STUDIES ON HUMAN GLYCOGEN

I. PREPARATION, PURITY, AND AVERAGE CHAIN LENGTH*

BY W. J. POLGLASE, EMIL L. SMITH, AND FRANK H. TYLER

(From the Laboratory for the Study of Hereditary and Metabolic Disorders, and the Departments of Biological Chemistry and Medicine, University of Utah College

of Medicine, Salt Lake City, Utah)

(Received for publication, February 5, 1952)

Many case histories of metabolic disturbances which are accompanied by increased storage of glycogen in liver, skeletal muscle, and other tissues have been reported. Mason and Andersen (1) have summarized and classi- fied these disturbances according to clinical and biochemical findings; how- ever, a definitive examination of the physicochemical constitution of the glycogen in these disorders has not been previously reported. Indeed, few studies on glycogen from human tissues are available.

The present investigation of human glycogen was begun as a result of clinical observations’ on a metabolic disorder in infants (subject N. D.) accompanied by unusually high glycogen deposition, particularly in skeletal muscle. For purposes of comparison, samples of glycogen were obtained from patients showing no symptoms of disturbed carbohydrate metabolism; these have been indicated to be normals (subjects P. F., J. H. W., J. K., B. K., G. G.). Also included (P. N.) is a biopsy sample from a typical case of glycogen storage disease similar to those described by von Gierke and others (1).

The properties of glycogen to be presented include specific rotation, yield of glucose, and comparative degree of branching as indicated by formic acid production following periodate oxidation. In the following paper (2), sedimentation studies are reported.

Materials-The major difficulty associated with a study of human gly- cogen is the problem of obtaining normal controls. Autopsy tissues are readily available, but seldom before considerable glycogenolysis has oc- curred and, occasionally, samples of liver and muscle tissue from autopsies fail to yield glycogen. Biopsies are to be preferred from this point of view but only small amounts of tissue can be made available. In neither autopsy nor biopsy samples can the glycogen be regarded as characteristic until sufficient material has been examined to establish normal properties. For this reason brief clinical summaries are given which will permit evalu-

* This investigation was aided by research grants from the National Institutes of Health, United States Public Health Service.

1 W. Krivit, W. J. Polglase, F. D. Gunn, and F. H. Tyler, in preparation.

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98 HUMAN GLYCOGEK. I

ation of the properties of glycogen described in this and in the following paper (2).

We are happy to acknowledge the cooperation of Dr. W. Krivit in obtaining many of the samples described below.

Normal; Autopsy (Subject P. F.)-A 10 month-old white female infant was admitted because of tachycardia and vomiting. A diagnosis of parox- ysmal auricular tachycardia was established and therapy consisted of 0.5 mg. of digitoxin over a period of 25 hours. Death occurred suddenly without signs of congestive failure and autopsy revealed no liver pathology. Liver and muscle samples were taken for study within 1.5 hours after death and immediately placed in dry ice.

Normal; Autopsy (Subject J. H. W.)-A 12 year-old white male with bulbar poliomyelitis expired in respiratory paralysis within 3 hours after admission. Autopsy specimens of liver were obtained within 1 hour after death and immediately frozen.

Normal; Biopsy (Subject J. K.)--rl 52 year-old white male with peptic ulcer of the stomach was subjected to gastroenterostomy under anesthesia. A biopsy of liver was obtained and immediately frozen. No evidence of hepatic disease was found.

Normal; Biopsy (Subject B. K.)-A muscle biopsy was obtained from a 36 year-old white female with peroneal muscular atrophy. The muscle was immediately frozen. Microscopic sections showed no evidence of glycogen storage disease.

Normal; Biopsy (Subject G. G.)-A 21 year-old white male was admitted for study of muscle disease and diagnosed as having paramyotonia con- genita (3). A biopsy of muscle was immediately frozen for glycogen isola- tion. Microscopic sections of the tissue revealed focal degeneration with no glycogen storage.

