THE

DETERMINATION
OF GLYCOGEN
MUSCLE
BY USE OF ANTHRONE
BY

NICHOLAS
(From

V. CARROLL,
JOSEPH

ROBERT
H. ROE

IN LIVER
REAGENT*
W.

LONGLEY,

the Department of Biochemistry, School

George Washington University, Washington,
(Received

for publication,

October

AND

AND

of Medicine,
D. C.)

28, 1955)

Method
Reagents1. Anthrone reagent. A solution containing 0.05 per cent anthrone, 1
per cent thiourea, and 72 per cent by volume HzS04 is used. For each
liter of reagent, place in a suitable flask 280 ml. of distilled water and add
cautiously 720 ml. of concentrated
H&04, sp. gr. 1.84, of highest purity.
Place in a flask 500 mg. of purified anthrone, 10 gm. of highest purity
thiourea, and 1 liter of the 72 per cent H,SOb. Warm the mixture to
80-90, occasionally shaking the flask to mix the contents.
Do not overheat the mixture.
Cool and store in a refrigerator.
This reagent will
keep for at least 2 weeks in a refrigerator.
2. 5 per cent trichloroacetic acid.
3:95

per

* Supported
ships, National

cent

ethanol.

in part by a grant from the Division

Institutes
of Health,
United
States
583

of Research
Grants
and FellowPublic
Health
Service.

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In currently used methods for the determination

of glycogen, tissue is
extracted either by boiling with 30 per cent potassium hydroxide solution
(KOH) or by homogenization
with trichloroacetic
acid solution (TCA).
The glycogen is precipitated from the extract by alcohol and determined
either by copper reduction after separation by centrifugation,
acid hydrolysis, and neutralization
(l-3) or by direct treatment
of the precipitated
An alternative
procedure is to
glycogen with anthrone reagent (4-6).
destroy alkali-labile carbohydrate
by boiling with KOH and then determine the glycogen in the alkali-treated
mixture with anthrone reagent
(5, 7).
Previously, we have reported that a method based upon the use of anthrone reagent gave results of a high degree of specificity and precision (8).
This method is a modification of procedures for the determination
of dextran (9) and biood sugar (10). In this paper we are reporting in detail our
anthrone-adapted
method, with critical studies of procedures based upon
extraction of tissues with 30 per cent KOH and with 5 per cent TCA.

584

GLYCOGEN

IN

LIVER

AND

MUSCLE

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4. Glucose standard.
(a) Stock solution. Dissolve 100 mg. of dry, highest purity glucose in 100 ml. of saturated benzoic acid solution. (b) Working standard. Place 5 ml. of the stock solution in a 100 ml. volumetric
flask and make up to volume with saturated benzoic acid solution. 2 ml.
of this solution, containing 0.1 mg. of glucose, are used as a standard.
Procedure-Place the t,issue sample in an efficient blendor under an appropriate volume of TCA and homogenize for 3 minutes. Pour the homogenate into a suitable centrifuge tube or bottle. Centrifuge and decant
the supernatant fluid upon an acid-washed filter paper placed in a funnel
draining into a graduated cylinder. Transfer the residue quantitatively
to the blendor with an appropriate volume of TCA and homogenize again
for 1 minute. Centrifuge the mixture and pour the supernatant fluid
through the same filter. Two more extractions may be made in the same
manner if it is desired to extract better than 97 per cent of the glycogen
present. Make up to the desired volume with 5 per cent TCA and mix
thoroughly.
The final volume should be a quantity that will contain 10
to 200 y of glycogen per ml.
1 ml. of the trichloroacetic acid filtrate is pipetted into a 15 ml. Pyrex
centrifuge tube. To obtain the most reliable results, duplicate samplesof
each unknown are analyzed. To each tube are added 5 volumes of 95 per
cent ethanol with careful blowing to effect thorough mixing. This should
be checked by noting the absence of an interface. The tubes are capped
with clean rubber stoppers and allowed to stand overnight at room temperature. (Alternatively, placing the tubes in a water bath at 3740 for 3
hours may be carried out.) After precipitation is complete, the tubes are
centrifuged at 3000 r.p.m. for 15 minutes. The clear liquid is gently decanted from the packed glycogen and the tubes are allowed to drain in an
inverted position for 10 minutes.
The glycogen is dissolved by addition of 2 ml. of distilled water, the water
being added in a manner that will wash down the sidesof the tube. If the
glycogen does not dissolve instantly, agitate the tube until solution is complete. A reagent blank is prepared by pipetting 2 ml. of water into a clean
centrifuge tube. A standard is prepared by pipetting 2 ml. of standard
glucosesolution, containing 0.1 mg. of glucose, into a similar tube.
At this point 10 ml. of anthrone reagent are delivered into each tube
with vigorous, but consistent, blowing. The stream of anthrone reagent
is directed into the center of the tube and should be sufficient to insure good
mixing. As each tube receives anthrone reagent, it is tightly capped with
an air condenser and placed in a cold tap water bath. The air condenser
is prepared by cutting off the small end of a size 0 rubber stopper and inserting a 4 inch length of glass tubing, 3 to 4 mm. in diameter. This
serves to prevent water from entering the tube from the water bath.

