RIOCHEMICAL MEDICINE 4, 171-180 ( 1970)

A Calorimetric Serum Glucose Determination Using

Hexokinase and Glucose-&phosphate

Dehydrogenase

JAMES J. CARROLL, NANCY SMITH, AND ARTHUR L. BABSON

Diagnostics Research Department, Warnedambert Research Institute, Morris Plains, New Jersey 07950

Received April 6, 1970

The quantitative determination of blood glucose has engrossed clinical chemists for more than a century as demonstrated by the vast number of published procedures. The methods developed generally follow one of two principles: ( 1) measuring the reducing power of glucose usually with copper as the oxidant, or (2) reacting glucose enzymatically with glucose oxidase or hexokinase. Henry (1) has reviewed the various glucose methods and the difBculties involved with each procedure.

Methods employing the reducing power of glucose require a protein- free filtrate and are usually not specific for glucose due to non-glucose reducing substances.

The glucose oxidase procedures usually measure the hydrogen per- oxide formed with peroxidase and a chromogenic oxygen acceptor. Although these procedures are more specific for glucose, they encounter difficulties when applied directly to serum because of the presence of inhibitors such as uric acid, ascorbic acid, bilirubin, catechols, and glutathione. Many of these inhibitors can be removed by preparing a protein-free filtrate, but this makes the assay as cumbersome as the copper reduction methods. Deproteinization techniques fail to remove uric acid, bilirubin, and ascorbic acid, and peroxides may be released in acid filtrates. (2) Glucose oxidase preparations may also contain catalase as a contaminant which competes with peroxidase for the hydrogen peroxide formed.

The enzymatic procedures employing hexokinase are usually coupled to a second enzymatic reaction employing glucose-Bphosphate de-

171

hydrogenase which measures thct formation of NADPFI’ at :GO VIII. I :J 1 This enzymatic procedure measures glucose t~xclusivcly o\\-ills LO tlrc complete specificity of glucose-&phosphate dt:llytlrogt~~lase but has the following disadvantages: ( a) it requires m ultraviolet spectrophoto- meter to measure the reduced pyridine dinuclcotidc, NADPH, (1~) individual serum blanks should be run because of the significant serum absorption at 340 nm, and (c) the cost of NADP is considerable.

The purpose of the present study was to investigate the possibility of developing a calorimetric serum glucose assay utilizing the hcxokinasc: glucose-8phosphate dehydrogenase reactions coupled with the con- comitant reduction of a tetrazolium salt, that would circumvent these limitations.

The following reactions summarize the assay: hexokinase

~llicone-6-phouphate Glucose-6-phosphate + SAD1 TV--------

dehydrogennse NAlJ PH + fi-phosphoglucclrlic ticid

(31

phenazine reduced NADPH + met,hosulfat,e - NADP + phenazine methosulfat c

(PMR) CPMSH) (S,

ioclonit.ro PMSH + tet,razolium chloride --) PM,S + formaznl) (520 tlm’~

(INT) (-rj

Reaction 3 regenerates NADP, therefore only catalytic quantities of this expensive ingredient should be required.

MATERIALS AND METHODS

Materials

The following substances were used without further purification: hexokinase and glucose-Sphosphate dehydrogenase from Nutritional Biochemicals Corporation (N.B.C.), Calbiochem or Seravac Laboratories Ltd.; nicotinamide adenine dinucleotide phosphate from P-L Biochemicals Inc.; adenosine-5triphosphate, disodium salt from Nutritional Biochem- icals Corporation; phenazine methosulfate from Mann Research Labora- tories; and 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride from Curtis L,aboratories.

1 The following abbreviations are used throughout the paper: NADP, NADPH = nicotinamide adenine dinucleotide phosphate oxidized and reducd, respectively; NAD, NADH = n&tin&de adenine dinucleotide oxidized and reduced, respec- tively; ATP = adenosine triphosphate; ADP = adenosine diphosphate: PMS, PMSH = phenazine methosulfate oxidized and reduced, respectively; INT = 2-n- iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride.

A SERUM GLUCOSE ASSAY 173

Methods

The enzymatic activity of glucose-g-phosphate dehydrogenase was measured by the spectrophotometric assay of Biicher et al. (4) and is reported as International Units. An International Unit is the amount of enzyme that will catalyze the reduction of 1 pmole of NADP per minute at 25”. Hexokinase was assayed similarly in the presence of an excess of glucose, ATT, and glucose-6-phosphate dehydrogenase.

Determination of Optimum Assay Conditions

The optimum conditions for the proposed assay were determined as described below. All variables were held at the final recommended level except for the one under study,

Glucose-8Phosphate Dehydrogenase

The quantity of glucose-6-phosphate dehydrogenase was varied from 0.2-0.6 IU per assay. A minimum activity of 0.06 IU per assay was required to cover a glucose level of 400 mg/lOO ml. The final reagent system contained 0.15 IU per assay to assure excess enzyme activity.

