Agric.

Biol.

Chem.,

44

(12),

Determination

2943`2949,

1980

of Glucose

2943

by a Modification

of Somogyi-Nelson

Method

Chitoshi HATANAKA
and Yoshiaki KOBARA
Departmentof AppliedBiochemistry,
Facultyof AppliedBiologicalScience,
HiroshimaUniversity,
Fukuyama720,Japan
ReceivedJuly 18, 1980
Nelson'sarsenomolybdate,the chromogenicreagent in Somogyi-Nelsonmethod, was
replaced by Folin-Ciocalteuphenol reagent. The major object was to remove the toxic
arsenic compoundsfrom the color reaction system. The color-producingability of the
phenolreagentwasconsiderablylowerthan that of Nelson'sreagent. However,the modified
methodwas favorablycomparableto Somogyi-Nelsonmethodin simplicity,reproducibility
and stability of color development. The error in both the modifiedand Somogyi-Nelson
methodcould be reducedto about one fourth by addingsodium benzoate(finalconcentra
tion, about 0.5%)to the test solutions.
The

arsenomolybdate

developed
still

by

being,

reagent

The

in

widely

for

reducing

reagent

Nelson1)
used

the

by

for

as

was

been,

the

and

determination

of

use

is probably

of

that

Nelson's

this

special

or

Chromogenic

compared

with

reagents
more

which
stable

nation

the

had
and

with

agents.1)

other

from

Katayama
2 N

been

of

This

far,

high

produces

reagent

deionized
a concentration

re

was

disadvantage.

The

to

present

and

by

Nelson's

eliminate

amount

of

ably
in

comparable
stability

to
of

the

color

replaced

reagent3)

is

Somogyi-Nelson

favor

method1)

developed

and

repro

ducibility.

from
out

Glucose

of

Nakarai
further

Chemicals
purification.

crystallized)

and

~ crystallized)
preparation
was from
from

were

maltose

Ltd.,

Kyoto,

Bovine

chicken
from

egg-white

Sigma

Chemical

were

Industries

grade)

used

albumin
lysozyme

Chemical

of glucoamylase
(NK-113,
Nagase
Sangyo
Co., Osaka.

Katayama

(special

serum

Co.

with
(1~
(3

A crude

1~104 GUN/g)
Other chemicals,
Co.,

latter

or

acidity

20-fold
acid

of the
0.1,

of

diluted

0.5

concentration

Table
so

I;

the

as

to

or 1

of
volume

of

contain

N.

the

re
each

an

equal

reagent.

reagent.

prepared

The

and

used

1937

with

reagent

the

of

modifications

Nelson.1)

Osaka,

were

glucoamylase

(500mg)

pended

in

room

stirred

temperature
5C

was

measured

paper.

The
at

acetate

volume

stopped

by

copper

of

reagent

present

method

method

the

) The
absorption

and
to

with

assay

buffer

of
the

0.5ml

reducing
the

with

obtained

those

glucoamylase

activity

was

by
was

and

0.5ml.

reaction

Somogyi's

results

4.8)

104g/ml).

the

power

compare

pH

mixture

of

filtrate

containing

protein,

period

at

Roshi

of the

(50mM,

reaction

a definite

The
1 hr

a Toyo

system

(as

sus

4.8.

about

activity

an

prepa
was

pH

for

filtered

enzyme

the

for
addition

order

iodometric

in

crude

3.2.1.3.)

buffer,

glucoamylase

of the

incubation

acetate

then

35

amount

The
(EC.

occasionally

and

(0.8mM),

total

activity.

glucoamylase
of 0.2M

was

No.

In

of

100ml

suspension

The

METHODS

and

the

chromogenic

by

Assay

After

Chemicals.

the

10-

hydrochloric

final

selected

copper

a suitable

AND

4-,

approximately

see

was

was

maltose

MATERIALS

the

of

acidity

the

Somogyi4)

in

was

phenol

2-,

a dilute

purchased

Co.;

this

modification

arsenomolybdate

Folin-Ciocalteu

its

reagent

ration

which

or
that

became

recommended

undertaken

diluted

combinations

Alkaline

serious

problem.

work

were

Industries

was

water

reagent
the

diluted

contains

is

acid)

with

agent

combi

copper

toxicity

in

arsenomolybdate

reagents

Chemical

such

For

in

alkaline
Nelson's

which

environmental

so

colors

Somogyi's

However,

arsenic

used

Nelson's
phenol

reagent,

phosphomolybdate

reproducible

grade.

reagent.