N.

V.

CARROLL,

R.

W.

LONGLEY,

AND

J.

,H.

ROE

585

After all tubes have reached the temperature of the cold water, they are
immersed in a boiling water bath to a depth a little above the level of the
liquid in the tubes for 15 minutes and then removed to a cold water bath
and cooled to room temperature.
The tubes and stoppers are wiped dry
and the contents of each tube are transferred
to a calorimeter tube and
read at 620 mp after adjusting the calorimeter with the reagent blank.
Care is taken to avoid introduction
of lint or contaminating
carbohydrate
into the anthrone reaction.
Calculation-The
calculation of glycogen is as follows:
DX

volume of extract
x 100 x 0.9
gm. of tissue
= mg. of glycogen per 100 gm. of tissue

where DU = optical density of the unknown, DS = optica density of the

standard, 0.1 = mg. of glucose in 2 ml. of standard solution, 0.9 = factor
for converting glucose value to glycogen value.
DISCUSSION

Methods of Extraction-In
applying anthrone reagent to the determination of glycogen the main problem was the method of extraction.
Good,
Kramer, and Somogyi (2) concluded that the original Pfhiger procedure,
which involves boiling the tissue in KOH solution, alcohol precipitation,
acid hydrolysis of the precipitate, and copper reduction of the neutralized
hydrolysate, is the only adequate method for the determination of glycogen. These authors introduced improvements into the Pfltiger procedure
which speeded up the method considerably.
Lower glycogen values have been found with acid extracts of tissuesthan
with extracts obtained with boiling alkaline solution (2, 6, 11, 12). Such
findings have led to the suggestion that glycogen exists in tissues in an
easily extractable or free form and a difficult,ly extractable or
fixed form (6, 13).
We made a comparative study of the acid and alkali extraction methods
applied to liver and muscle. With liver the procedure was to divide the
organ, homogenize one half with 5 per cent TCA, and treat the other half
with boiling 30 per cent KOH for 15 minutes. In experiments on muscle
one gastrocnemius muscle was homogenized with 5 per cent TCA and the
other was boiled with 30 per cent KOH. Glycogen was precipitated by
adding 1.2 volumes of 95 per cent ethanol to the KOH solution and 5 volumes of et.hanol to the TCA extract. The 1.2 volumes of ethanol were
added because this concentration is employed in currently used methods
(2, 3, 5) and higher concentrations have been found to precipitate non-glycogen carbohydrate (2, 3). For the precipitation from TCA solution 5

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D-u x 0.1 x

GLYCOGEN

586

IN

LIVER

AND

MUSCLE

volumes of 95 per cent ethanol were found to be optimal.

The glycogen
was determined by the anthrone method described.
The results with liver, shown in Table I, are related to the level of liver
glycogen.
At glycogen levels of 3 to 7 per cent the values by TCA extraction were 94 to 102 per cent of those obtained by boiling the liver with 30
per cent KOH.
With fasted rats the values from homogenization with 5
per cent TCA ranged from 7 to 48 per cent of those obtained by boiling
with 30 per cent KOH.
TABLE

Nutritional

state

of

Mg. glycogen
TCA

Fed
I

...........................
...........................
...........................
...........................
...........................
...........................
...........................
...........................
Fasted ........................

........................
I
........................
I
........................

........................
I
.........................

........................
I
........................