Herokinase

The range of hexokinase activity studied was 0.01-2.5 IU per assay. A minimum activity of 0.32 IU per assay was retired for a glucose level of 400 mg/lOO ml. The final reagent system contained 0.9 IU to assure excess enzyme activity.

Various commercial preparations of glucose-Sphosphate dehydrogenase and hexokinase were evaluated for their utility in the proposed assay. All the preparations investigated, except N.B.C. lyophilized hexokinase, sp act 600 KM units/gm, could be used interchangeably in the proposed assay.

Adenosine-S-Triphosph, (ATP)

The quantity of ATP was varied from 0.25-S poles per assay. Quantities greater than 2 pmoles ATP per assay reduced the final color developed and those less than 0.5 poles per assay were insufllcient for the hexokinase reaction utilizing a 400 mg/lOO ml glucose sample. Consequently, 0.75 pmole ATP per assay was used.

Nicotinumide Adenine Dinucleotide Phosphate (NADP)

Quantities ranging from 0.0254 pmoles per assay were investigated. The minimum quantity required to recover 400 mg/lOO ml glucose was 0.05 pmole NADP per assay. In order to minimize the quantity of costly NADP used in the final reagent system and still maintain a convenient

174 C:ARROIL. SMITH, AND BARSOK

reaction time, 0.2 ,mole NADP per assay was used (SW (:olur, L?c?celo/~- ment Time j .

Magnesium Chloride

Th e presence of Mg’+ is essential for the enzymatic activity of hexokinase (5). The quantity of magnesium was varied from 5-50 pmoles per assay. The minimum quantity required for the hexokinase reaction was 10 pmoles per assay. Quantities greater than 30 pmoles per assay resulted in reduced final color. The quantity selected for use was 25 pmoles per assay.

Ethyiknediaminetetraacetic acid (EDTA)

Glucose-6-phosphate dehydrogenase is extremely sensitive to heavy metals, but can be protected with EDTA (6). Concentrations studied ranged from 0.6 mM to 0.1 M. A slight reduction in final color development occurred with concentrations less than 2.5 mM and greater than 0.05 M

EDTA. The concentration selected for use in the proposed assay was 5 mM EDTA.

Phenuzine Methosulfate

The quantity studied ranged from 0.05-0.2 mg per assay. No apparent differences were observed throughout the range studied, except an excess of phenazine methosulfate increased the reagent blank signi- ficantly. The quantity selected for use was 0.1 mg PMS per assay.

2-p-Zodophenyl-3-p-nitroplwnyl&phenyl tetrazolium chloride (ZNT)

The quantity studied ranged from 0.2-0.8 mg per assay, and 0.4 mg per assay was found to be quite adequate.

PH

The pH optimum was found to be 7.8 with a fairly broad peak ranging from 7.5-8.3 using a 0.1 M Tris buffer.

The molarity of the Tris buffer (pH 7.8) varied from 0.012-0.5 M. !%rnilar results were obtained from 0.025-0.5 M, and 0.1 M Tris buffer

was used. A 0.1 M phosphate buffer (pH 7.8) was substituted for the Tris buffer

and the final color was almost completely inhibited. Glucose-&phosphate dehydrogenase is reportedly inhibited by phosphate (7) as was readily demonstrated by using the phosphate buffer.

A SERUM GLUCOSE ASSAY 175

Color Development Time

The time required for the reactions to go to completion was related to the concentration of NADP. Lyophilized buffered-enzyme reagents were prepared incorporating 0.1, 0.2, and 0.6 mole NADP per assay. The time required for complete color development for 306 mg/lOO ml glucose in the proposed assay was: 5 minutes with 0.6 pmole NADP per assay, 10 minutes with 0.2 pmole NADP per assay, and 15 minutes with 0.1 mole NADP per assay. The final reagent system contained 0.2 pmole NADP per assay, since incubation time was convenient.

Reagents and Procedure

Reagents 2

Buffered-Enzyme Reagent. The following constituents are contained in 100 ml of 0.1 M Tris buffer pH 7.8: 50.8 mg magnesium chloride hexahydrate, 18 mg NADP, 50 mg ATP, disodium salt, 10.4 mg EDTA, tetrasodium salt, 44,500 IU glucose-&phosphate dehydrogenase, 280,000 IU hexokinase, 150 mg protein as dialyzed plasma, and 20 mg methyl paraben as a preservative. Additional protein was required to stabilize the enzymes during lyophilization and to solubilize the formazan.

This reagent is stable for several days when refrigerated. However, the enzymes cannot be frozen and thawed repeatedly without signi- ficant enzyme inactivation. A stable reagent was obtained by lyophilizing lo-ml aliquots.

