CLIN. CHEM. 19/9.

959-962 (1973)

Single Glucose Oxidase-Peroxidase Reagent for Two-Minute

Determination of Serum Glucose

Leo G. Morin and Jerome Prox

The glucose oxidase-peroxidase procedure for de- Stock glucose standard. Dissolve and dilute to
termining glucose in serum or plasma has been 100 ml 1 g of anhydrous primary standard dextrose
modified by changing the assay pH to 5.5, altering in distilled water containing 0.1 g of benzoic acid per
the ratio of glucose oxidase to peroxidase, and using deciliter. Appropriate dilutions are made with dis-
a soluble chromogen, p-diphenylamine sulfonate, to tilled water. Store refrigerated.
prepare a single complete reagent that develops sta-
Procedure. To 2.5 ml of reagent
(prewarmed to
ble color within 2 mm without deproteinization. Most
interferences were inconsequential. Serum that
room temperature, 22-32#{176}C),
add 50 pl of serum
clearly is icteric or hemolytic is Somogyi-precipitat- and mix. Let stand at room temperature for 2 mm
ed. Recovery was 99% and the CV was 3.3%. Cor- and read at any wavelength between 460 nm and 470
relation with results obtained by the neocuproine nm vs. a reagent blank. Incubation at 30#{176}
or 37#{176}C
procedure, with use of the SMA 12/60, averaged 12 does not measurably shorten test time. Determine
mg/dl lower values (r2 = 0.98). the concentration of unknown from a standard curve
or from standards run along with the determination.
Additional Keyphrases: p-diphenyiamine sulfonate as glu- All data reported in this paper, unless otherwise in-
cose reagent #{149}neocuproine method compared #{149}
colorimetry of glucose
dicated, were obtained with the Model 44 spectro-
photometer (Coleman Instruments, Maywood, Ill.
Owing to the frequency with which glucose deter- 60153), with use of 13 x 100 mm cuvets. In cases
minations are requested from the clinical laboratory where the serum or plasma is clearly icteric or hemo-
and the consequent desirability for a rapid yet accu- lytic, it is advisable to deproteinize with Somogyi re-
rate and precise method, we have investigated the agents.
glucose oxidase-peroxidase ($-D-glucose :oxygen
oxidoreductase, EC 1.1.3.4; and hydrogen peroxide Results and Discussion
oxidoreductase, EC 1.11.1.7) reaction and have pre- Reagent development. The optimum pH for glu-
pared a single reagent for the determination of glu- cose oxidase activity was determined to be 5.1 (Fig-
cose in 2 mm. Currently used colorimetric proce- ure 1), that for peroxidase 6.8 (Figure 2). Because
dures require from 30 mm (1-3) to 20 mm (4). Fre- catalase (hydrogen peroxide: hydrogen peroxide
quently, the necessary time is decreased by stopping oxidoreductase, EC 1.11.1.6) is a usual contaminant
the reaction with sulfuric acid at a precise time be- of glucose oxidase preparations and could conceiva-
fore completion, usually 10 mm (5). bly result in nonspecific loss of peroxide, the opti-
mum pH for catalase was determined and found to
Materials and Methods be 6.8. In increasing the amount of glucose oxidase
Complete single reagent. Dissolve 60 mg of glucose used to decrease total test time we found that linear-
oxidase (19000 U/g; Sigma Chemical Co., St. Louis, ity became progressively worse from pH 5.9 to 7.0.
Mo. 63178), 30 mg of peroxidase (160 Purpurogallin This loss of linearity, attributed to increased
Units/mg, RZ 1.6, Sigma Chemical Co.), and 90 mg amounts of catalase, was corrected by selecting a
of reduced sodium p-diphenylamine sulfonate (ACS lower pH, one closer to the optimum for glucose oxi-
grade; less nearly pure material is available but not dase, so that less glucose oxidase would be required
suitable) in 100 ml of citrate buffer (0.1 mol/liter, and consequently less catalase would be introduced
pH 5.5). Stable for at least 3 months if kept refriger- into the system at a pH less favorable to catalase ac-
ated; at room temperature, stable for about 4 h. tivity. To compensate for the less favorable pH for
peroxidase activity, we increased the amount of per-
From the Chemistry Department, Research Section, Kiess In-
struments, Inc., 8768 S.W. 131st St., Miami, Fla. 33156. oxidase used. Figures 1 and 2 show the activities of
Received April20, 1973; accepted May 25, 1973. various amounts of enzyme at various pH values for