TCA and KOH Methods of Extraction

from Fed and Fasted Rats

of Liver

per 100 gm. liver

KOII

2944

2980

4360
4330
4000
7162
3067
3950
4980
101
42
15
103
20
138
8
21

4440
4560
4260
6975
3233
4025
5044
210
118
82
652
147
987
107
307

I gg

x 100

Di%.XWX

36
80
230
260
187
166
75
64
109
76
67
549
127
849
99
286

99
98
95
94
102
95
98
98
48
35
17
16
13
15
8
7

Results of comparative studies upon muscle are shown in Table II. The
values found by TCA extraction ranged from 92 to 96 per cent of those
obtained by KOH extraction.
Bloom, Lewis, Schumpert, and Shen (6) made a study of the amounts
of glycogen obtained from liver and muscle by boiling with 30 per cent
KOH and by homogenizing with 10 per cent TCA. These authors found
that the amounts of TCA-extractable glycogen in the livers of fed rats,
rats fasted for 12 hours, and rats fasted for 24 hours were 84.9, 56.4, and
7.9 per cent, respectively, of the values obtained in each case by KOH
The per cent of TCA-extractable glycogen in liver observed
extraction.
by these authors varied directly with the glycogen level. Our findings
are in good agreement with the data of Bloom et al. However, we do not,

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Comparison

N.

V.

CARROLL,

It.

W.

LONGLEY,

AND

J.

H.

587

ROE

TABLE

Comparison

of TCA

and

KOH

Methods

Mg. glycogen

II
of Extraction

of

gH
TCA

in Fed Rats

per 100 gm. muscle

Experiment
No.

2
3
4
5
6

Muscle

433
493
470
651
678
567

KOH

453
538
507
693
717
609

x 100

Difference

20
45
37
42
39
42

96
92
93
94
94
93

shown in Table II, our recoveries of glycogen from rat muscle with cold
TCA, compared with those with KOH extraction, are as high as those obtained by Kemp and Kits van Heijningen with boiling TCA, a result probably due to the high speed and repeated homogenizations
we used.
Kits van Heijningen and Kemp (13) made a comparative study of the
free and %xed glycogen content of rat muscle.
They considered the
values obtained by extracting with cold TCA as <free glycogen and those
found by extracting the cold TCA residue wit,h boiling TCA as ((fixed
glycogen.
They observed a mean ratio of free to fixed glycogen of
1.74 f 0.26. The (free glycogen thus averaged 63 per cent of the total
glycogen.
Dialysis Experiments-To
obtain evidence that might explain the discrepancy between the data obtained by the two methods of tissue extraction, experiments were designed to show whether anthrone-sensitive
materials, other than glycogen, are present in the precipitates
obtained by
treatment of the two types of extracts with alcohol.

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agree with their assumption that the value obtained by KOH extraction
represents the total and therefore the true glycogen content of the liver,
since, as will be shown later, the KOH extract contains non-glycogen carbohydrate.
Bloom et al. (6) found that the TCA-extractable
glycogen of muscle in
normal fed rats was 55 per cent of that obtained by KOH extraction, a
value that is considerably
lower than the TCA recoveries we obtained
(92 to 96 per cent).
Kemp and Kits van Heijningen (12) developed a method for the determination of glycogen in which the TCA homogenate of tissue is warmed at
100 for 15 minutes.
These authors found that extracts prepared with
boiling TCA gave values for rat muscle that ranged from 89 to 111 per
cent of those obtained by the Pfliiger alkali extrachion procedure.
As

588

GLYCOGEN

IN

LIVER

AND

MUSCLE

Livers were removed from anesthetized animals and either boiled with 30
per cent KOH or homogenized with 5 per cent TCA.
To the KOH and
TCA extracts were added 1.2 and 5 volumes of 95 per cent ethanol, respectively.
After centrifuging,
decanting, draining, and washing with 95 per
cent ethanol, the precipitates were taken up in distilled water, placed in
Visking
cellophane tubing, and dialyzed.
Anthrone-sensitive
material
could not be demonstrated in the 24 hour dialysate from the glycogen prepared by TCA extraction,
but, within an hour from the starting of the
dialysis with the glycogen isolated from the boiling KOH extract, material
responding to ant#hrone reagent was found in the dialysate.
The cello-

