Agricultural and Biological Chemistry

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Determination of Glucose by a Modification of

Somogyi-Nelson Method

Chitoshi Hatanaka & Yoshiaki Kobara

To cite this article: Chitoshi Hatanaka & Yoshiaki Kobara (1980) Determination of Glucose by a
Modification of Somogyi-Nelson Method, Agricultural and Biological Chemistry, 44:12, 2943-2949,
DOI: 10.1080/00021369.1980.10864408

To link to this article: https://doi.org/10.1080/00021369.1980.10864408

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 Agric. Bioi. Chem., 44 (12), 2943 ~ 2949, 1980 2943

Determination of Glucose by a Modification of Somogyi-Nelson Method

Chitoshi HATANAKA and Yoshiaki KOBARA

Department of Applied Biochemistry, Faculty of Applied Biological Science,
Hiroshima University, Fukuyama 720, Japan
Received July 18, 1980

Nelson's arsenomolybdate, the chromogenic reagent in Somogyi-Nelson method, was

replaced by Folin-Ciocalteu phenol reagent. The major object was to remove the toxic
arsenic compounds from the color reaction system. The color-producing ability of the
phenol reagent was considerably lower than that of Nelson's reagent. However, the modified
method was favorably comparable to Somogyi-Nelson method in simplicity, reproducibility
and stability of color development. The error in both the modified and Somogyi-Nelson
method could be reduced to about one fourth by adding sodium benzoate (final concentra-
tion, about 0.5 %) to the test solutions.

The arsenomolybdate reagent which was of special or guaranteed grade.

developed by Nelson!) in 1944 has been, and is
still being, widely used as the chromogenic
reagent for the quantitative determination of
reducing sugars by Somogyi-Nelsonmethod.!,2)
The reason for the extensive use of Nelson's
arsenomolybdate is probably that this reagent,
as compared with the other phosphomolybdate
reagents which had been used so far, produces
more stable and reproducible colors in combi-
nation with Somogyi's alkaline copper re-
agents.!) However, Nelson's reagent contains
arsenic of which high toxicity is a serious
environmental problem.
This work was undertaken to eliminate this
disadvantage. The present modification in
which Nelson's arsenomolybdate was replaced
3
by Folin-Ciocalteu phenol reagent ) is favor-
ably comparable to Somogyi-Nelson method!)
in stability of the color developed and repro-
ducibility;
MATERIALS AND METHODS
Chemicals. Glucose and maltose (special grade)
from Nakarai Chemicals Ltd., Kyoto, were used with-
out further purification. Bovine serum albumin (1 x
crystallized) and chicken egg-white lysozyme (3 x
crystallized) were from Sigma Chemical Co. A crude
preparation of glucoamylase (NK-I13, 1 x 10' GUN/g)
was from Nagase Sangyo Co., Osaka. Other chemicals,
from Katayama Chemical Industries Co., Osaka, were
Chromogenic reagent. Nelson's arsenomolybdate
and Folin-Ciocalteu phenol reagents were purchased
from Katayama Chemical Industries Co.; the latter
(about 2 N in acid) was diluted 2-, 4-, 10- or 20-fold
with deionized water or a dilute hydrochloric acid of
such a concentration that the final acidity of the diluted
phenol reagent became approximately 0.1, 0.5 or 1 N.
For the combinations of the concentration of the re-
agent and its acidity see Table I; the volume of each
diluted reagent was selected so as to contain an equal
amount of the chromogenic reagent.
Alkaline copper reagent. The 1937 reagent of
Somogyi4) was prepared and used with the modifications
recommended by Nelson. I )
Assay of glucoamylase activity. The crude prepa-
ration (500 mg) of glucoamylase (EC. 3.2.1.3.) was sus-
pended in 100 ml of 0.2 M acetate buffer, pH 4.8. The
suspension was stirred occasionally for about 1 hr at
room temperature and then filtered with a Toyo Roshi
No. 5C paper. The glucoamylase activity of the filtrate
was measured at 35°C in an assay system containing
maltose (0.8 mM), acetate buffer (50 mM, pH 4.8) and
a suitable amount of the enzyme (as protein, 104 ,ug/ml).
The total volume of the reaction mixture was 0.5 m!.
Mter incubation for a definite period the reaction was
stopped by addition of 0.5 ml of Somogyi's alkaline
copper reagent and the reducing power was determined.
In order to compare the results obtained by the
present method with those obtained by another standard
method the glucoamylase activity was assayed by the
iodometric method. 5)
The amount of protein was determined by the UV
absorption method. 6)
2944 C. HATANAKA and Y. KOBARA

