THE EFFECT OF DIFFERENT METHODS OF PRECIPITATION OF PROTEIN ON THE ENZYMATIC DETERMINATION OF BLOOD GLUCOSE

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1 THE AMERICAN JOURNAL OF CLINICAL PATHOLOGV Vol. 38, No. 3, pp September, 1902 Copyright 19C2 by The Williams & Wilkiiis Co. Printed in U.S.A. THE EFFECT OF DIFFERENT METHODS OF PRECIPITATION OF PROTEIN ON THE ENZYMATIC DETERMINATION OF BLOOD GLUCOSE NORMAN L. WELCH, M.D., AND WAYNE H. DANIELSON, PH.D. Department of Pathology, Denver Children's Hospital, Denver, Colorado There has been a great deal of interest rely in the use of glucose oxidase for the determination of glucose in body fluids. This interest has arisen chiefly from a desire for a specific method for glucose, in order to replace the widely used nonspecific reducing methods. In studying some of the many methods that have been proposed, it was observed that the values obtained for blood glucose varied significantly, depending on the technic used for precipitation of protein. Zinc hydroxide has been the most frequently used precipitating agent; however, there is no agreement on the exact technic to be used. Marks 4 states that the zinc hydroxide should be formed before the blood is added. Middleton and Griffiths 5 diluted the blood in 0.9 per solution of sodium chloride, and then added zinc sulfate, followed by sodium hydroxide. In the procedure as described by Fales and co-workers 2 blood is lysed in water first, and then sodium hydroxide and zinc sulfate are added, in that order. It was found that each of these variations in technic resulted in a different value for blood glucose when it was determined by means of the glucose oxidase method. Only 1 reference pertaining to this effect could be found in the literature. Middleton and Griffiths 5 observed that the levels for glucose were higher if the order of adding the precipitating agents was sodium hydroxide followed by zinc sulfate, rather than the reverse. This paper is a report of the differences in levels of blood glucose found in proteinfree filtrates prepared by means of 3 different technics, a study to determine which of the technics yields a value most nearly representing true blood glucose, and an effort to Received, December 26, 1961; accepted for publication April 30, Dr. Welch is Resident in Pathology, and is presently at Presbyterian Hospital. Dr. Danielson is Clinical Chemist at Denver Children's Hospital. 251 explain some of the causes of different values observed. METHODS Reagents 1. Zinc sulfate solution. 5 per ZnS04-7H 2 0 in water (weight per volume). 2. Sodium hydroxide solution. 0.3 N NaOH in water. The zinc sulfate and sodium hydroxide conrations were adjusted so that 10.8 to 11.2 ml. of sodium hydroxide solution would neutralize 10 ml. of zinc sulfate solution diluted to 60 ml., using phenolphthalein as the indicator. Procedures 1. Preparation of protein-free filtrates. Three technics for preparing protein-free filtrates were used: A. Three and one-tenth milliliters of water was placed in a rifuge tube, and 0.1 ml. of whole blood, 0.4 ml. of sodium hydroxide solution, and 0.4 ml. of zinc sulfate solution were added, in that order, with very thorough mixing after each addition. After 5 min., the mixture was rifuged and the supernatant fluid was used for analysis. B. The same procedure was used as in A, with the exception that the order of adding the sodium hydroxide and zinc sulfate was reversed. C. The same volumes were again used, but the zinc sulfate and sodium hydroxide were mixed with the water to form a suspension of zinc hydroxide before the blood was added. 2. Determination of glucose. The procedure described by Marks, 4 utilizing a single mixed enzyme reagent composed of glucose oxidase, peroxidase, and o-tolidine in an acetate buffer, was used for the determination of glucose in the protein-free filtrates. This technic was chosen because it is simple, rapid, and reliable, and the mixed enzyme