Showing

Dialyzable
Anthrone-Sensitive
from KOH
Digests
by 1.2 Volumes

Exp%:?=t

Mg. glycogen

Material
in Glycogen
Precipitated
of 95 Per Cent Ethanol
I

per 100 gm. liver

Per cent dialyzable

Before

dialysis

After

dialysis
(2)

2060
4625
3535
2160
1486
77
81
100

2040
4480
3400
2040
1358
57
58
60

Difference
(1) -

20
145
135
120
128
20
23
40

(2)

1
3
4
6
9
26
28
40

phane used was shown to be impermeable to clinical dextran! with an average molecular weight of 70,000.
Further experiments were performed to determine t.he amount of dialyzable mat,erial in glycogen isolated from boiling KOH extracts.
The precipitate obtained in the usual KOH procedure, after washing with 95 per
cent ethanol, was taken up in 10 ml. of water.
5 ml. of this solution were
transferred
to a section of Visking cellophane tubing for dialysis and bhe
other 5 ml. were retained for a control.
Dialysis was carried out against
running water for 24 hours, after which the contents of the cellophane tubing were decanted into a volumetric
flask, the tubing was thoroughly
washed, and the solut,ion was made up to volume.
A carbohydrate
determination by the anthrone method was made on the dialyzed portion and
upon the control which was handled in the same manner except that dialysis was omitted.
The difference between the two values was considered
the amount of dialyzablc material.
The results are shown in Table III.
These data show that the apparent glycogen precipitated by alcohol from

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III

TABLE

Data

N.

V.

CARROLL,

1~. \V.

LONGLEY,

AND

J.

589

1%. ROE

.55

. 25

FASTED

0-b
o--o

GLYCOGEN
GLYCOGEN

t BLOOD

----------o------a

---------I
2

I
3
TIME

1. Effect

DIGEST

+ KOH
+ KOH

1
I

FIG.

LIVER

.15r
-05

O--e

of heating

with

I
4
IN

30 per

I
5

I
6

-0
I
7

I
8

HOURS
cent

KOH

on rat

liver

glycogen

remained at the same concentration,

but the apparent glycogen content of
the KOH digest diminished.
It is well known that the content of pure
glycogen is not diminished by boiling in KOH solution.
Therefore, the
decrease in the KOH digest, shown in Fig. 1, was due to decomposition of
a non-glycogen complex into fragments not precipitable by ethanol.
The
decrease in apparent glycogen in this experiment was marked, because
liver from fasted rats was used in which a relatively high proportion
of
non-glycogen carbohydrate is present.
Nature of Insoluble Residue in KOH Extracts-As
is well known to
workers with the Pfltiger procedure, after boiling liver with 30 per cent
KOH solution, cooling, and allowing the mixture to stand for a time, an
insoluble material separates from the solution.
Analysts are cautioned
not to remove t,his residue as it contains glycogen (5). This material was
examined as follows.
The apparent glycogen content of the solution ob-

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KOH digests of liver contained water-soluble,

alcohol-precipitable,
anthrone-sensitive,
dialyzable material which obviously was not glycogen.
E$ect of Prolonged Boiling with KOH-The
effect of prolonged boiling
of the 30 per cent KOH digests of liver was next studied.
1 ml. aliquots
of KOH extract of liver from fasted rats were pipetted into centrifuge
tubes, and the tubes were capped with condensers and placed in a boiling
water bath.
At various intervals tubes were removed from the water bath
and 1.2 volumes of 95 per cent ethanol were added. 1 ml. aliquots of a
solution of purified glycogen were treated similarly.
The glycogen in the
precipitates
was determined by the anthrone method.
The results are
shown in Fig. 1. During the 8 hour boiling period the purified glycogen

590

GLYCOGEN

IN

LIVEIL

.1ND

MUSCLE

tamed by boiling liver in 30 per cent KOH, after cooling but before the
insoluble residue had appeared, was determined.
After the residue had
separated and the mixture was centrifuged, determinations
of the glycogen
content of the supernatant
fluid and the insoluble residue were made.
The results are shown in Table IV. The amount of glycogen in the supernatant fluid plus that in the residue accounted completely for the quantity
of glycogen found in the original extract.
These experiments suggested
that the insoluble residue that forms in KOH extracts of tissue is not glycogen, since it is insoluble in KOH solution.
However, it may be argued
that the insoluble residue is glycogen separat,ing from a supersaturated