Folin-Ciocalteu

phenol

as

guaranteed

and

(about

method.1,2)

extensive

of

is

chromogenic

Somogyi-Nelson

the

arsenomolybdate

which
has

quantitative

sugars
reason

1944

was

was
alkaline

determined.

obtained

by

another

standard

assayed

by

the

the

method.5
amount

of

method.6)

protein

was

determined

by

the

UV

2944

C. HATANAKA and Y. KOBARA

TABLE I.

a A commercial
reagent was diluted with water or a dilute
The values of N are approximate .

Measurement

of

procedure
In

of

a test

tions

tube

0.5ml
tubes

for

20min1,4)

running

less

in

fourfold
and

with

1 mol

of

5ml

water

in

and

reagent.

The

and

heated

cooling

0.5

with

in

reagent

N in

solutions

acid)

were

absorbances

cell

solu
mixed

phenol

about

their

test
was

After

resultant

10-mm

follows:

the

marbles

water.

water,

The

of

660nm

copper
glass

standard
as

glucose

Folin-Ciocalteu

with

stirred.

of

of

alkaline
with

boiling

2ml

The
is

0.5ml

than

covered

water,

added

power.
modification

(16.5~165mm),

were

(diluted

reducing
present

of Somogyi's

test

at

the

containing

with

PROCEDURES IN THE PRESENT METHOD

was

In

the

case

of the

determination

the

reactions

were

described

by

procedure

was

test

Nelson,1)

was

alkaline

copper

cooling,

0.5ml

and

the

11ml

to

124

reagent

and

scale

heated

of

for

solution

same

tube

the

of

the

After
was

added

diluted

used

Stability of the color developed with Folin

- Ciocalteu phenol reagent
Under the present conditions the color reac
tion with the phenol reagent was very fast

Somogyi's

20min.

was
was

as
of

0.5ml

0.5ml

arsenomolybdate

colored
The

essentially

the

Namely,

with

of Nelson's

water.

Somogyi-Nelson
out

that

half.

mixed

resultant

of

except

reduced

solution

by
carried

about 0.1 N in acid) was somewhat difficult.

This is probably due to the low acidity of the
phenol reagent because the precipitates were
easily dissolved with the reagent having an
acidity of about 0.5 N (diluted 20-fold with a
dilute hydrochloric acid, procedure E). In the
following experiments we used procedure C
as the standard method.

read

Type

spectrophotometer.

method,

acid.

diluted

were

Hitachi

hydrochloric

as

with

described

above.

RESULTS

Effect

of

the

phenol

concentration

reagent

on

of
the

Folin-Ciocalteu

calibration

curve

of

glucose
Alterations
reagent
effect
of

the

found

the

linearlity

glucose
straight

phenol

concentration
to

have
of

the

and
F,

of

passed
however,

cuprous
reagent

of

no

all
through

curves
the

(diluted

by
20-fold

curves

the

dissolving
oxide

the

appreciable

calibration

(0`1.8mol/ml);

procedure

cipitates

the

was
on

were
In

in

with

1.

Absorption

with

Folin-Ciocalteu

The

values

Spectra

of the

Phenol

Reagent.

Colors

Developed

origin.
the

mixing

FIG.

pre
with
water,

tration
a Hitachi
see

text.)

in

parentheses

(mol/ml).
Type

The
340

show
spectra

spectrophotometer.

the
were

glucose
determined
(For

concen
with
details

Modified
and,
a

upon

few

mixing,

broad

The

spectrum

500nm,

was
for

solutions

mol/ml)
to

the

800nm

at

observed

Effect
of

intervals

any

wavelengths

wavelengths

from

1.2,

1.6

repeatedly

in

2.0

5 hr

400
but

absorbances
(Fig.

on

five

and
from

for

their

the

above

no
were

1).

calibration

curve

absorbance

at

glucose

The
given

relation

was

absorption
lines,

between

wavelength

glucose

the

determined

except

following

the

and

spectra

passed

of

scanned

2945

absorption

produced
0.8,

changes
at

of

colors

the

Method

in

giving

region

stable;

30-min

maximum
color,

visible

(0.4,

were

detectable

the

quite

glucose

its

resultant

in

spectra

reached

seconds.