Color Developer. Color developer is prepared by dissolving 200 mg INT in approximately 80 ml of distilled water, to which 50 mg PMS and 20 mg of methyl paraben are added and the final solution is diluted to 100 ml with distilled water, This reagent must be stored in an amber- colored bottle.

PMS and INT can be added to buffered-enzyme reagent resulting in a one-reagent system. However, this material cannot be lyophilized and must be refrigerated and stored in an amber-colored bottle.

Hydrochloric Acid, 0.1 N

Procedure A

1. Add 20 d of serum to 1.0 ml of the buffered-enzyme reagent and warm to 37” in a water bath.

‘Prepared reagents are available commercially as GlucoStrate from General Diagnostics Division, Morris Plains, New Jersey.

2. Add 0.2 ml of the color developer and mix. 3. Incubate for 10 minutes at 37O ( cstc~ridcd inc~iIbation periods

beyond. 10 minutes result in higher blanks 1. 1. Add 5 ml of 0.1 N HCI and mix. S. Read samples versus a reagent blank at 520 nm. 7’1~ color is stabk

for at least rj0 minutes.

Procedure B

Reconstitute the lyophilized buffered-enzyme reagent with 8 ml of water and 2 ml of color developer. The combined reagent is stable for 3 days when refrigerated and stored in an amber bottle.

1. Warm 1 ml of combined reagent at 37” 2-3 minutes, add 20 ~11 serum and mix.

2. Proceed as in Procedure A steps 35. Both procedures can be performed utilizing half-quantities of all

reagents and a 10-J sample or by making a prior l/10 sample dilution and using 0.1 ml of diluted sample with half-quantities of reagents. All procedures gave identical results.

Standardization

The assay procedure can be standardized by either aqueous glucose standard solutions or serum-calibration references.

RESULTS

The standard curve was linear with (a) aqueous glucose standards up to 400 mg/lOO ml, (b) V ersatol,3 Versatol-A, Versatol-A Alternate, or (c) Calibrate3 1, 2, and 3 was linear as illustrated in Fig. 1.

The proposed glucose reagent system was evaluated using 27 fresh nonfasting normal sera, and the results were compared with those obtained using a commercial glucose oxidase reagent system4 on whole serum as summarized in Table 1. The range observed (mean i 2 SD) was 57-105 mg/lOO ml for the proposed assay and ES-104 mg/IOO ml for the glucose oxidase procedure.

A buffered-enzyme reagent was prepared without ATP to determine the contribution of serum to the blank. The optical density obtained with this reagent and serum was about 0.030 using a Beckman DB and

a l-cm cuvette. The complete reagent blank without serum was 0.022.

‘Versatol and Calibrate are commercial human serum controls and serum calibration references available from General Diagnostics Division, Morris Plains, New Jersey.

’ Glucostat, Worthington, Biochemical Corporation, Freehold, N. J.

A SERUM GLUCOSE ASSAY 177

MG % GLUCOSE

FIG. 1. Glucose standard curve. Aqueous glucose standards ( n ). Serum-calibra- tion reference samples: Calibrate-l, ‘75 mg/lOO ml, Calibrate-2 150 mg/lOO ml, Calibrate-3, 300 mg/lOO ml (0); V ersatol, 86.4 mg/lOO ml, Versatol-A, 204 mg/lOO ml, Versatol-A Alternate, 304 mg/lOO ml ( A ).

The difference in optical density represented only 3 mg/lOO ml glucose. In another study both enzymes and ATP were eliminated from the

buffered-enzyme reagent to evaluate any nonglucose reducing con- stituents in sera samples and void any contribution of endogenous ATP. Fifteen hospital sera samples were assayed, several of which were hemolyzed. The absorbance difference between the reagent blank and the serum blanks represented only 4 mg/lOO ml glucose. The same dif- ference was observed with serum standards. Consequently, a reagent blank could be used safely in the proposed assay, since the small effect

1% CARROLL. SMITH, AND RAHSOS

Sample

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 2” I 2s 24 25 26 27

Mean rk SD

GillCObl:il jmg/lOO n1l)

S6

02 !Ji 85 .xJ 7s 94 7s ss sa s7 484 SJ 8,i

76 61 7’ 1) !Jl 5x2 69 66 97 5s SO 70 84 86

80 + 12

of nonglucose serum constituents would be negligible in the proposed assay system when serum calibration is used.

DISCUSSION

A rapid, simple and specific calorimetric serum glucose assay has been described which utilizes lo- or 20-d samples in a coupled hexo- kinase reaction and exhibits a minimal reaction blank. The major ad- vantages of the proposed glucose reagent system are: (1) a protein-free filtrate is not required in the assay procedure, (2) it is extremely sensi- tive and flexible, (3) it is specific for glucose, and (4) the enzymes employed are not inhibited by physiological serum constituents that can inhibit glucose oxidase.