of KOH

TABLE

IV

Digests

of Liver

and

Mg. per cent of

Muscle

apparentglycogen

Material

After
Original

extract

Supernatant
fluid
Liver.
I

Muscle.
I

20.4
27.4
19.0
39.7
123.9
19.2
567.5
717.0
452.7
507.0

centrifugation

I-

13.9
13.4
15.8
16.6
87.0
14.3
552.2
705.0
424.2
499.0

Residue

6.8
11.1
5.1
20.9
25.8
3.9
17.4
16.0
28.5
8.0

KOH solution.
That this was not the case was shown by further study of
the nature of the residue.
It was found that this residue is not soluble in water which proved that
it was not glycogen.
After washing this substance several times with
water, it was found to react characteristically
with anthrone reagent, and,
after hydrolysis,
to reduce alkaline copper solution.
These observations
showed that the insoluble residue that forms in KOH digests of liver is not.
glycogen and is a material that responds to both anthrone and copper reduction techniques.
Completeness of Extraction with TCA-It
is difficult to remove all of the
glycogen from tissue by grinding with TCA because of the requirement for
completely disrupting the cells and thoroughly dispersing the proteins that
are precipitated by TCA.
To study the recoveries by TCA extraction, rat livers were homogenized

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Analysis

N.

V.

CARROLL,

R.

W.

LONGLEY,

AND

J.

H.

591

ROE

in a Servall omnimixer at 14,000 r.p.m.

The first homogenization
was
The
for 3 minutes, after which the mixture was centrifuged and decanted.
residue was returned to the blendor for further homogenization for 1 or 2
minutes.
This procedure was repeated until five homogenizations
had
been carried out. The supernatant liquid obtained aft.er each homogenizaThe residue after the fifth TCA extraction was analyzed for glycogen.
tion was taken up in 30 per cent KOH and treated by the Pfliiger proceTABLE
Recovery

of Glycogen

Extraction

No.

Liver
by Repeated
Homogenizations
Acid with Servalb Omnimixer
at 14,000

I-

ICime homogenized

5iingle

with 6 Per
R.p.m.

Cent

Per cent recovery

extraction

Cumulative

min.

Residue

Residue

Residue

1
2
3
4
5
by KOH
I
2
3
4
5
by KOH

3
4
5
by KOH

?IIg.

3
1
1
1
1
3
1
1
1
2
3
1
I
1
2

3367.0
374.8
63.2
24.2
15.1
142.3
718.3
87.0
12.6
11.0
12.6
164.5
64.9
14.7
5.0
2.6
5.4
112.4

87.5
9.7
1.7
0.7
0.4

87.5
97.2
98.9
99.6
100.0

85.4
10.3
1.5
1.3
1.5

85.4
95.7
97.2
98.5
100.0

70.1
15.8
5.4
2.8
5.9

70.1
85.9
91.3
94.1
100.0

dure, the apparent glycogen being determined by the anthrone method.

The results are shown in Table V. The amounts of glycogen in the fractions analyzed after the fifth homogenization,
per 100 gm. of liver, were
15.1, 12.6, and 5.4 mg. for livers containing 3367,718, and 64.9 mg. per 100
gm., respectively.
Since these amounts were small, it was assumed for
purposes of comparison that the glycogen obtained by the five homogenizaBased upon this assumption, the per cent
tions was all that was present.
in each extraction is shown in the fourth column of Table V and the cumulative recoveries after each extraction in the last. The cumulative recoveries show that 10 to 15 per cent of the total glycogen was present in

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Trichloroacetic

from

592

GLYCOGEN

IN

LIVER

AND

MUSCLE

SUMMARY

1. A method has been developed

liver and muscle.

for the determination

of glycogen in

Downloaded from http://www.jbc.org/ by guest on September 3, 2016

the second extraction.