Somogyi-Nelson

only

through

in

on
Fig.

one
the

concentration
the
1.

All

determined
origin

experiments

we

(Fig.
used

basis

of

the

straight

FIG. 3.

at

800nm,

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

2).
a

a
of

In

the

the

wavelength

Curves

obtained by Somogyi-Nelson
see text.)

660nm.

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

Calibration

was

considerably

lower

than

that

of

of Glucose

method.

Determined

(For

details

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, 1ml of glucose solution
was mixed with 1ml of Somogyi's alkaline
copper reagent and heated for 20min. After
cooling, the mixture was treated with 4ml of
the phenol reagent (diluted fourfold with water,
about 0.5 N in acid) and the resultant color
was measured at 660nm without dilution with
water. Procedure H was the same as procedure
G, except that 2ml 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

All the concentrations

Glucose
blank

fering

amount

presence
amount

EFFECTS OF PROTEINS AND BUFFERS ON THE PRESENT

AND SOMOGYI-NELSON METHODS

of the substances

was determined

absorbance

substances

II.

were expressed

from

from that of the mixed solution

on

both

methods.

the
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
by each of the buffers

TABLE

III.

EFFECT
OF THE

Test
2.0,

3.0

solutions
or

water;

the

except

those

were

4.0ml
final
of

of

5%

prepared
sodium

concentration
the

The average

errors,

values

as

follows:

benzoate
of the

are

OF SODIUM

PRESENT

AND
To

benzoate

expressed

of triplicates.

as

being
the

of glucose

BENZOATE

by subtracting

and interfering

ON THE

SOMOGYI-NELSON

a glucose

solution

obtained

.
the corresponding

substance.

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

In the

of the interfering
substances,
of glucose was determined
from

from similar interferences

as those in the test solutions

the absorbance

and
0.25,

absorbance

solution
the

mixture

0.50,
at

1.00,
660nm.

REPRODUCIBILITY
METHODS

(1.0mol/ml,
was
1.50

diluted
or

2.00%,

5ml)
to

was

a volume
respectively.

added
of

0.5,
10ml
All

1.0,
with

values,

Modified

Somogyi-Nelson

led to a marked
increase in the blank absorb
ance but the glucose
amount
obtained
was
almost
Tris.

equal

to that

of the control

without

In

this

Method

case

2947

the

prepared

in

(50mM)

and

the
the

calibration
presence
enzyme

curve
of

the

protein

of glucose
acetate

was
buffer

(104g/ml).

DISCUSSION
Effect

of

of

sodium

the

The

present

and

accuracy

judged

the

absence

determination

of

excellent

Nelson

method

concentration
tendency

to

Assay

drolysis

of

ration.

by

4.

the
a slight

absorbance.

course

curves

of

obtained

the

of

the

time

hy

prepa
by

methods

Course

the

glucoamylase
the

pres

were

closely

course

tested.

Hydrolysis

of

Maltose

Glucoamylase.
conditions:

50mM

acetate,

total

%.

Somogyi

Increasing

time

throughout

Time

0.3`0.4

showed

blank

the

iodometric

Assay

per

two

concen

activity

maltose

the

of

the

solution

in

III).

the

the

The
and

errors

an

hand,

glucose

benzoate

shows

superposed

by

the

increase

other

obtained

of glucoamylase

Figure

FIG.

with

mean

gives

(final

the

(Table
of

This

to

were

having

had

benzoate

In

typical

each

the

be

III.
a

0.5ml,

On

results

results

Table

replicates,
per

sodium

Similar

ent

five

0.25`2.0%)

gave

in

can

benzoate,

1.2%.

of

tration,

methods

0.554}0.0068.

about

addition

reproducibility

method

shown

glucose
of

the

present

sodium
of

of

absorbance
error

the

results

of

0.25mol

on

Somogyi-Nelson

of

from

the

benzoate

ml

of

reaction

volume,

method; ,

substrate,
pH

4.8;

mixture;

0.5ml. ,
the

0.8mM
enzyme

iodometric

(as

maltose;

buffer,

protein),

104g

incubation,
Procedure
method.