2 252 WELCH AND DANIELSON Vol. 38 reagent is stable for at least 2 weeks at refrigerator temperature. The observation of Solomon and Johnson 7 that the chloride ion has an inhibitoryeffect on the enzymatic determination of glucose was confirmed. For this reason, water was used instead of 0.9 per sodium chloride solution in the preparation of protein-free filtrates. The blood samples were obtained at random from adults, children, and infants. Capillary blood was taken from the infants, and venous blood, with and without oxalate as an anticoagulant, was taken from the older persons. RESULTS Protein-free filtrates were prepared from 50 samples of whole blood, using the 3 different technics described above. The glucose was then measured by means of the method described. Without exception, when the filtrate was prepared by adding sodium hydroxide first ( A), the highest value for glucose was obtained. When zinc sulfate was added first ( B), an intermediate value was found, and when the blood was added to the zinc hydroxide suspension ( C), the lowest value was obtained. The results, as summarized in Table 1, demonstrate that there are statistically significant differences in the glucose values obtained with the use of the 3 methods of protein precipitation. The reason for using the intermediate value as a basis for comparison will be discussed. On 6 of these 50 samples of blood, proteinfree filtrates were also prepared with barium hydroxide and zinc sulfate, as described by Somogyi. 10 The same 3 variations in the order of adding the precipitating reagents were used, and the glucose was determined by means of the same method. It was found that the 3 technics of protein precipitation again produced 3 different glucose values. Also, the values were the same as when zinc sulfate and sodium hydroxide were used. Thus, barium hydroxide apparently offers no advantages over sodium hydroxide and is somewhat more difficult to use. In order to determine which of the values for glucose obtained by means of the 3 methods of protein precipitation represented the true level of blood glucose, the following experiment was performed. Blood glucose was determined by the glucose oxidase method on 10 samples of whole blood, using the 3 technics of protein precipitation for each sample. From the same samples of blood, the proteins were precipitated with tungstic acid and 1 half of each filtrate was incubated with yeast in order to remove the glucose, using the technic of Somogyi. 8 Then the reducing values of both the untreated and yeastfermented filtrates were determined by means of the Folin-Malmros method. 3 The difference in the reducing values of these 2 filtrates is assumed to represent true blood glucose. From the results listed in Table 2, it is evident that the true values for blood glucose obtained by the yeast fermentation method correspond very closely with the values found when zinc sulfate was added first ( B) in preparing the filtrate for the glucose oxidase method. Also, the values of the means of these 2 groups are almost identical. An attempt was made to determine the cause of the differences in the glucose values TABLE l GLUCOSE VALUES IN THREE TYPES OF ZINC HYDROXIDE FILTRATES OF WHOLE BLOOD (FIFTY SAMPLES) Technic of Preparation of Hltrate Blood and Water Mixed, Followed by: NaOH, then ZnS04 ( A) ZnSO (, then NaOH ( B) Water, NaOH, and ZnSO., Mixed, then Blood Added ( C) Mean glucose value Mean per greater or less than B 80.5 mg. per 100 ml. 7.5 per greater Standard deviation, 3.57 per.0 mg. per 100 ml mg. per 100 ml. 7.8 per less Standard deviation, 4.50 per

3 Sept ENZYMATIC ANALYSIS FOR GLUCOSE 253 TABLE 2 COMPARISON OP GLUCOSE VALUES OBTAINED BY MEANS OF THE GLUCOSE OXIDASE METHOD, USING THREE TYPES OF ZINC HYDROXIDE FILTRATES AND THE YEAST FERMENTATION METHOD (Values expressed as mg. per 100 ml.) Sample No Mean Glucose Oxidase Zinc hydroxide filtrates prepared by means of: A B C Yeast Fermentation (Folin-Malmros) Tungstic acid filtrates Untreated Yeast fermented Difference (true blood glucose) found when the 3 different technics of protein precipitation were used. First, the possibility that inhibiting substances were present in some of the filtrates was investigated. The glucose was removed from each of the 3 types of zinc hydroxide filtrate by yeast fermentation. 8 A known amount of glucose equivalent to mg. per 100 ml. was then added to each filtrate, and the resulting glucose levels were measured by means of the glucose oxidase method described above. Sixteen samples of blood were tested in this manner, and the results are listed in Table 3. An inhibitory factor is evidently present in the filtrates produced by B and, to a greater extent, in those produced by C. Of possible inhibitory substances present in the blood, only glutathione was investigated in this study. When reduced glutathione was added to pure glucose solutions in a conration equivalent to that of whole blood (30 mg. per 100 ml.), no inhibitory effect was produced on the glucose oxidase method described herein. Glutathione is probably not a factor any- TABLE3 RECOVERY OF GLUCOSE ADDED TO THREE TYPES OF YEAST-FERMENTED PROTEIN-FREE FILTRATES OF WHOLE BLOOD (SIXTEEN SAMPLES) Technic of Preparation of Filtrate Recovery Standard deviation Blood and Water Mixed, Followed by:. NaOH, then ZnS04 ( A) 100 per 1.57 mg. ZnSO<, then NaOIT ( B) 94.6 per 1.96 nig. Water, NaOIT, and ZnS0 4, Mixed, then Blood Added ( C) 90.0 per 3.50 nig. way, inasmuch as it is quite effectively removed from blood by zinc hydroxide. The possibility that the higher glucose values found in some of the filtrates may be greater than the actual blood glucose levels must also be considered. The increased glucose levels could have been caused by the production of a factor that enhanced the color development, or the release of glucose itself, from some combined form by the precipitating reagents or the hemolyzed blood. The most obvious possible enhancing factor would be hydrogen peroxide produced from something other than glucose. If hydrogen peroxide were formed in the precipitating process, it would react with the peroxidase and o-tolidine to increase the color density. This possibility was investigated by adding peroxidase and o-tolidine to each of the 3 types of proteinfree filtrates described above and measuring any color produced. No color was formed, indicating that no hydrogen peroxide or other similar substance was present in any of the filtrates. In order to determine whether or not glucose was being released from some combined form and producing the elevated values for glucose, substances which could possibly release glucose were added to samples of whole blood, and protein-free filtrates were prepared by means of the 3 technics described above. Levels of glucose were then measured by means of the glucose oxidase method. Glucose-6-phosphate, glucose-1-phosphate, glucosamine, glucuronic acid, sucrose, maltose, and lactose were

