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THE JOURNALOFBIOLOQICAL HEMISTRYVol. 236, No. 5, May 1961

Printed in U.S.A.

Complete Removal of Glycogen from Tissues by Extractionwith Cold Trichloroacetic Acid Solution*

JOSEPH H. ROE, J. M. BAILEY, R. RICHARD GRAY, AND JOHN N. ROBINSON

From the Department of Biochemistry, School of Medicine, George Washington Universi ty, Washington, D. C.

(Received for publication, November 25, 1960)

Much of the experimental work upon the state of glycogen inanimal tissues has been interpreted as showing that glycogenexists in tissues in two forms. Early workers (l-4) found thatextraction of animal t issues with water or cold trichloroaceticacid solution yielded less glycogen than was obtained by boilingthe tissue in an alkaline solution. Various names were used to

designate the two fract ions. Easily extractable, free,lyo-, and trichloroacetic acid-extractable are terms thathave been used for the glycogen fraction extracted with wateror cold trichloroacetic acid solution; and di ffi cult ly extractable,fixed, bound, desmo-, and residual are designationsthat have been used for the form that required alkaline digestionto extract it from the tissues. Many workers assumed that thedifficult ly extractable glycogen was bound to protein, but Pfliiger(5) and Meyer (6) found no basis for the assumption of such abinding.

Bloom et al. (7) found that in resting, normal, fed rats, theglycogen of the liver extractable with trichloroacetic acid solutioncomprises 85% of the total glycogen obtained by boiling with

30% KOH solution. The same extraction of the livers of theseanimals, after they had been fasted for 24 hours, yielded only7.9% of that obtained by alkaline digestion. These authorsfound that in the muscle of normal rats the trichloroacetic acid-extractable glycogen was 55 y0 of that obtained by alkaline diges-tion; and 4 hours after subcutaneous epinephrine injection thisfraction was 16.4% of the amount obtained by alkaline digestion.Other workers have observed a shifting in the distribution ofglycogen between the two fractions as a result of fasting (4, 8-lo), epinephrine injection (8, ll-14), insulin administration (12,14, 15)) thyroxin injection (16)) adrenalectomy (9), hypophysec-tomy (9), and muscle work (17).

In our laboratory it was found that extraction of heart muscle

by thorough grinding with 5% trichloroacetic acid solution(Servall Omnimixer once, and the Potter-Elvehjem apparatusthree times) yielded an average of 57% of the total glycogen(18). Alkaline digestion of the residue after the four homoge-nizations with trichloroacetic acid solution, followed b y alcoholicprecipitation, yielded an additional 43 % of glycogen. The latterwas shown to be an essentially pure product by analysis withanthrone reagent and, after hydro lysis , by copper reduction andglucose oxidase techniques (18).

By grinding the tissue in cold trichloroacetic acid solution andthen boiling the homogenate for 15 minutes, Kemp, Kits, and

* This investigation was supported in part by a research grant(A-2802) from the Division of Research Grants,National Insti-

tutes of Health, United States Public Health Service.

Van Heijningen (19) found glycogen values as high as were ob-tained by boiling with 30% KOH solution. These authorsassumed, however, that glycogen exists in tissues in two formsbecause cold trichloroacetic acid solution would not extract allof the compound.

Carroll, Longley, and Roe (20) found that repeated homoge-

nization with 5y0 trichloroacetic acid solution increased the re-covery of glycogen from liver and muscle. Based upon theirimproved recoveries from repeated homogenizations, these au-thors suggested that the distinction between free and fixedglycogen may be simply the result of inadequate homogenization.With the apparatus then available, however, these authors werenever able to effect complete removal of glycogen from tissuesby extraction with trichloroacetie acid solution.

Recent ly, we have employed homogenizing apparatus withsuff icient grinding power to permit removal of all the glycogenfrom the tissues with cold trichloroacetic acid solution. Thetissue residues, after the vigorous homogenization now employed,do not yield appreciable glycogen after being boiled with 30%

KOH solution. It is concluded that a distinction between freeand bound glycogen, based upon nonextractability with coldtrichloroacetic acid solution, is no longer tenable.