Glycogen Storage Disorder; Biopsy and Autopsy (Subject N. D.)-An 18 month-old white female was admitted because of muscular weakness since the age of 6 months. A year earlier a sibling who had had a similar clinical picture was found at autopsy to have glycogen storage disease of liver and striated muscle. Epinephrine and glucose tolerance tests of IX. D. were normal. Liver and muscle biopsy samples showed excessive gly,cogen storage and these were placed in dry ice immediately. Several weeks later the patient expired with pneumonia and autopsy was performed within 1 hour after death. Tissues were frozen immediately. Autopsy studies con- firmed the diagnosis of glycogen storage disease. No chemical differences were found between earlier samples of glycogen obtained by biopsy and the later ones from tissues secured at autopsy. The detailed clinical find- ings will be described e1sewhere.l

Glywgen Storage Disorder (von Gierke’s Disease); Biopsy (Subject P. N.)-

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W. J. POLGLASE, E. L. SMITH, AND F. H. TYLER 99

A 14 month-old white female was admitted because of hepatomegaly. Studies demonstrated constant hypoglycemia, acetonuria, normal epineph- rine, and glucose tolerance tests. Biopsy specimens of liver and muscle were rapidly frozen. Definite evidence of glycogen storage disease of live1 was found in microscopic sections.

Preparation of Glycogen-All samples were prepared by a modification of the classical method of extraction with aqueous potassium hydroxide followed by alcohol precipitation. Purification was carried out by acetic acid precipitation (4). The procedure was as follows. The frozen tissue was heated with 30 per cent aqueous pot,assium hydroxide solution on a boiling water bath2 until completely disintegrated (about 2 hours). The mixture was clarified by filtering through glass wool and the glycogen was precipitated by the addition of 1.2 volumes of 95 per cent ethanol. The crude glycogen was collected by centrifugation and reheated with fresh 30 per cent potassium hydroxide solution for 2 hours. The glycogen was precipitated with alcohol, washed three times with 52 per cent alcohol, dissolved in water, and centrifuged to remove suspended impurities. The glycogen was again precipitated with alcohol and washed thoroughly by centrifugation with 52 per cent alcohol. The glycogen was then dissolved in water and precipitated by the addition of 4 volumes of glacial acetic acid, as recommended by Bell and Young (4). This acetic acid precipita- tion was repeated four times in rapid succession, the glycogen being redis- solved in water without heating after each precipitation. After the final acetic acid precipitation, the glycogen was washed several times with 52 per cent ethanol, redissolved in water, and again precipitated with ethanol.3 The precipitated material was washed repeatedly with 52 per cent alcohol, followed by further washing with gradually increasing concentrations of alcohol, and finally with absolute alcohol and acetone. Samples were dried to constant weight at 94” in vacua.

Glycogen is known to be hygroscopic and most workers equilibrate sam- ples for analytical study in the atmosphere and correct for moisture by a separate determination. Because of the limited amount of material avail- able, all analytical determinations were performed on material previously dried to constant weight at 94” in vacua. In the relatively dry atmosphere in Utah it is possible to weigh glycogen samples for analyses without any special precautions other than completing the weighing within 2 or 3 min- utes.

Yield and Putity-These are shown in Table I. Glycogen samples were characterized by the specific rotations in aqueous molar sodium chloride

* The boiling point of water is 94” at the altitude of this laboratory. 3 It was usually necessary to add a trace of electrolyte (lithium chloride) to induce

coagulation at this point.

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100 HUMAN GLYCOGEN. I

solution. This solvent was chosen for two reasons. First, human liver glycogen gives solutions of high opalescence, and transmission of light in molar saline is somewhat better. Nevertheless, in most cases the maxi- mum concentration of’ liver glycogen which could be used in a 1 dm. polarimeter tube was 1 per cent. Second, aqueous molar sodium chloride has been used in most of the published ultracentrifuge studies on glycogen

TABLE I

Yield and Purity of Human Glycogen Samples

Tissue Yield

I ysist

Liver, normal, a. (P. F.). _. “ “ “ (J. H. W.). 0.1 +192 f 5+48.5 92.4 “ “ b. (J. K.) _. “ glycogen storage, b. (N. D.) 1.8 0.17 9.1 $202 f 5. “ “ “ “ 35 2.2 6.3 f198 f 5f49.9 95.0 “ “ “ it (P. N.).‘.‘.:: 2.1 0.29 13.8 +192 f 2+49.7 94.7