4 254 WELCH AND DANIELSON Vol. 88 tested in this manner, and no increase in the value for glucose was obtained in any of the samples. Furthermore, no inhibition was produced. Other possible sources of glucose, which were not tested, are glycogen and mucoproteins. DISCUSSION A study of some of the methods of preparing protein-free filtrates to be used in the enzymatic determination of blood glucose has led to several interesting observations. Acid-precipitating agents can not be used because they liberate hydrogen peroxide from the blood, which causes falsely high glucose values. 7 The present study demonstrated that the barium hydroxidezinc sulfate precipitation described by Somogyi 10 offers no advantages over the sodium hydroxide-zinc sulfate method of Somogyi. 9 At present, the latter method seems to be the most acceptable; however, variations in the order of addition of blood, sodium hydroxide, and zinc sulfate in preparing the filtrate produce different glucose values. As Somogyi originally described the method, blood is first added to water, and this is followed by zinc sulfate, and finally, sodium hydroxide. 9 When this technic is used, one obtains blood glucose values equal to true blood glucose levels, as determined by yeast fermentation studies; however, further studies demonstrated that "factors" inhibiting the enzyme reactions were present in the filtrates prepared in this manner. Certain blood constituents, such as hemoglobin, bilirubin, ascorbic acid, and uric acid are known to interfere with the glucose oxidase-peroxidase-chromogen reaction. 1,2 ' It would seem that 1 or more of these, or some other interfering substances, are incompletely removed by the precipitating process. If there is an inhibiting substance present in the protein-free filtrates that yield true values for blood glucose, then there must also be an enhancing factor present in the same filtrates. This enhancing factor could either be additional glucose released from some combined form, or production or release of some substance other than glucose, which has a positive effect on the glucose oxidaseperoxidase-o-tolidine reaction. The study described in this paper was not successful in demonstrating the nature of such an enhancing factor. SUMMARY This paper deals with the findings in a study of various methods of preparing protein-free filtrates to be used in the determination of blood glucose by means of the glucose oxidase method. The sodium hydroxide-zinc sulfate method of Somogyi 9 was found to be the most acceptable; however, the order in which the precipitating reagents are added has a significant effect on the value for glucose. In order to obtain true values for blood glucose, as determined by means of fermentation with yeasts, the blood must be lysed in water first, and then zinc sulfate and sodium hydroxide added, in that order. Some of the possible causes of the falsely high and low values for blood glucose obtained with some of the filtrates were also investigated. SUMMAEIO IN INTEKLINGUA Iste communication reporta le constatationes in un studio de varie methodos de preparar filtratos libere de proteina al uso in le determination de glucosa sanguinee per medio del methodo de oxydase de glucosa. Le methodo de Somogyi a hydroxydo de natrium e sulfate de zinc se monstrava como le plus acceptabile; nonobstante, le ordine in que le reagentes precipitante es addite exercite un significative effecto super le valores obtenite pro glucosa. Pro obtener le ver valores de glucosa sanguinee, como Ulos es determinate per medio de fermentation con levatura, le sanguine debe primo esser lysate in aqua, e postea sulfato de zinc e hydroxydo de natrium debe esser addite in iste ordine. Esseva etiam investigate varie possibile causas del falsemente alte e basse valores de glucosa sanguinee que esseva obtenite con certes del filtratos. REFERENCES 1. CAWXEY, L. P., SPEAR, F. E., AND KENDALL, R.: Ultramicro chemical analysis of blood glucose with glucose oxidase. Am. J. Clin. Path., 32: 195-0, FALES, F. W., RUSSELL, J. A., AND FAIN, J. N.: Some applications and limitations of the

5 Sept ENZYMATIC ANALYSIS FOR GLUCOSE 255 enzymio, reducing (Somogyi), and anthrone methods for estimating sugars. Clin. Chem., 7: 3-403, FOLIN, 0., AND MALMKOS, H.: An improved form of Folin's micro method for blood sugar determination. J. Biol. Chem., 83: 115-1, MARKS, V.: An improved glucose-oxidase method for determining blood, C.S.F., and urine glucose levels. Clin. Chim. Acta, 4: , MlDDLETON, J. E., AND ORIFFITHS, W. J.: Rapid colorimetric micro-method for estimating glucose in blood and C.S.F. using glucose oxidase. Brit. M. J., 2: , SAIFER, A., AND GERSTENFELD, S.: The photometric microdetermination of blood glucose with glucose oxidase. J. Lab. & Clin. Med., 51: 448^160, SOLOMON, L. L., AND JOHNSON, J. E.: Enzymatic microdetermination of glucose in blood and urine. Anal. Chem., 31: , SOMOGYI, M.: The distribution of sugar in normal human blood. J. Biol. Chem., 78: , SOMOGYI, M.: A method for the preparation of blood filtrates for the determination of sugar. J. Biol. Chem., 86: , SOMOGYI, M.: Determination of blood sugar. J. Biol. Chem., 160: 69-73, 1945.