EXPERIMENTAL PROCEDURE

1. Determination of Glycogen in Trichloroacetic Acid ExtractRats of both sexes, fed on standard chow, were used. The

animals were anesthetized with vinyl ether and the tissues foranalysis were removed within 1 to 2 minutes after beginningsurgery. Tissues were ground under 10% trichloroacetic acidsolution in a VirTis homogenizer,r at speeds of 36,000 to 45,000r.p.m., with and without glass homogenizing beads, for periodsof 5 minutes. The homogenizing flask was placed in an ice-water bath, and the temperature of the contents was maintainedbelow 15 during the treatment. After the initial homogeniza-tion, the mixture was centrifuged, the fluid was decanted, andthe procedure was repeated upon the residue once or three times.The supernatant fluids from each homogenization were pooledand made up to volume. Aliquots of this solution were pipettedinto centr ifuge tubes, 5 volumes of ethyl alcohol were added,and the mixture was allowed to stand overnight to precipitatethe glycogen. The tubes were then centrifuged, the fluid wasdecanted and drained, and the glycogen was determined by theanthrone method of Carroll, Longley, and Roe (20).

1 Macro model 45,250..m l flask, The Vir Tis Company, Gardiner,

New York.

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May 1961 J. H. Roe, J. M. Bailey, R. R. Gray, and J. N. Robinson 1245

2. Determination of Glycogen by Alkaline TABLE IDigestion of Tissue Residue Study of methods of extraction of glycogen from tissu es

The tissue residue after the final homogenization under tri -chloroacetic acid solution was transferred to a flask, 30% KOHsolution was added, and the mixture was boiled for 2 hours. The

alkaline solution was made up to volume, aliquots were pipettedinto centrifuge tubes, and 1.2 volumes of ethyl alcohol wereadded. After standing overnight the tubes were centrifuged,and the fluid was decanted and drained. The insoluble precipi-tate in the tubes contained a dark material which would haveinterfered in the anthrone method, and it was necessary to repre-cipitate the glycogen. To each centrifuge tube was added ameasured volume of 10% trichloroacetic acid solution and theprecipitate was thoroughly dispersed with a mechanical agitator.This step dissolved the glycogen but not the protein-like, pig-mented material remaining after alkaline digestion of tissues.The tubes were centrifuged, aliquots of the supernatant wereremoved to other centrifuge tubes, and 5 volumes of ethyl alcohol

were added to each measured aliquot. After standing overnightthe tubes were centrifuged, the fluid was decanted and drained,and the glycogen was determined by the anthrone method (20).

RESULTS

The results of these studies are summarized in Table I. Thedata of primary interest appear in the column headed Residueboiled with KOH.

In this table previous work is cited in which rat liver and heartwere homogenized under trichloroacetic acid solution with agrinder having a speed of 14,000 r.p.m. and the residue was boiledwith 30% KOH solution. The glycogen was determined in thetrichloroacetic acid and the KOH extracts. The results showinability to remove all the glycogen from liver and heart by ex-traction with cold trichloroacetic acid solution when homogeniz-ing apparatus with speeds up to 14,000 r.p.m. is used.

When homogenizing apparatus with a maximal speed of 45,000r.p.m. was used in conjunction with glass homogenizing beads,strikingly different results were obtained. With four homoge-nizations under trichloroacetic acid solution and the inclusion ofhomogenizing beads, essentially all the glycogen was transferredfrom the tissues to the extracting fluid . Negligible amounts o fglycogen were found in the KOH extract of the tissue after ithad been extracted with trichloroacetic acid solution. Whenthe number of homogenizations with the high speed grinder

under trichloroacetic acid solution was reduced to two andhomogenizing beads were not used, the values in the analysis ofthe residue boiled with KOH, in mg per 100 g, were 110 and 112for heart, 182 and 57 for liver, and 127 for gastrocnemius muscle.When two homogenizations under trichloroacet ic acid solutionwere carried out with the inclusion of homogenizing beads, thevalues found in the alkaline solution obtained by boiling theresidue with KOH solution, in mg per 100 g, were 2 and 7 forheart, 5 and 14 for liver , and 8 for gastrocnemius muscle.

