An Enzymatic Method for Glucose Quantitation in Normal Urine Daniel M. Keller A method to quantitate the glucose concentration in normal urine is described and evaluated. It utilizes the enzymes hexokinase and glucose-6-phosphate dehydrogenase and depends on measuring NADPH2 absorbance at 340 mis. 1 (JRING INVESTIGATIONS of the renal reabsorptive mechanism for glucose, a method to quantitate adequately the glucose of normal urine (1-3) was required. The nonspecificity of classic methods for reducing sugars makes them inadequate for application to normal urine. Specific glucose oxidase methods have not yielded adequate recoveries when applied to normal, dilute urine. I have adopted the enzymatic method for glucose mentioned by Slein et at. (4) and by Con and Lamer (5), which uses hexokinase and glucose-6-phosphate dehydrogenase. The method is here described in detail as used in this laboratory, and its application to dog and human urine is evaluated. The method involves the following 2 reactions: hexokinase glucose + ATP glucose-6-phosphate + ADP (1) glucose-6-phosphate glucose-6-phosphate + NADP (2) dehydrogenase -g1uconolactone-6-phosphate + NADPH2 The over-all reaction is followed by measuring the absorbance at 340 mp., an absorption peak for reduced nicotinamide adenine dinucleotide phosphate (NADP). Oxidation of 1 mole of glucose leads to formation of 1 mole of NADPH2; thus, the quantity of glucose oxidized can be calculated by using the molar extinction coefficient of NADPHO at 340 m (6). From the Department of Physiology and Bioplmysics, University of Tennessee Medical Ummits, Memphis, Tenn. 38103. This investigation was supported by Research Grant AM-04871 from the National Institute of Arthritis and Metabolic Diseases, U. S. Public Health Service, and by a General Research Support Grant from the University of Tennessee. I am grateful to Mrs. Jeri Jaggar and Miss Margaret Wiley for valuable technical assistance. Received for publication Aug. 7, 1964. Accepted for publication Nov. 10, 1964. 471
472 KELLER Clinical Chemistry Equipment and Reagents Experimental A Beckman Model.1)U spectropliotometer with cuvets of 1 cm. light path may be used. However, an instrument which automatically records absorbance as a function of time would considerably facilitate the measurements. For small samples, the Pyrocell microadapter (Arthur H. Thomas Company) with 1 cm. light path cuvets is satisfactory. All chemicals were reagent grade or as specified. 1. Glycyiglycine buffe-r 0.25 M glycylglycine free base (Sigma Chemical Co.) is mixed with 0.25 M NaOH to give a solution of ph 7.4, ionic strength 0.037. Store in refrigerator; use within 2 weeks. 2. MgC12 Prepare as a 0.2 M solution. 3. Glucose-6-phosphate dehydrogen-ase Suspend the ammonium sulfate preparation (Sigma, Type V, from yeast) to contain approximately 50 Kornberg units (7) per 0.1 ml. of water. This may be kept in the refrigerator at 1-4#{176}for several months with little loss of activity. Dilute this stock suspension 1:200 with tile glycylgiycine buffer; keep in ice and use on the same day. 4. NADP-hexokinase solution To a flask add each of the following: NAD1, monosodium salt (Sigma), 64 mg.; hexokinase, from yeast, glucose free (Sigma, Type III), 1.6 mg.; glycyiglycine buffer, 40 ml.; MgC10 solution, 8 ml.; distilled water, 24 ml. Stopper and mix by gentle inversion; keep in ice and use on the same day. 5. NADP-hexokinase-ATP solution To 24 rng. of disodium adenosine triphosphate. 4 1120 (Sigma), add 36 ml. of solution No. 4 above. Dissolve by gentle inversions. Keep in ice and use Ofl tile same day. These reagents are sufficient for 40 determinations. The concentration of these reagents in the final reaction mixture is as follows: adenosine triphosphate, 0.33 mm; hexokinase (8), 40 Kunitz-McDonald units/mi.; glucose-6-phosphate dehydrogenase, 0.008 Kornberg units/ml.; NADP, 0.33 mm; MgCl2, 6.7 mm; glycylglycine, 43 mm. Procedure Using a pair of matched cuvets, add 0.90 ml. of NADP-hexokinase solution No. 4 to the reference cuvet and 0.90 ml. of NADP-hexokiriase-ATP solution No. 5 to tile second cuvet. Add 2.00 ml. of the solution to be analyzed for glucose (0.01 to 0.15 M of glucose per ml.) to each cuvet. Add 0.10 ml. of G6PD solution No. 3 to each cuvet and mix by inversion; this is zero time. These volunies may be reduced by a factor of 10 if the Pyrocell adapter is used. Read the absorbance at 340 nip, with the first cuvet as the reference. The absorbance generally reaches a maximum in about 20 mm. Occa-
Vol. II, No. 4, 1965 GLUCOSE QUANTITATION 473 sionally, it may slowly decline in value after this point. The temperature of the reaction mixture will influence the rate at which the maximum absorbance is reached, but it does not detectably change the value of the maximum. The glucose concentration as M/ml. in the solution which was added is calculated as: - mnaximmmumn absorbance X 3 )- 6.22X2 The molecular extinction coefficient incorporated into this formula, 6.22 X 10#{176} sq. cm./mole, was reported by Horecker and Kornherg (6), and was found suitable for our conditions of measurement. Results and Discussion Kinetics In Fig. 1 is a plot of the change in absorbance when aqueous glucose is analyzed as a function of time after onset of the reaction. Following an initial, nearly rectilinear region of the curve, the rate of reaction rapidly slowed to essentially zero by 15 mm. In this example, the absorbance re- Mmnus., mamed constant until 29 mm., after which it decreased 0.010 by time 55 mill. I)ata from a typical canine urine analysis are also plotted in Fig. 1. Maximum absorbance was reached at 21 mm. in this instance, after which it slowly declined. Specificity With the reagents used, this method is not specific for glucose. The absorbance also increases with fructose as substrate (Fig. 1) apparently because of the small amount of phosphohexoisomerase present. At equivalent concentrations, the initial rate of reaction for fructose is slower than for glucose. However, the maximum absorbance finally attained with fructose solutions is equal to that attained with glucose.