Muscle, normal, a. (P. F.) 125 0.076 0.06+191 f 2 “ “ b. (B.K.) 5.7 0.027 0.5 +2OO f 2 I‘ “ “ (G. G.) ._...._ .._ 7.1 0.051 0.7 +isS f 2 “ glycogen storage, b. (N. D.) 2.7 0.24 8.9 +191 f 2 “ “ “ a. “ A.. 4.4 : 0.31 7.1 f192 f 2+49.1 93.5 ‘I I‘ ‘I “ “ B.. 1.9 0.12 6.4 +200 f 2f52.5100 “ I‘ ‘I ‘I “ ‘25. 2.5 0.21 8.4 +199 f 2 “ “ “ b. (P. N.) 0.50 Trace

Heart “ “ a. (N. D.). .I 1.2 ~ 0.083 2.5 +191 rt 2,

* Determined at a concentration of 1 per cent in 1 M sodium chloride. t Calculated on basis of liberated n-glucose. $ Calculated from rotation of hydrolyzed glycogen as compared to equilibrium

rotation of n-glucose (+52.59). $ a., autopsy; b., biopsy. Tissue was permitted to stand at room temperature

(about 23”) for 17 hours before isolation of glycogen.

and was used as the solvent in the ultracentrifuge studies reported on the same samples (2). In order to conserve material, the same solution of glycogen was used for both rotation and sedimentation studies. Solutions of human muscle glycogen show less light scattering than those containing human liver glycogen and the rotation of a 1 per cent solution is readily determined in a 2 dm. polarimeter tube.

Whenever sufficient material was available, the rotation of an acid hy- drolysate was determined. A dry sample of glycogen (25 mg.) was dis- solved in 1 M hydrochloric acid and the volume adjusted to 2.50 ml. The

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W. J. POLGLASE, E. L. SMITH, AND F. H. TYLER 101

solution was heated at 94” in a sealed tube for 6 hours. The specific rotation (D line) was determined in a 2 dm. tube.

The specific rotation of the glycogen sample and the rotation of the acid hydrolysate indicated about the same degree of purity for the glycogen and further showed that pure human glycogen has a specific rotation between +203” and +208’. This value is consistent with previous studies of glycogen samples from various sources (5). By these criteria, prepara- tions varied in purity from 92 to 100 per cent.

The yields of glycogen from liver biopsy in both instances of glycogen storage disorder (N. D. and P. N.) are much higher than from the single normal liver biopsy (J. K.). Insufficient muscle tissue was available from P. N. for glycogen isolation but a calorimetric determination (6) indicated 0.6 per cent glycogen.

A sample of normal muscle obtained at autopsy (P. ?.) gave little or no glycogen when permitted to stand at room temperature for some hours. Muscle tissue obtained from N. D. at autopsy and frozen within 1 hour after death gave yields of 6.4 and 7.1 per cent glycogen. A similar tissue sample which was permitted to stand at room temperature for 17 hours yielded 8.4 per cent glycogen. Within the variability of these samples, it may be concluded that the muscle of N. D. was incapable of destroying glycogen.

Solutions of all human liver glycogens were of a higher turbidity than either rabbit liver glycogen or human muscle glycogen, with the exception of the liver sample from the patient showing von Gierke’s syndrome. This glycogen sample gave an almost clear solution.

Average Chain Length-Since glycogen is known to possess a highly rami- fied structure consisting of a-n-glucopyranosyl units joined 1,4 with occa- sional branching at Ca, it was of interest to determine the average chain length by the method of periodate oxidation. Because in most cases only small amounts of glycogen were available, the periodate method was studied with the objective of developing a technique suitable for handling 25 to 50 mg. samples.