A SERUM GLUCOSE ASSAY 179

The effect of non-glucose reducing constituents in whole serum was shown to be only about 4 mg/lOO ml and was compensated by the use of serum calibration references.

The glucose reagent system is extremely sensitive and flexible in that lo- or 20-,pl samples are employed and the reaction goes to comple- tion in the lo-minute incubation period. It may be simplified by incor- porating the color developer constituents with the buffered-enzyme reagent thereby eliminating one reagent manipulation. Half-quantities of reagents could be used with a 10-J sample or 0.1 ml of a l/10 prediluted serum sample. The molar extinction coefficient of the forma- zan was 14.6 X lo3 versus 6.22 X 10” for NADPH. Therefore the colori- metric procedure is 2.3 times more sensitive than the spectrophoto- metric procedure.

Although hexokinase catalyses the transfer of phosphate from adeno- sine triphosphate to D-ghCOSe, n-fructose, n-mannose, n-glucosamine, or 2-deoxy-n-glucose (8), th e secondary enzyme reaction utilizes glucose- 6-phosphate and NADP exclusively (7).

The enzymes employed in the proposed procedure are not inhibited by physiological serum constituents. Fluoride, which is often added to serum samples to preserve glucose by inhibiting glycolysis, had no effect on the proposed reagent system up to 8 mg sodium fluoride per milliliter of blood. Glucose oxidase procedures are affected by samples containing 1 mg sodium fluoride per millileter of blood (9).

SUMMARY

A calorimetric serum glucose assay was developed utilizing hexokinase: glucose-g-phosphate dehydrogenase reactions coupled with the con- cam:tant reduction of a tetrazolium salt. The rapid, simple and specific assay requires only 10 ~1 or 20 ~1 of serum and exhibits a minimal re- action blank. Standard curves obtained with aqueous glucose standards or commercial serum calibration references were linear up to 400 mg/lOO ml glucose. A variety of alternative procedures gave identical results, and the method correlated well with a glucose oxidase procedure.

REFERENCES

1. HENRY, R. J., “Clinical Chemistry Principles and Technics,” pp. 625-641. Harper (Hoeber), New York (1968).

2. HENRY, R. J., “Clinical Chemistry Principles and Technics.” p. 634. Harper ( Hoeber), New York ( 1968).

3. MAGER, M., AND FARESE, G., Amer. J. Clin. Pathol. 44, 104 (1956). 4. BERSFNHERZ, G., BOLTZE, H. J., BUCHER, T. H., CZOK, R., GARBADE, K., MEYER-

ARENDT, E., AND F’LERDERER, G., Z. Naturforsch. 86, 555 (1953).

180 CARROLL, SMITH, AND BABSOPi

5. BERGER, L., SLEIN, &I. W., COLOWICK, S. P., AND CORI, C. I'.. Gen. Physiol. 29, 379 (1946).

6. GLASER, L., AND BROWN, II. H., J. Biol. Chem. 216, 67 (1955). 7. KORNBERG, A., AND HORECKER, B. L., in “Methods in Enzymology” ( S. P. Colowiek

and N. 0. Kaplan, eds.), Vol. I, pp. 323-325, r\cademic Press, New York (1955).

8. DAFtROW, R. A., AND COLOWICK, S. P., in “Methods in Enzymology,” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. V, pp. 22&235, Academic Press, New York ( 1962).

9. MARKS, V., Clin. Chim. Acta 4, 395 (1959).

Announcement

Third International Meeting of the International

Society for Neurochemistry

Budapest, Hungary, 5-9 July 1971

The Meeting is open to members of the Society and all other persons interested in neurochemistry. Communications on original work on any aspect of neurochemistry may be submitted for presentation. Those re- lated to the topics listed below will be especially welcome.

1. CNS proteins. 2. Molecular basis of drug action on CNS. 3. Biochemistry of myelin. 4. Biochemistry of neuronal membranes.

The program will include specific symposia, round-table discussions, and general sessions with free communications. Presentations will have to be limited to no more than 10 minutes each. The abstracts of communica- tions, preferably in English, should be submitted before February 1971. The Organizing Committee reserves the right to select the communica- tions to be presented at the meeting.

Registration Fees are ISN members US $40.00; nonmembers US $45.00; Affiliates, students, and nonprofessionals US $10.00. In due course a second announcement and registration forms will be distributed,

Secretaries for the meeting are Prof. J. Folch-Pi, McLean Hospital, Bel- mont, Mass. 02178, USA; Prof. P. Mandel, Facultk de M&mine, 67-Stras- bourg, France; and Prof. I. Husz&k, Inst. for Brain Research, Szeged, Hungary.