474 KELLER Clinical Chemistry This defect in specificity can be rectified if necessary by utilizing purified glucose oxidase enzyme (9). By incubating 5 ml. of the glucose and fructose containing solution with 0.1 mg. glucose oxidase (Sigma, Type III) and 85 Sigma units of catalase (Sigma C-100) in 0.1 ml. of ph 7.3 phosphate buffer at 37#{176} in oxygen for 2 hr., the urinary glucose will be completely destroyed. Analysis of this glucose oxidase treated urine should indicate the concentration of fructose and fructose-like substances. The glucose concentration may then be calculated by difference. Analysis of 36 urine samples from fasting dogs after glucose oxidase treatment indicated 5.9% ± 3.5 (S.D.) of the original reacting material was not glucose. Table 1 lists data from an experiment designed to test a variety of Table 1. INTERFERENCE OF SEVERAL AGENTS WITH DETERMINATION OF GLUCOSE IN A NORMAL URINE % Original % Recovery after Agent glucose gi ucose ox-zdase Added agent concentration recovered treatment None 5.8 l(+)arabinose.2 mm 100.0 4.4 d(-)arabinose.2 mm 100.0 0.0 Galactose.2 mm 100.0 6.6 Glucuronic acid lactone.2 mm 99.1 2.2 d(±)glucosamine hydrochloride.2 mm 101.3 43.0 i-inositol.1 mm 99.1 5.8 d(+)mannose.1 mm 103.5 3.4 Mannoheptulose.1 mm 99.1 5.8 l(+)rhamnose.1 mm 100.0 3.5 Sucrose.1 mm 105.7 34.5 Sucrose.1 gm./ml. >>100 >>100 d-ribose.1 mm 99.1 15.6 Na glucuronate.1 mm 100.0 4.4 n(+)xylose.1 mm 99.1 2.2 I(-)Xy1ose.1 mm 100.0 0.9 a Chloralose.1 mg./ml. 99.1 3.8 Pentobarbital.13 ing.fml. 102.2 3.4 Fumaric acid.1 mg./ml. 100.0 0.9 I-Malic acid.74 mm 100.0 2.2 Malonic acid.1 mg./nil. 100.0 2.2 Pyruvic acid.1 nig./ml. 100.0 5.8 Suecinie acid.1 mg./m]. 102.2 0.9 Phlorizin.106 mm 71.4 Sedoheptulose.12 mm 102.2 5.8 Mannitol 60 mg.fml. 100.0 10.1 Creatinine 1 mg./ml. 100.0 0.9 Na p-aminoimippurate 5 mg./ml. 102.2 0.9 Na citrate 1 mg./ml. 98.7 0.0 Inulin 5 mg./ml. 107.9 31.0
Vol. II, No. 4, 1965 GLUCOSE QUANTITATION 475 agents sometimes present in urine, either naturally (10) or experimentally, for possible interference with the method. Each agent was added to a normal human urine specimen (with an analyzed glucose concentration of 0.0227 mm) in the concentrations indicated, the specimen analyzed, and the per cent recovery of the original glucose of the urine calculated. Of the agents tested, sucrose, inulin, mannose an(l phlorizin interfered. Phlorizin at this concentration, ph, and wave length contributes very significantly to tile absorbance of the solutions. Analysis of the same samples after treatment with glucose oxidase plus catalase reveals that d (+ ) glucosamine hydrochloride, d-ribose, and mannitol interfere with the glucose oxidase tecimique for measuring fructose-like substances. Reproducibility and Recovery Table 2 lists the coefficient of variation of analyses on several glucosecontaining fluids. Recovery of glucose from aqueous solution is generally excellent. The original analyzed glucose concentration in 23 cainne Table 2. REPRODUCIBILITY OF GLUCOSE ANALYSIS Analyzed glucose Coejllcient concentration No. of of variation Sample (MM) determinations (%) Aqueous glucose, 150 zm/l. 149.2 10 0.169 Aqueous glucose, 90 M/L. 89.5 10 0.311 Dog urine No. 1 32.7 8 1.11 Dog urine No. 2 27.7 8 2.28 Dog urine No. 3 27.0 8 339* *Micronletll(mml urine samples was 10.0-51.2 p.m; the concentration of added glucose was 27.8-69.4 p.m. Average recovery was 98.4%, with a standard deviation of 2.42%. Application The data in Table 3 show the relationship between urinary glucose concentration, as determined by this method, and rate of water diuresis. The subject was a 35-year-old, 77-kg., healthy male who had been fasting 11 hr. at the beginning of the experiment. Physical activity during the experiment was limited to sitting and occasionally walking al)out. Urine samples were collected by voiding. Following emptying of tile bladder at zero time (5:30A.M.), the subject drank 1200 ml. of water. Tmmediately after each voiding, through the collection at 225 mm., the subject drank