at
of

the

35;
present

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
of the reaction.7) Under the present experi
mental conditions, maximum reduction of cop
per with glucose was obtained at a heating
period of about 17min. A 20-min heating
period, which was adopted by Nelsons1)and
Somogyi,4)was also used in this experiment.
The color reaction with Folin-Ciocalteu
phenol reagent was very fast similarly to that
with Nelson's arsenomolybdate.
Nelsons1)
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
phosphotungstatee8) also may be used as the
chromogenic reagent for the reducing sugar
assays. This reagent, however, requires more
than 10min for the maximum color develop
ment and, moreover, the resultant color is
likely to be much less stable than the color
developed with Nelson's arsenomolybdate or
with Folin-Ciocalteu phenol reagent.
The color developed with the phenol reagent
gave a broad spectrum in the visible region
above 500nm and use of the different wave
lengths above 500nm does not affect on the
linearlity of the calibration curve of glucose;
except only one curve determined at 800nm,
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 720nm.
The sensitivity of the present method was

2948

C. HATANAKA and Y. KOBARA

considerably lower than that of SomogyiNelson 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. However, 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 eggwhite lysozyme were comparable to those in
Somogyi-Nelson method.
The fact that both the present and SomogyiNelson 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 decreases 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.

Although

the

200g/ml
weight)
of

II),

and

such

scarcely
enzyme

interferences

the

the

the

by

methods

were

the

of

case

amount

was

acetate

can

ibility

of

addition

of

solution.
stock

the

was

sodium

antiseptic.
of

sodium

sulfate

the

some
is

to

In

that
by

Somogyi's

alkaline

suggest

copper

oxide.
a

On

of
the

reagent
the

as

copper

tration
The

of

of
follows:

about

method

0.5

of
we

result

to

but

the

not

of

cuprous

we

propose

alkaline
sodium

in

copper

benzoate
a final

to

results

benzoate

results

give

was

benzoate

These

Somogyi's

to

sodium

reoxidation

reoxidation

A1)

be

alkaline

the

of

of these

to

of

reagent

Add

reagent

property

that

reagent.

the

basis

modification

this

com

experiment,10)

of

participation

prevention

to

of

above-

this

satisfactory

phenol

some

curve

Somogyi's
the

addition

an

appears

another

a similar
the

Folin-Ciocalteu

the

prevent

oxide.4)

obtained

for

respects
to

the

effect

the

property

to
as

the

of

reason

added

reagent

confirmed

added

property

the

glucose

standards

incidentally

this

in

cuprous

usually

calibration

which

copper

the

the

understood,

reproduc

by

to

examined

on

of

Somogyi-Nelson

we

found

comparable

the

Therefore,

Although

not

is

the

enzyme

improved

acid

excellent

pound.
is

and

to

the

glucose

and

mentioned

and

III,

in

precence

of

benzoate

glucose

the

benzoate

Benzoic

4);
glucose

reference

significantly

solutions

iodometric

the

in

present

curves

(Fig.

by

Table

of

two

the

(50mM)

of
ex

hydrolysis

method

prepared

pre

For

the

and

(104g/ml).
be seen from
both

in
by

the

present

most

presence

superposed

buffer

methods

the

of

present

curve

protein
As

in

determined

calibration

In

substances.

closely
the

may

extremely

avoided

glucoamylase,

the

protein

used.

be

course

each

components

curve

time

by

obtained

the

probably

interfering

in

of

are

calibration

maltose

on

unless

the

by

methods

amount
practice

from

will

expected

ample,

in
solutions

solution

of

amount,

influences

a large

cases,

paring

a concentration

glucose

Somogyi-Nelson

used

crude

test

the

considerable

present

(Table

at

times

gave

the

be

proteins

(2.2

to

concen

%.

presented

in

this

paper

is

Modified

Somogyi-Nelson

favorably
comparable
to Somogyi-Nelson
method in simplicity of operational
procedure
and in stability
of the color developed.
In
addition,
the present
over Somogyi-Nelson
toxic problem
Nelson method
Folin-Ciocalteu
Nelson's

method has an advantage

method;
namely,
the

of the arsenic
in Somogyican easily be solved by use of
phenol
reagent
instead
of

arsenomolybdate.

Acknowledgments.
We wish to express our thanks
to Professor T. Imamura of this department for his
encouragement during this work.

Method

2949

REFERENCES
1) N. Nelson, J. Biol. Chem., 153, 375 (1944).
2) M. Somogyi, ibid., 195, 19 (1952).
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10) C. Hatanaka and Y. Kobara, unpublished data.