes (Mode 3). Similarly, neither nonenzymatic destruction of H202 nor bleaching was observed, and any inhibition of peroxidase was not great enough to be significant. Nature of the Interference by the Fraction Containing Protein of 500 000 Molecular Weight When the glucose oxidase reaction was followed increasing amounts of interference by this high-molecular-weight fraction caused the rate of the reaction to decrease markedly. Table 3 summarizes data for three rate measurements made in the absence of this high-molecularweight inhibitor (to check the reproducibility of the measurement procedure) and in the presence of two different concentrations of the inhibitor. Table 3 with the oxygen electrode, (last column) shows a linearly decreasing reaction rate with increasing concentration of the interference, indicating that the high-molecular-weight material is a glucose oxidase inhibitor operating through Mode 1. The above experiment does not rule out the possi- bility that catalase, as an impurity immediate chromogen oxidation by the H202 produced by glucose oxidation. Thus, uric acid inhibition of peroxidase is not completely ruled out by the preceding experiments-its inhibition could be masked by the large excess of peroxidase used. Chinh (12) observed that uric acid caused bleaching (Mode 5) when 2,2’-azine-diethylbenzthiazoline sulfonic acid was used instead of o- dianisidine. When uric acid was added to the H202-peroxidase-odianisidine system after color development was complete, no such bleaching was observed, indicating that Mode 5 was inoperative. Nature of the Interference by the Fraction Containing Protein of 40 000 Molecular Weight Experiments similar to those for uric acid were performed by using the chromatographically separated fraction containing the protein of 40 000 molecular weight, concentrated by as much as eight-fold as compared with its concentration in normal serum. Results were similar to those observed for uric acid. We saw no effect on the rate of the glucose oxidase reaction as followed with the oxygen electrode. Interference with the peroxidase reaction alone was in- in the glucose oxi- dase preparation, is the target of the interfering agent. Activation of catalase by the high-molecularweight material would appear to cause an inhibition of glucose oxidase. Because cyanide is known to inactivate catalase but not glucose oxidase, we repeated the experiment in the presence of 1 mmol of NaCN per liter. We obtained the same results as before, confirming that the interfering fraction actually inhibits glucose oxidase, and not artifactually through an impurity in the catalase. Some additional work was done that indicated that the high-molecular-weight fraction also interfered with the peroxidase reaction. Two milliliters of the fraction (corresponding to a concentration about 15fold greater than that encountered in serum), was added to H202-peroxidase-.-odianisidine mixture, and the resulting absorbance was 84% of that observed in the absence of the interference. This degree of interference, when combined with that operating on the glucose oxidase reaction alone, accounts for all of this interference by the high-molecular-weight fraction, as measured directly on the overall reaction. The mechanism of interference with the peroxidase reaction was not resolved further, mainly because the turbidity of the fraction made quantitative measurements difficult. CLINICAL CHEMISTRY, Vol. 21, No. 1, 1975 123 This work was supported in part by the National Science Foundation (Grant GP-40694 X). During 1973, J. M. U. was the recipient of a Du Pont Summer Fellowship. Special thanks go to Ronald Laessig, Director, Wisconsin State Laboratory of Hygiene, for providing the serum samples, and to Professor Stuart Updike, University of Wisconsin School of Medicine, for helpful discussions and for the use of the oxygen electrode. References 1. Keston, A. S., Colorimetric enzymatic reagents for glucose. Abstracts of Papers, 129th Meeting, American Chemical Society, April 1956, p 31C. 2. Teller, J. D., Direct quantitative colorimetric determination of serum or plasma glucose. Abstracts of Papers, 130th Meeting, American Chemical Society, Sept. 1956, p 69C. 3. Miskiewicz, S. J., Arnett, B. B., and Simon, G. E., Evaluation a glucose oxidase-peroxidase method adapted to a single-channel AutoAnalyzer and SMA 12/60. Clin. Chem. 19, 253 (1973). 4. Saifer, A., and Gerstenfeld, nation of blood glucose with 51, 448 (1958). S., The photometric microdetermiglucose oxidase. J. Lab. Clin. Med. 5. Kingsley, G. R., and Getchell, G., Direct ultramicro dase method for determination of glucose in biological Chem. 6, 466 (1960). 124 CLINICAL CHEMISTRY, of Vol. 21, No. 1, 1975 glucose oxifluids. Clin. 6. Salomon, L. L., and Johnson, J. E., Enzymatic microdeterminain blood and urine. Anal. Chem. 31,453 (1959). 7. Cramp, D. G., New automated method for measuring glucose glucose oxidase. J. Clin. Pathol. 20, 910 (1967). tion of glucose by 8. Hill, J. B., and Kessler, G., An automated determination of glucose utilizing a glucose oxidase-peroxidase system. J. Lab. Clin. Med. 57, 970 (1961). 9. Getchell, G., Kingsley, G. R., and Schaffert, R. R., An automated direct determination of glucose by the glucose oxidase-peroxidase system. Clin. Chem. 8,430 (1962). 10. Robin, M., and Saifer, A., Determination of glucose in biological fluids with an automated enzymatic procedure. Clin. Chem. 11, 840 (1965). 11. Caraway, W., Carbohydrates. In Chemistry. N. W. Tietz, Ed. Saunders, 159. Fundamentals Philadelphia. 12. Chinh, N. H., Mechanism of interference glucose oxidase/peroxidase method for serum 20,499 (1974). of Clinical Pa., 1970, p by uric acid in the glucose. Clin. Chem. 13. Blaedel, W. J., and Olson, C., Continuous analysis by the amperometric measurement of reaction rate. Anal. Chem. 36, 343 (1964).