TABLE 1. PROCEDURES IN THE PRESENT METHOD

Measurement of reducing power. The standard about 0.1 N in acid) was somewhat difficult.
procedure of the present modification is as follows: This is probably due to the low acidity of the
In a test tube (16.5 x 165 mm), 0.5 ml of the test solu-
phenol reagent because the precipitates were
tions containing less than 1 flmol of glucose was mixed
with 0.5 ml of Somogyi's alkaline copper reagent. The easily dissolved with the reagent having an
test tubes were covered with glass marbles and heated acidity of about 0.5 N (diluted 20-fold with a
for 20 min,,4) in boiling water. After cooling in dilute hydrochloric acid, procedure E). In the
running water, 2 ml of Folin-Ciocalteu phenol reagent following experiments we used procedure C
(diluted fourfold with water, about 0.5 N in acid) was
as the standard method.
added and stirred. The resultant solutions were diluted
with 5 ml of water and their absorbances were read
at 660 nm in a lO-mm cell with a Hitachi Type 124 Stability of the color developed with Folin-
spectrophotometer. Ciocalteu phenol reagent
In the case of the determination by Somogyi-Nelson Under the present conditions the color reac-
method, the reactions were carried out essentially as
tion with the phenol reagent was very fast
described by Nelson,!) except that the scale of the
procedure was reduced to half. Namely, 0.5 ml of the
test solution was mixed with 0.5 ml of Somogyi's 2.4
( 2.0
alkaline copper reagent and heated for 20 min. After
cooling, 0.5 ml of Nelson's arsenomolybdate was addedj
2.0
and the resultant colored solution was diluted with ( I .6 )
11 ml of water. The same tube was used as described
above. '"
" I. 6
c: ( I.2
o
.0
~ 1.2

RESULTS '"
.0
(

«
0.8
Eifect of the concentration of Folin-Ciocalteu (0.4 J
phenol reagent on the calibration curve of 0.4
glucose
Alterations in the concentration of the
400 500 600 700 800
reagent was found to have no appreciable ~ Wavelength (nm)
effect on the linearlity of the calibration curves
of glucose (0", 1.8 ,umoljml); all the curves J FIG. 1. Absorption Spectra of the Colors Developed
with Folin-Ciocalteu Phenol Reagent.
were straight and passed through the origin.
The values in parentheses show the glucose concen-
In procedure F, however, dissolving the pre- tration (flmol/ml). The spectra were determined with
cipitates of cuprous oxide by mixing with a Hitachi Type 340 spectrophotometer. (For details
the phenol reagent (diluted 20-fold with water, see text.)
 Modified Somogyi-Nelson Method
and, upon mixing, reached its maximum in
a few seconds. The resultant color, giving a
broad spectrum· in the visible region above·
500 nm, was quite stable; the absorption
spectra for the colors produced from five
glucose solutions (0.4, 0.8, 1.2, 1.6 and 2.0
,umol/ml) were scanned repeatedly from 400
to 800 nm at 30-min intervals for 5 hr but no
detectable changes in their absorbances were
observed at any wavelengths (Fig. 1).
•
Effect of wavelengths on the calibration curve
of glucose
The relation between the absorbance at a
given wavelength and the concentration of
glucose was determined on the basis of the
absorption spectra in Fig. 1. All the straight
lines, except only one determined at 800 nm,
passed through the origin (Fig. 2). In the
following experiments we used a wavelength
of 660 nm.
2.
""c 1.5
c
.c
~
o
'"
.01
<t
0.5
0.4 0.8 1.2 1.6
° Glucose (j.lmol/ml)
FIG. 2. Calibration Curves of Glucose Determined
at Different Wavelengths.
All the curves were reconstructed from the absorption
spectra in Fig. 1. The values in parentheses show
the wavelength (nm) used.
Calibration curves obtained by the present and
Somogyi-Nelson methods
The color-producing ability of the phenol
reagent was considerably lower than that of
2945
E
oc 15
.
w
w
-
c
"I.")f-
"c
o 2
.a
~
a
'" I
~o
0. I 0.3 0.4 0.5
° 0.2
Glucose (jJmol/ml)
FIG. 3. Calibration Curves of Glucose Determined
by the Present and Somogyi-Nelson Methods. Curves
1, 3 and 4 were obtained by procedures C, G and H
of the present method, respectively. Curve 2 was
obtained by Somogyi-Nelson method. (For details
see text.)
Nelson's arsenomolybdate. The slope of the
curve 1 obtained by the standard procedure in
the present method was about 2/3 of that of
curve 2 obtained by Somogyi-Nelson method;
at the same level of the final volume of the
colored solution, the slope of curve 1 would
become only about one half of that of curve 2
(Fig. 3). However, the curves obtained by
procedure G and H, respectively, increased
1.6 and 2.4 times in slope compared to that
obtained by Somogyi-Nelson method. In the
case of procedure G, 1 ml of glucose solution
was mixed with 1 m! of Somogyi's alkaline
copper reagent and heated for 20 min. After
2.0
cooling, the mixture was treated with 4 ml of
the phenol reagent (diluted fourfold with water,
about 0.5 N in acid) and the resultant color
was measured at 660 nm without dilution with
water. Procedure H was the same as procedure
G, except that 2 mI of the phenol reagent
(diluted twofold with water, about 1 N in acid)
was used as the chromogenic reagent.
Effect of interfering substances on the present
and Somogyi-Nelson methods
Table II shows the effects of several inter-
2946 C. HATANAKA and Y. KOBARA