CLINICAL CHEMISTRY, Vol. 19, No. 9, 1973 959

 20 20

16 16

12 12
U,
Li
U, I-
Li
I-

z E
8 8
E

100 80 60 40 20
UNITS X 100 GLUCOSE OXIDASE MG/DL

Fig. 1. The effect of glucose oxidase concentration and Fig. 3. Effect of chromogen concentration on the time re-
pH on the time required to transform 350 mg/dl glucose quired to complete oxidation of chromogen from con-
Glucose oxidase concentration indicated as fixed activity, based on sup- sumption of 350 mg/dI glucose
pliers assay under optimum conditions, per 100 ml of reagent. Peroxi- All variables, except chromogen, are as in the proposed procedure. A,
dase and chromogen concentrations and method as in proposed proce- o-dianisidine; B, p-diphenylamine sulfonate; C, o-tolidine
dure. A, pH 5.1; B, pH 5.0; C, pH 5.3; D, pH 5.5; E, pH 5.8; F, pH 6.8

20
kit form from Boehringer Mannheim Corp., N. Y.
10017) were not investigated, but, based on the liter-
16 ature (3, 6, 7), they should be suitable.
Several buffers-including acetate, maleate, glu-
tarate, succinate, and citrate-were examined, and
12 citrate was selected because it appeared to enhance
U,
Li glucose oxidase activity while supporting the lowest
z activity of maltase (EC 3.2.1.20), a frequent contam-
inant of glucose oxidase preparations. Differences in
results obtained with the buffer systems we tested
4 were neither conspicuous nor statistically significant,
and our choice of citrate was essentially subjective.
Glycerol (40 ml/dl) in the reagent extended the sta-
10 8 6 4 2 bility of the reagent at room temperature from 4 h to
UNITS X 1000 PCROXIDASE at least 10 days, but unfortunately also had an in-
Fig. 2. The effect of peroxidase concentration and pH on hibitory effect that increased the total reaction time
the time required to react with hydrogen peroxide result- from 2 mm to 6 mm. Because we encountered no
ing from transformation of 350 mg/dI glucose
Peroxidase concentration indicated as fixed activity, based on supplier’s
problem in storing the reagent under refrigeration,
assay (in Purpurogallin Units) under optimum conditions, per 100 ml of we omitted glycerol.
reagent. Glucose oxidase and chromogen concentrations and method as
in proposed procedure. A, pH 6.8; B, pH 7.0; C, pH 6.5; 0, pH 5.5; E,
Linearity and reaction rate. Measurements are lin-
pH 5.3; F, pH 5.1 ear to 350 mg/dl, and a representative standard
curve is expressed by the equation y = 0.0018x,
where y is absorbance and x is glucose concentration
in milligrams per deciliter. The reaction rate is rapid
glucose oxidase and peroxidase, respectively. From and color development reaches virtual completion
these data, 1140 U (based on supplier’s assay) of glu- within 2 mm (Figure 4).
cose oxidase and 4800 Purpurogallin Units (sup- Accuracy and reproducibility. Within-run preci-
plier’s assay) of peroxidase at pH 5.5 were selected. sion of the method was checked with 40 replicate as-
With the increased peroxidase activity, it was nec- says each of “Calibrate” (General Diagnostics, Mor-
essary to increase the chromogen beyond a rate-lim- ris Plains, N. J. 07950) “1,” “2,” or “3” (reference
iting concentration (Figure 3). Both o-tolidine and sera containing 75, 150, or 300 mg, respectively, of
o-dianisidine were found to be unsuitable at these added glucose). The mean results (±1 SD) were 75
concentrations because of their poor solubility and (±2), 150 (±2), and 300 (±3) mg/dl. The coefficient
consequent turbidity. Of many sulfonic acid salts of of variation for all determinations (120) was 1.3%.
phenolic amines investigated, sodium p-diphenyl- Day-to-day precision was checked in the same man-
amine sulfonate was selected and used at a concen- ner, with 20 replicate assays each, six times over a
tration of 90 mg/dl. Salts of 2,2’-azino-di-[3-ethyl- six-month period. The coefficient of variation for all
benzo-thiazoline-(6) -sulfonic acid] (available only in 360 determinations was 3.3%. Recovery was 99%,