The values obtained from the tissue residues boiled with KOHsolution are not considered to represent a significant quantity ofglycogen. They are ver y small in comparison with the valuesobtained by the same procedure without the use of beads, andit is known that in alkaline digests of animal tissues there areanthrone-sensitive, nonglycogen substances (20).

Tissue Alimentation

Liver*Liver*Liver*HeartfHearttHeartHeartHeartHeartHeartLiver

LiverLiverLiverLiverLiverMuscle1Muscle

FedFedFastedFedFastedFastedFastedFastedFastedFastedFed

FastedFedFedFastedFastedFedFed

-

No.

5

554

44222

24

4222

222

r.p.?i

14,000

14,00014,00014,000

14,00045,00045,000

45,00045,00045,000

45,000

45,00045,000

45,00045,00045,00045,000

45,000

-----

-

-

-

-

-

3844

84193

172

207175

154130291240

4052

6544902

4913201

56

501483

142

164112141

18202

1107

112

10

85

18214

578

127I I* From the data of Carroll, Longley, and Roe (20).t One extraction in Servall grinder at 14,000 r.p.m., three ex-

tractions with Potter-Elvehjem apparatus.Gastrocnemius.

_-

-Homogenizations

Maximalspeed

-

Beads

-

E:1

ch1

Glycogen content

DISCUSSION

The results in Table I show clearly that all the glycogen inanimal tissues can be extracted with cold trichloroacetic acidsolution when the homogenization is adequate. The crucial ex-periments in this table are those in which two homogenizationswith the high speed grinder were carried out, with and withouthomogenizing beads. In the experiments without beads, twohomogenizations with the VirTis grinder did not remove all theglycogen. With beads in the homogenate, essentially all theglycogen was transferred to the trichloroacetic acid solution bythe same number of homogenizations at the same speed. Failureto recover all the glycogen from tissues by trichloroacetic acidextraction, in these and other experiments that we have reported(20), is thus shown to be due to inadequate homogenization.

Our experiments suggest that the so called bound glycogenis not held in the tissues by a chemical binding but rather byphysical entrapment. Incomplete recovery of glycogen fromtissues by extraction with cold trichloroacetic acid solution ap-pears to have been due to a failure to effect complete dispersionof the tissues.

That glycogen does exist in the tissues bound to protein tosome extent must be accepted as true. Accepted theory of en-zyme action postulates that some glycogen in tissues is bound tophosphorylase; and the Michaelis, K,, values (21, 22) reportedfor glycogen-phosphorylase association support this assumption.Nonenzymatic association of glycogen with proteins also prob-ably occurs (23). However, there is no evidence that there is abond between glycogen and proteins which is resistant to coldtrichloroacetic acid. The results in Table I show that such abond does not exist.

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246 State of Glycogen in Tissues Vol. 236, No. 5

SUMMARY

1. Glycogen in liver , heart, and skeletal muscle has been ex-acted completely with cold trichloroacetic acid solution by

making use of a high speed homogenizer and homogenizing beads.2. Data have been obtained which suggest that failure to re-

over all glycogen from tissues with cold trichloroacetic acidolution has been due to inadequate homogenization.

3. It appears from the results reported that glycogen is notbound in the tissues in the sense of being held by a bond that

s not broken b y cold trichloroacetic acid solution.4. The data of this report suggest that the distinctions and

nterpretations in the literature concerning (free and boundlycogen in tissues are no longer tenable.

REFERENCES

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Hoppe-Se ylers, 226, 103 (1934).5. PFL~~GER, E. F. W., Das Glycogen, M. Hager, Bonn, 1905.6. MEYER, K. H., in F. F. NORD (Editor), Advance s in enzymol-

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