Table II. EFFECTS OF PROTEINS AND BUFFERS ON THE PRESENT

AND SOMOGYI-NELSON METHODS

Present method Somogyi-Nelson method

Substance" Absorbance Glucose Absorbance Glucose
at 660 nm found b at 660 nm found b
(.umol/ml) (ltmol/ml)
Nothing 0.020 0.030
Citrate (50 mM, pH 7.8) 0.010 0.019
Phosphate (50 mM, pH 7.0) 0.012 0.023
Tris (50 mM, pH 9.0) 0.134 0.180
Albumin (200 ,ug/ml) 0.052 0.060
Lysozyme (200 Itg/m!) 0.068 0.088
Glucose (0.50 "mol/ml) 0.540 0.50 0.802 0.50
Glucose + Citrate 0.469 0.44 0.708 0.45
Glucose + Phosphate 0.498 0.47 0.746 0.47
Glucose + Tris 0.668 0.51 0.940 0.49
Glucose + Albumin 0.628 0.55 0.900 0.54
Glucose + Lysozyme 0.658 0.57 0.996 0.59
" All the concentrations of the substances were expressed as those in the test solutions.
b Glucose amount was determined from the absorbance obtained by subtracting the corresponding
blank absorbance from that of the mixed solution of glucose and interfering substance.

fering substances on both methods. In the
presence of the interfering substances, the
amount of glucose was determined from the
absorbance obtained by subtracting the cor-
responding blank absorbance from that of the
mixed solution of glucose and interfering sub-
stance. Both methods were found to suffer
from similar interferences by each of the buffers
and the proteins tested (Table II). The addi-
tion of citrate or phosphate resulted in a
decrease not only in the blank absorbance but
also in the glucose amount determined. On the
other hand, in the presence of bovine serum al-
bumin or chicken egg-white lysozyme, the appar-
ent glucose amount was somewhat higher than
that of actually present. The addition of Tris

TABLE III. EFFECT OF SODIUM BENZOATE ON THE REPRODUCIBILITY

OF THE PRESENT AND SOMOGYI-NELSON METHODS
Test solutions were prepared as follows: To a glucose solution (1.0 Itmol/ml, 5 ml) was added 0.5, 1.0,
2.0, 3.0 or 4.0 ml of 5 % sodium benzoate solution and the mixture was diluted to a volume of 10 ml with
water; the final concentration of the benzoate being 0.25,0.50,1.00,1.50 or 2.00%, respectively. All values,
except those of the errors, are expressed as the absorbance at 660 nm.

Present method Somogyi-Nelson method

Benzoate (%) Benzoate (%)

0 0.25 0.50 1.00 1.50 2.00 0 0.50 1.00

1 0.561 0.555 0.559 0.554 0.564 0.569 0.732 0.726 0.722
2 0.544 0.553 0.558 0.559 0.559 0.560 0.713 0.719 0.728
3 0.560 0.554 0.556 0.553 0.564 0.563 0.704 0.724 0.722
4 0.559 0.560 0.554 0.557 0.563 0.564 0.728 0.723 0.726
5 0.548 0.557 0.558 0.555 0.561 0.561 0.712 0.729 0.727
Average 0.554 0.556 0.557 0.556 0.562 0.563 0.718 0.724 0.725
Standard deviation 0.0068 0.0022 0.0016 0.0020 0.0018 0.0024 0.0098 0.0026 0.0024
Error (%) 1.23 0.40 0.29 0.36 0.32 0.43 1.36 0.36 0.33
Blank" 0.020 0.021 0.023 0.021 0.024 0.026 0.032 0.034 0.035

" The average values of triplicates.