A second blending of the tissue was necessary to
obtain 97,96, and 86 per cent of the glycogen with liver contents that were
average, low, and at the fasting level, respect,ively, and two more homogenizations of 1 minute each were necessary to recover essentially all of t,hc
glycogen.
The liver glycogen values in Table V for the residues boiled with KOH
after five extractions
with TCA are not considered true glycogen values.
The evidence from Tables II, III, and IV support this assumption.
Concerning l+ee and Rxed
Glycogen-Our
studies led us to the conclusion that the distinction between free and fixed glycogen, proposed
in certain reports in the literature, is an incorrect assumption.
It is our
opinion that the glycogen value obtained by extraction with TCA, when
the extraction procedure is adequate, is the true glycogen content of the
tissues.
In our data of Table V there is no reason to assume that the glycogen obtained in each successive extraction existed in the tissues in a form
differing from that in the preceding extraction, although it might be said
to be more difficultly
extractable.
Further evidence against this assumption is our demonstration
of the presence of non-glycogen carbohydrate in KOH extracts of tissues, which largely accounted for the difference
between the values obtained by TCA and KOH extractions.
Specificity of Proposed Method-The
proposed method, which involves
extraction by TCA and isolation of the glycogen by alcohol precipitation,
is on a sound basis. Excellent recoveries mere obtained in analyses of
blood to which purified glycogen was added. Methods (12) in which the
TCA extract is boiled are subject to the objection that losses may occur
from hydrolysis of the glycogen.
Isolation of the glycogen by alcohol precipitation
is a preferable step.
Boiling with alkali may remove interfering sugars, but usually this procedure leaves a solution that is not ideal for direct color production.
Seifter et al. (5) observed interference with the anthrone reaction when applied
to KOH solutions of liver from which glycogen had been removed by alcohol precipitation.
The interference, amounting to 1.5 per cent for livers
containing 1 per cent of glycogen, was not considered serious at normal or
high glycogen levels.
However, this observed error is in addition to that
produced by other interfering substances which are precipitated from KOH
digests by alcohol.
The time required for the technical manipulations
involved in the proposed method, in which anthrone reagent is used, is much less than that
required for copper reduction procedures.

N.

V.

CARROLL,

R.

W.

LONGLEY,

AND

J.

I-1.

ROE

593

BIBLIOGRAPHY

1. Pfliiger,
E. F. W., Das Glykogen
und seine Beziehungen
zur Zuckerkrankheit,
Bonn, 2nd edition
(1905).
2. Good,
C. A., Kramer,
H., and Somogyi,
M., J. Biol.
C&m.,
103, 485 (1933).
3. Cori, G. T., J. Biol.
Chem., 96, 259 (1932).
4. Morris,
D. I,., Science,
107, 254 (1948).
5. Seifter,
S., Dayton,
S., Novic,
B., and Muntwyler,
E., Arch.
Biochem.,
25, 191
(1950).
6. Bloom,
W. L., Lewis,
G. T., Schumpert,
M. Z., and Shen, T.-M.,
J. BioZ. Chem.,
188, 631 (1951).
7. Hanson,
It. W., Schwartz,
H. S., andBarker,
S. B., Abstracts,
American
Chemical
Society,
128th
meeting,
Minneapolis,
73C (1955).
8. Carroll,
N. V., Longley,
R. W., and Roe, J. H., Abstracts,
American
Chemical
Society,
127th meeting,
Cincinnati,
23C (1955).
9. Roe, J. H., J. Biol. Chem., 208, 889 (1954).
10. Roe, J. H., J. Biol. Chem., 212, 335 (1955).
11. Willstiitter,
R., and Rohdewald,
M., 2. physiol. Chem., 226, 103 (1934).
12. Kemp,
A., and Kits van Heijningen,
A. J. M., Biochem.
J., 66, 646 (1954).
13. Kits van Heijningen,
A. J. M., and Kemp,
A., Biochem.
J., 59, 487 (1955).

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2. Glycogen is precipitated from trichloroacetic

acid filtrate of tissues
by ethanol and determined by an anthrone procedure previously used for
dextran and blood sugar.
3. Studies have shown that extracts made by boiling liver with 30 per
cent, KOH contain material that is not glycogcn.
This material is dialyzable, anthrone-sensitive,
and decomposed to some extent by prolonged
boiling in KOH solution, and, after hydrolysis,
it, reduces alkaline copper
solution; it is, therefore, a source of error in glycogen methods based upon
alkali extraction of tissues.
4. The studies made indicated that the distinction between free and
fixed glycogen, reported in the literature, is an incorrect assumption.

THE DETERMINATION OF GLYCOGEN

IN LIVER AND MUSCLE BY USE OF
ANTHRONE REAGENT
Nicholas V. Carroll, Robert W. Longley and
Joseph H. Roe
J. Biol. Chem. 1956, 220:583-593.

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