Methylation studies (7,8) have indicated that the length of the repeating chain in rabbit liver glycogen is about 12 anhydroglucose units. Oxidation studies with potassium periodate (9) have confirmed thii value for most preparations (5) and have indicated a chain length of 11 for human muscle glycogen. During the course of the present study two publications ap- peared in which the chain length of rabbit liver glycogen by sodium peri- odate oxidation was found to be about 18 (10, 11). In one of these publi- cations (lo), a new enzymatic method for the determination of chain length was reported which indicated a value of 14.7 for rabbit glycogen. However, other workers (12) have since reaffirmed the earlier value of 12

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102 HUMAN QLYCOGEN. I

by oxidation with dilute sodium periodate. It is apparent that the prob- lem of unit chain length of glycogen is not completely settled and that the results may depend on the method of analysis. The data obtained by periodate oxidation presented herein are, therefore, considered to be com- parative but not necessarily true average chain lengths.

Periodate Oxidation-A 50 mg. sample of dried glycogen was weighed rapidly and dissolved in 1 ml. of water in a 5 ml. volumetric flask. The solution was cooled in an ice water bath and 4 ml. of 0.2 M sodium periodate (previously cooled to 2”) added. Reaction mixtures were then placed in a refrigerator maintained at 2“. Aliquots of 1 ml. were transferred at suit- able intervals to 10 ml. flasks containing about 0.1 ml. of ethylene glycol. All glassware used up to this point was precooled in the refrigerator. It was found necessary to take the usual steps to insure absolutely clean glassware by washing in chromic acid and distilled water, followed by steaming and drying.

The solutions were allowed to stand at room temperature for 1 hour prior to titration. Formic acid was measured by titration to the methyl red end-point with 0.01 N barium hydroxide from a Machlett micro burette fitted with a No. 23 hypodermic needle. The quantity of barium hydrox- ide solution required for the titration was usually in the range of 0.4 to 0.7 ml., depending on chain length. A 25 mg. sample could be treated as above by the use of half the quantity of reagent. Controls of sodium periodate solution without glycogen were run parallel to the oxidation test. These did not show any increase in acidity. All glycogen samples were similarly found to be free of acidity.

Results-The results of the periodate oxidat.ions are listed in Table II as average chain lengths at different time intervals. The data obtained by the method described above (Method A) show that, with increasing reac- tion times, progressively lower values are obtained for the average chain length. The procedure of Potter and Hassid (13) for amylose and amy- lopectin has been shown by Schlamowitz (11) to reach a constant value for glycogen after a reaction time of 24 hours. As already noted (1 l), this method (Method B) is capable of detecting a glycogen of low chain length and is, therefore, satisfactory for comparative purposes. Method B does, however, yield higher values for the chain length of rabbit liver glycogen than other published procedures (9). The results with Method A after a 24 hour oxidation show good agreement with those obtained by the enzy- matic method of Cori and Larner (10).

The essential difference between Method A and Method B is that in the former a larger excess of oxidant is provided, although the concentration is lower. As noted by Abdel-Akher and Smith (12), the presence of a sub- stantial excess of periodate at a low concentration appears to be important

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W. J. POLQLASE, E. L. SMITH, AND F. II. TYLER 103

for insuring quantitative oxidation of terminal glucose residues to formic acid without extensive oxidation of primary oxidation products. It ap- pears that the enzymatic method (lo), by virtue of its specific nature, is probably the most reliable procedure for obtaining true average chain lengths of glycogen preparations. The periodate technique, although of great comparative value, cannot be regarded as completely specific.

A comparison of the chain lengths determined at 24 hours of glycogens from different individuals (Table II, Method A) shows that only the prep-

TABLE II Chain Length of Glycogen

Chain length

Source --

5 hrs.

Human liver normal Autopsy (P. F.). . . . . . . . . . .

“ (J. H. W.) . . 18.0 Heavy fraction (P. F.) (2). . Light “ “ (2) . . . . . . . . . . 18.0

Human liver, glycogen storage Biopsy (P. N.) . . . . . . . . . 18.0

“ (N. D.) . . . . . . . . . . . . . . Autopsy heavy fraction (N. D.) (2). . . 14.0

‘< light “ I‘ (2). . 14.0 Rabbit liver..............................., Human muscle, normal

Autopsy (P. F.). . . . . . . . . . . . . . . . . Biopsy (G. G.). . . . . . . . . . .