960 CLINICAL CHEMISTRY, Vol. 19, No.9,1973

 by Marks and Lloyd (13). For serum or plasma pre-
0.5 pared promptly, the error for glucose is about 3%, not
usually clinically significant (Table .1). Although our
study of glycolysis was not as extensive as that of
0.4
Overfield et al. (14), our results do not suggest the
gross levels of glycolysis reported by them. Routine
Li
LI 0.3 anticoagulants such as oxalate (2 mg/dl), citrate,
z
ethylenediaminetetraacetate (10 mg/dl), and hepa-
S
U,
rin salts (2500 USP units) do not interfere. Bilirubin
has no effect if present in concentrations of less than
8 mg/dl; at greater concentrations there is a 1.4
mg/dl increase in apparent glucose per 100 mg of
0.1
glucose per deciliter for each 1 mg/dl increase in bili-
rubin. Uric acid has no effect at concentrations of
2 4 6 less than 12 mg/dl; at greater concentrations the ap-
MINUTES parent glucose is depressed by 1.8 mg/dl per 100 mg
Fig. 4. Rate of color development with the proposed sin- of glucose for each additional 1 mg of uric acid per
gle reagent deciliter. Ascorbic acid has no effect in concentra-
A, 75 mg/dl glucose; B, 150 mg/dl; C, 300 mg/dl tions of less than 10 mg/dl; higher concentrations
produce marked depression in apparent glucose (as
much as 40%-50%). Creatinine (20 mg/dl), reduced
glutathione (50 mg/dl), gentisic acid (10 mg/dl), or
acetylsalicylic acid (10 mg/dl) do not interfere. Less
ranging from 96% to 100% with 20 serial additions of than 200 mg of hemoglobin per deciliter does not in-
20 mg of glucose per deciliter to a glucose-free di- terfere; at greater concentrations, apparent glucose
alyzed serum base. increases by 4.6 mg/dl per 100 mg of glucose for each
Sixty sera from a general hospital population were 100 mg/dl increase in hemoglobin. To remove inter-
assayed with this reagent on the “DMA 16/P” ference from hemoglobin and bilirubin, clearly icteric
(Kiess Instruments, Inc.) and simultaneously by the or hemolytic serum should be Somogyi-precipitated.
neocuproine procedure (8, 9) on the “SMA
12/60” While this paper was in preparation, the report of
(Technicon Instruments Corp., Tarrytown, N. Y. Meites and Saniel-Banrey (4) appeared, in which
10591). The regression equation was y = 0.98x - they reported the results of increasing the glucose
12.57, = 3.4, r2 = 0.98. Although uremic speci- oxidase-peroxidase concentration. Unfortunately,
mens were not deliberately omitted, close correlation they used a commercial kit (“Glucostat”; Worthing-
with the copper-neocuproine procedure suggests that ton Biochemical Corp., Freehold, N. J. 07728) and
no uremic specimens were included (10). made no attempt to vary the glucose oxidase:perox-
Interferences. Contrary to the report of Meites and idase ratio or alter the pH of the system. It is diffi-
Saniel-Banrey (4), use of isotonic sodium fluoride cult to make comparisons with their results, because
(1.78 g/dl) produced a 32% depression in values. By they report neither the pH of their system (presum-
preparing the reagent without glucose oxidase and
adding various appropriate amounts of hydrogen
peroxide, we determined that fluoride has only a
slight (<3% depression) effect on the peroxidase
reaction. Because the pH used in our test was 5.5
and the amount of peroxidase 60 times greater than Table 1. Glucose Determined in Preserved,
that used by Gochman and Schmitz (11), our results Fresh, and Aged Serum and Plasma from
are not directly comparable with theirs. Consequent- Six Subjectsa
Preserved
ly, the 32% depression observed was attributed to plasma”
diminished glucose oxidase activity. This conclusion Fresh Fresh Aged Aged
(c) (d) serumc plasmac serum’1 plasma”
was also supported by two other observations: (a)
the 32% depression reflected a slowed reaction rate, 96/97 94 95 94 93
in that no depression was observed if the test was 102/102 99 99 101 101
138/137 135 137 135 136
read after 12 mm; and (b) the peroxidase reaction,
146/146 142 141 140 141
both with and without fluoride, is almost instanta-
220/222 214 214 212 210
neous, requiring less than 30 s, an observation also 227/225 222 224 224 221
reported by Sharp (12). If the cells can be separated
O Each value represents mean of 10 assays, in mg/dl.
from the plasma or serum within 2 h, the sample re- Preserved with sodium iodoacetate (2 mg/mi of blood), then treated
frigerated, and the test performed within 24 h, there as described in footnotes C or d
C Separated from cells or clot within 15 mm of collection, and assayed
is no real need to add an inhibitor or fluoride. How- within 30 mm thereafter.
ever, if an inhibitor is to be used, sodium iodoacetate “Separated from cells or clot after 2 h, then refrigerated for 24 h before
assay.
(2 mg/ml of blood) should be used as recommended