 Modified Somogyi-Nelson Method
led to a marked increase in the blank absorb-
ance but the glucose amount obtained was
almost equal to that of the control without
Tris.
Effect of sodium benzoate on the reproducibility
of the present and Somogyi-Nelson methods
The accuracy of the present method can be
judged from the results shown in Table III. In
the absence of sodium benzoate, a typical
determination of five replicates, each having
0.25,umol of glucose per 0.5 ml, had a mean
absorbance of 0.554±0.0068. This gives an
error of about 1.2 %. On the other hand, the
addition of sodium benzoate (final concen-
tration, 0.25", 2.0 %) to the glucose solution
gave excellent results with errors of 0.3 ",0.4 %.
Similar results were obtained in Somogyi-
Nelson method (Table III). Increasing the
concentration of the benzoate showed a slight
tendency to increase the blank absorbance.
Assay of glucoamylase activity
Figure 4 shows the time course of the hy-
drolysis of maltose by the glucoamylase prepa-
ration. The two curves obtained by the pres-
ent and the iodometric methods were closely
superposed throughout the time course tested.
--E 0.8
'-
-0
E 0.6 •
=>.
- developed with Nelson's arsenomolybdate or
.,
'" 0.4
o
u
::l
<:;; 0.2
o 60 120 180
Reaction time Imin)
FIG. 4. Time Course of the Hydrolysis of Maltose
by Glucoamylase.
Assay conditions: substrate, 0.8 mM maltose; buffer,
50 mM acetate, pH 4.8; enzyme (as protein), 104 flg
per ml of reaction mixture; incubation, at 35°C;
total volume, 0.5 ml. 0, Procedure C of the present
method; ., the iodometric method.
2947
In this case the calibration curve of glucose was
prepared in the presence of the acetate buffer
(50 mM) and the enzyme protein (104 ,ug/ml).
DISCUSSION
The rate of copper reduction with different
sugars is closely associated with their chemical
structures, and the alkalinity of the copper
reagent greatly affects the rate and the extent
7
of the reaction. ) Under the present experi-
mental conditions, maximum reduction of cop-
per with glucose was obtained at a heating
period of about 17 min. A 20-min heating
period, which was adopted by Nelson1 ) and
Somogyi,') was also used in this experiment.
The color reaction with Folin-Ciocalteu
phenol reagent was very fast similarly to that
with Nelson's arsenomolybdate. Nelson 1 )
stated that the colors developed with the
various phosphomolybdate reagents he tried
lacked the desired stability. However, the
color developed with Folin-Ciocalteu phenol
reagent was quite stable. The phenol reagent,
containing a large amount of phosphotungstate
besides phosphomolybdate, might not be in-
cluded in the phosphomolybdate reagents he
tried. According to Somogyi,2) Benedict's
phosphotungstate S ) also may be used as the
chromogenic reagent for tl;te reducing sugar
assays. This reagent, however, requires more
than 10 min for the maximum color develop-
ment and, moreover, the resultant color is
likely to be much less stable than the color
with Folin-Ciocalteu phenol reagent.
The color developed with the phenol reagent
gav,e a broad spectrum in the visible region
above 500 nm and use of the different wave-
lengths above 500 nm does not affect on the
linearlity of the calibration curve of glucose;
except only one curve determined at 800 nm,
all the straight lines passed through the origin,
although the slope of each line increases with
increasing the wavelength (Fig. 3). Accord-
ingly, we may use any wavelengths from 500
to 720 nm.
The sensitivity of the present method was
2948 C. HATANAKA
considerably lower than that of Somogyi-
Nelson method. The slope of the calibration
curve of glucose (curve 1) obtained by pro-
cedure C, the standard one in the present
method, was about 2/3 of that of curve 2
obtained by Somogyi-Nelson method. How-
ever, procedure C will probably be adequate
for most analyses as it is. If necessary, we
may use procedure G or H as a more practical
one; the slopes of curves 3 and 4 were, respec-
tively, 1.6 and 2.4 times higher than that of
curve 2.
Folin-Ciocalteu phenol reagent is widely
used as the chromogenic reagent for the deter-
mination of proteins so that somewhat high
interferences by proteins were expected in the
present method. However, the interferences
by bovine serum albumin and chicken egg-
white lysozyme were comparable to those in
Somogyi-Nelson method .
•
The fact that both the present and Somogyi-