Human muscle, glycogen storage Biopsy (N. D.). . . . . . . .

Method A

24 hrs. 48 hrs. 12 hrs.

13.5 14.0 13.5 15.0

13.5 9.5

11.0 10.5 14.5

14.5 14.5

13.0

12.5 13.0 12.5 14.0

12.5 8.5

10.5 9.5

14.0

11.5

7.0

11.5

T- Method B (11)

17.0

12.0

The numbers in parentheses refer to bibliographic citations.

arations from the liver of N. D., which gave values of 9.5 to 11, differed significantly from the results obtained on other samples, which varied from 13 to 15. The fractions separated on the basis of particle weight (2) from the liver of P. F., a normal individual, did not show significant differences in chain length. Likewise, the light and heavy fractions from the liver of N. D. were essentially identical and with a shorter average chain length. Determinations by Method R also show the difference in chain length for the glycogen samples of the two individuals, although the absolute values are higher than those obtained by Method A.

It is of interest that the liver glycogen of the patient (P. N.) with von Gierke’s disease had a normal chain length.

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104 HUMAN GLYCOGEN. I

An interpretation of the significance of these data for human glycogen storage diseases cannot be presented with the limited data available at this time. The important observation is that glycogen disorders may be accom- panied by abnormal glycogen structures. These abnormalities may lx associated with chain length, as in the case of N. I)., or, as noted in the following paper (2), with particle size.

SUMMARY

1. Glycogen prepared from several samples of human tissue, including two cases of glycogen storage disorder, has been characterized by optical rotation and by rotation of acid hydrolysates.

2. Glycogen from human liver gives a more opalescent solution than rabbit liver glycogen or human muscle glycogen. A sample of glycogen from one case of glycogen storage disorder (von Gierke’s) shows less tur- bidity in molar saline than does normal liver glycogen.

3. A periodate technique is described for determining comparative aver- age chain lengths of glycogen with 25 or 50 mg. samples.

4. Several normal human glycogen preparations from both liver and muscle tissue have been observed to have about the same average chain length. Liver glycogen from a patient having a glycogen storage disease primarily affecting muscle was about 25 per cent shorter in chain length than normal liver glycogen preparations. The muscle glycogen from this patient was not definitely different in chain length from the glycogen of normal individuals. A preparation of glycogen obtained by biopsy from the liver of a patient with von Gierke’s disease showed normal chain length.

BIBLIOGRAPHY

1. Mason, H. H., and Andersen, D. H., Am. J. Dis. Child., 61, 795 (1941). 2. Polglase, W. J., Brown, D. M., and Smith, E. L., J. Biol. Chem., 199, 105 (1952). 3. Tyler, F. H., Witten, T. A., and Stephens, F. E., Am. J. Med., 6, 391 (1949). 4. Bell, D. J., and Young, F. G., Biochem. J., 28,882 (1934). 5. Bell, D. J., Biol. Rev. Cambridge Phil. Sot., 23, 256 (1948). 6. Good, C. H., Kramer, H., and Somogyi, M., J. Biol. Chem., 100, 485 (1933). 7. Haworth, W. N., and Percival, E. G. U., J. Chem. Sot., 2277 (1932). 8. Haworth, W. N., Hirst, E. L., and Smith, F., J. Chem. Sot., 1914 (1939). 9. Halsall, T. G., Hirst, E. L., and Jones, J. K. N., J. Chem. Sot., 1399 (1947).

10. Cori, G. T., and Lamer, J., J. Biol. Chem., 188, 17 (1951). 11. Schlamowitz, M., J. Biol. Chem., 188, 145 (1951). 12. Abdel-Akher, M., and Smit,h, F., J. Am. Chem. Sot., 73, 994 (1951). 13. Potter, A. L., and Hassid, W. Z., J. Am. Chem. Sot., 70.3488 (1948).

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TylerW. J. Polglase, Emil L. Smith and Frank H.  AVERAGE CHAIN LENGTHPREPARATION, PURITY, AND STUDIES ON HUMAN GLYCOGEN: I.ARTICLE:

1952, 199:97-104.J. Biol. Chem. 

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