CLINICALCHEMISTRY, Vol. 19, No.9, 1973 961

ably, 7.0) nor the activities of either glucose oxidase method for determination of glucose in whole blood. Clin. Chem.
19,308(1973).
or peroxidase. They were able to shorten incubation
5. Cawley, L. P., Spear, F. E., and Kendall, R., Ultramicro
time at 37#{176}C
to a minimum of 20 mm. They should chemical analysis of blood glucose with glucose oxidase. Amer. J.
be able to improve this by decreasing the pH and Clin. Pathol. 32, 195(1959).
possibly by increasing the amount of peroxidase 6. Gawehn, K., Wielinger, H., and Werner, W., Screening von
used. In contrast with their report, we observed that Chromogenen fur Blutzuckerbestimmung nach der GOD/POD-
Methode. Z. Anal. Chem. 252, 224 (1970).
bilirubin interfered positively rather than negatively,
7. Kahie, K., Weiss, L., K., and Wieland, 0., Klinischchemische
and that uric acid by our procedure has a threshold Erfahrungen mit einem neuen Chromogen f#{252}r
die Blutzuckerbes-
of interference (12 mg/dl). Otherwise, with the ex- timmung nach der GOD/POD-Methode unter Verwendung eines
automatischen Analysiergerates. Z. Anal. Chem. 252, 228 (1970).
ception of fluoride and hemoglobin (for which they
8. Brown, M. E., Ultra.micro sugar determinations using 2,9-
report no interference), the effects of interfering sub-
dimethyl-1,10-phenanthroline hydrochloride (neocuproine). Dia-
stances are fundamentally similar. betes 10,60(1961).
Currently used preparations requiring 30 to 20 mm 9. Bittner, D., and McClearly, M., The cupric-phenanthroline
apparently are not optimized for rapid determination chelate in the determination of monosaccharides in whole blood.
Amer. J. Gun. Pathol. 11,423(1963).
in terms of glucose oxidase:peroxidase ratio and
10. Powell, J. B., and Djuh, Y.-Y., A comparison of automated
operational pH. methods for glucose analysis in patients with uremia before and
after dialysis. Amer. J. Clin. Pathol. 56, 8 (1971).
References 11. Gochman, N., and Schmitz, J. M., Application of a new per-#{149}
oxide indicator reaction to the specific, automated determination
I. Washko, M. E., and Rice, E. W., Determination of glucose by of glucose with glucose oxidase. Clin. Chem. 18, 943 (1972).
an improved enzymatic procedure. Clin. Chem. 7,542(1961). 12. Sharp, P., Interference in glucose oxidase-peroxidase blood
2. Lloyd, J. B., and Whelan, W. J., An improved method for en- glucose methods. Clin. Chim. Acta 40, 115 (1972).
zymatic determination of glucose in the presence of maltose. 13. Marks, V., and Lloyd, K., Preservation of blood samples for
Anal. Biochem. 30, 467 (1969). glucose analysis by glucose oxidase. GUn. Chim. Acta 8, 326
3. Werner, W., Rey, H.-G., and Wielinger, H., Iiber die Eigen- (1963).
schaften eines neuen Chromogens f#{252}r
die Blutzuckerbestimmung 14. Overfield, C. V., Savory, J., and Heintges, M. G., Glycolysis:
nach der GOD/POD-Methode. Z. Anal. Chem. 252, 222 (1970). A re-evaluation of the effect on blood glucose. Clin. Chim. Acta
4. Meites, S., and Saniel-Banrey, K., Modified glucose oxidase 39,35(1972).

962 CLINICAL CHEMISTRY, Vol. 19, No.9, 1973