Nelson methods suffer from similar inter-
ferences by commonly used buffering agents
and proteins should be kept in mind when
enzyme activities are measured by the reducing
sugar assays. Depressing effects of citrate on
Somogyi-Nelson method has already been de-
scribed by Paleg,9) who suggested that the more
effective property of citrate than that of tartrate
of forming a chelate complex with copper
might be responsible for its depressing activity.
In the case of Tris, which increased markedly
the blank absorbance (Table II), also its chelate-
forming ability might be responsible for the
increasing blank absorbance, because, on addi-
tion of Tris, the blue color of Somogyi's
alkaline copper reagent was immediately
changed to a violet color characteristic of the
formation of a Tris-copper complex. The
reason why the absorbance increases or de-
creases in the presence of Tris or citrate,
respectively, is obscure. In the case of Tris,
in spite of the high blank absorbance, the
glucose amount obtained was almost equal to
that of actually present. However, high blank
absorbances would tend to cause poor repro-
ducibility of the assay. Such was the case
with Tris in the present experiment.
and Y. KOBARA
Although the proteins at a concentration of
200 ,ug/ml (2.2 times the glucose amount, by
weight) gave considerable influences on each
of the present and Somogyi-Nelson methods
(Table II), such a large amount of protein may
be scarcely used in practice unless extremely
crude enzyme solutions are used. In most
cases, interferences from the components in a
test solution will probably be avoided by pre-
paring a calibration curve in the presence of
the expected interfering substances. For ex-
ample, in the time course of the hydrolysis of
maltose by the glucoamylase, the two curves
obtained by the present and the iodometric
methods were closely superposed (Fig. 4); in
the case of the present method the glucose
amount was determined by reference to the
calibration curve prepared in the precence of
the acetate buffer (50 mM) and the enzyme
protein (104 ,ug/ml).
As can be seen from Table III, the reproduc-
ibility of both the present and Somogyi-Nelson
methods was significantly improved by the
addition of sodium benzoate to the glucose
solution. Benzoic acid is usually added to the
stock solutions of glucose standards as an
antiseptic. Therefore, we examined the effect
of sodium benzoate on the calibration curve of
glucose and found incidentally the above-
mentioned excellent property of this com-
pound. Although the reason for this property
is not understood, this property appears to be
comparable in some respects to that of sodium
sulfate which is added to Somogyi's alkaline
copper reagent to prevent the reoxidation of
cuprous oxide. 4 ) In another experiment/Oj we
confirmed that a similar satisfactory result was
obtained by the addition of the benzoate to
Somogyi's alkaline copper reagent but not to
Folin-Ciocalteu phenol reagent. These results
suggest some participation of the benzoate in
the prevention of the reoxidation of cuprous
oxide. On the basis of these results we propose
a modification of Somogyi's alkaline copper
reagent as follows: Add sodium benzoate to
the copper reagent AI) to give a final concen-
tration of about 0.5 %.
The method presented in this paper is
 Modified Somogyi-Nelson Method
favorably comparable to Somogyi-Nelson
method in simplicity of operational procedure
and in stability of the color developed. In
addition, the present method has an advantage
over Somogyi-Nelson method; namely, the
toxic problem of the arsenic in Somogyi-
Nelson method can easily be solved by use of
Folin-Ciocalteu phenol reagent instead of
Nelson's arsenomolybdate.
Acknowledgments. We wish to express our thanks
to Professor T. Imamura of this department for his
encouragement during this work.
2949
REFERENCES
1) N. Nelson, 1. Bioi. Chern., 153, 375 (1944).
2) M. Somogyi, ibid., 195, 19 (1952).
3) O. Folin and V. Ciocalteu, ibid., 73, 627 (1927).
4) M. Somogyi, ibid., 117, 771 (1937).
5) C. Hatanaka, Nippon N6geikagaku Kaishi, 41, 448
(1967).
6) V. F. Kalb, Jr. and R. W. Bernlohr, Anal. Bio-
chem., 82, 362 (1977).
7) P. H. Shaffer and M. Somogyi, J. Bioi. Chem.,
100, 695 (1933).
8) S. R. Benedict, ibid., 51, 187 (1922).
9) L. G. Paleg, Anal. Chern., 31, 1902 (1959).
10) C. Hatanaka and Y. Kobara, unpublished data.