476 KELLER Clinical Chemistry Table 3. GLUCOSE CONCENTRATION IN NORMAL HUMAN URINE AS A FUNCTION OF WATER DIURESIS Fructose-like Glucose Rate of material remaining Urine concen- glucose after glucose Time flow rate Osmolality trat ion excretion ocidase treatment (mm.) (ml.imin.) of urine (e11) (i1/nmin.) (#{231} 0-45 0.93.699 226.0.210 0.1 45-75 7.53.107 33.1.249 1.5 75-105 12.16.065 23.0.280 2.2 105-135 11.77.063 21.7.255 2.3 135-165 11.17.061 22.0.246 2.3 165-195 10.67.062 24.0.256 2.1 195-225 11.00.063 22.2.244 0.0 225-255 11.17.064 23.7.265 3.4 255-285 9.47.068 26.8.254 1.9 285-315 8.07.072 29.5.238 2.7 315-345 7.17.091 33.8.242 0.6 345-375 2.97.209 75.2.223 1.1 375-405 1.18.451 199.5.235 0.9 405-435 0.78.726 319.3.249 0.5 435-466 0.81.632 271.3.220 0.2 AVERAGE.2445 1.4 a volume of water equal to the volume of the urine sample. Maximal diuresis was present about 1 hr. after the initial water ingestion and persisted until 255 mm. when it began to subside. Urine flow rate varied from a low of 0.78 to 12.16 ml./min.-a 15-fold change. Similarly, the osmolality (measured by freezing point depression) varied from 61 to 726 milliosmoles/kg. water-a 12-fold change. In contrast, the quantity of glucose excreted per unit of time changed relatively little, the lowest rate being 0.210 p.m/mm., and the highest being 0.280 p.m/mm. The higher rates of glucose excretion occurred when the glucose concentration in the urine was least, i.e., during maximal water diuresis. In order to determine whether any of the glucose measured in this urine was actually fructose or fructose-like substances, each urine sample was treated with glucose oxidase and analyzed as before, the resuits being listed in the last column (Table 3) as per cent of the original glucose concentration. These values are negligibly small. References 1. Harding, V. J., Nicholsomm, T. F., and Archibald, R. M., Some properties of the reducing material in certain fractions of normal urines. I. The nature of the free fermentable sugars and the fermentable sugars produced on hydrolysis in fasting urines. Biochem. J. 30, 326 (1936). 2. Eastham, M., Paper.partition clmromatography of the sugars in urine. Biochem..1. 45, xiii (1949). 3. Froeseh, E. R., and Renold, A. F., Specific enzymatic determination of glucose in blood and smrino using glucose oxidase. Diabetes 5, 1 (1956).
Vol. II, No. 4, 1965 GLUCOSE QUANTITATION 477 4. Slein, M. W., Con, G. T., and Con, C. F., A comparative study of hexokinase from yeast and animal tissues. J. Biot. Chem. 186, 763 (1950). 5. Con, G. T., and Lamer, J., Action of amylo-1,6-glucosidase and phosplsorylase on glycogeim and amylopectin. J. Biot. Chenm. 188, 17 (1951). 6. Horeeker, B. L., and Konnberg, A., The extinction coefficients of the reduced band of pynidine nueleotides. J. Biol. Chern. 175, 385 (1948). 7. Kornberg, A., Enzymatic synthesis of tniphosphopyridine nucleoticle. J. B jot. Chern. 182, 805 (1950). 8. Kunitz, M., and McDonald, M. R., Crystalline hexokinase (heterophosphatese). J. Gen. Physiot. 29, 393 (1945-46). 9. Adams, E. C., Jr., Burkhart, C. F., and Free, A. H., Specificity of a glucose oxidase test for urine glucose. Science 125, 1082 (1957). 10. White, A. A., and Hess, W. C., Paper chromatographic detection of sugars in normal and dystrophic unines. Arch. Biochem. Biophys. 64, 57 (1956).