THE EFFECT OF pH ON CARBOHYDRATE CHANGES IN ISOLATED ANAEROBIC FROG MUSCLE*

BY MARGARET KERLY AND ETHEL RONZONI

(From the Laboratory of Biological Chemistry, Washington University School of Medicine, St. Louis)

(Received for publication, August 5, 1933)

One means of studying the transformations of carbohydrate which occur in muscle is the attempt to balance the loss of gly- cogen with its products when isolated muscles are kept under anaerobic conditions and oxidation is thereby avoided. Accord- ing to Meyerhof’s experiments (1920) nearly all of the glycogen which disappears under such conditions is accounted for by the lactic acid formed. More recently Eggleton and Eggleton (1929- 30) observed an increase of a phosphate ester which Cori and Cori (1931-32) found to be hexosemonophosphate, thus making it nec- essary to include this substance in a balance sheet of the carbo- hydrate changes during anaerobiosis.

We find that the reaction of the medium in which the muscle is suspended, and presumably the reaction of the muscle itself, has a marked effect on the fate of glycogen broken down. At an alkaline reaction, as in Meyerhof’s experiments, the increase of lactic acid approximates the glycogen breakdown, while the hex- osemonophosphate content changes little. As the reaction is made progressively more acid, the amount of lactate formed is less and is much below the equivalent of the glycogen loss; at the same time the hexosemonophosphate increases significantly.

Still other products of glycogen appear when the muscles are made acid by equilibrating them with high tensions of CO, with- out oxygen. Both glucose and a non-fermentable reducing car- bohydrate are formed. During anaerobiosis at alkaline or near neutral reaction little or no formation of these sugars takes place

* This work was aided by a grant from the Rockefeller Foundation to Washington University for research in science.

161

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162 Carbohydrate Changes in Muscle

in isolated muscle. Although it appears to be well established that muscle glycogen is not directly convertible into glucose to an extent permit.ting its diffusion into the blood and that the con- centration of muscle glucose is normally definitely lower than the blood sugar, it would seem from our observations that the forma- tion of some glucose may take place in muscle if its interior reaches a certain acidity. The possibility also exists that glucose forma- tion may occur likewise under normal conditions and be prevented from accumulating by more rapid conversion back to glycogen, hexosephosphate, lactate, or other products.

Methods

For a reasonably satisfactory balance of the changes taking place in carbohydrate constituents of muscle it seemed essential to develop analytical methods permitting all determinations on a single muscle sample; otherwise variat.ions are apt to obscure the changes searched for. After a number of preliminary experi- ments, the following procedure was adopted. All of the lactate and hexosephosphate, together with a portion of the glycogen, are extractable and are determined in extracts. The carbohy- drate left in the muscle residue is separately determined and added to that found in the extract. After weighing, the muscles of one side were frozen with COn snow and analyzed as stated below for the initial value. Those from the other side were placed in a flask in Ringer’s solution (2 to 3 times the muscle volume), con- taining 0.02 M NaHCOs and equilibrated with (a) nitrogen to give pH 9, (b) nitrogen plus 5 per cent COZ for pH 7.2, and (c) Cot for pH 6.0. The flasks were shaken in a water bath at 25’ the desired length of time, after which the muscles were removed and frozen.

The frozen muscles were ground in a chilled mortar with a solu- tion of HgCh in HCl, the quantities being such that the 6nal concentration became 3 per cent HgCla in 0.5 N HCl when the Ringer’s solution was later added. The extract was separated from the precipitat,e by centrifugation and the residue reextracted three times, the final volume of extract giving a dilution of from 5 to 10. This procedure extracts 40 to 55 per cent of the glycogen, the soluble carbohydrate, lactic acid, and acid-soluble phosphorus compounds.

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M. Kerly and E. Rmuoni

E&nation of Total Carbohydrate-The whole of the residue and an aliquot portion of the extract were separately hydrolyzed by heating 4 hours in 0.5 N HCl. After preliminary treatment with HgS04 and BaCOa (West, Scharles, and Peterson, 1929) the total and non-fermentable material in each solution was determined by the Shaffer-Somogyi (1933) Reagent 50 with 2 gm. of KI, the fermentable sugar being obtained by difference. The non-fer- mentable reducing substance was fairly constant in amount, about 100 mg. per cent. The data given for total carbohydrate are the sum of that found in the residue and extract and represent yeast-fermentable sugar expressed as glucose.

The carbohydrate of hexosemonophosphate is included only to a negligible extent in the results for total carbohydrate. That portion of the ester which withstands hydrolysis is removed in the treatment by HgSOa-BaCOo in the presence of chloride. If no chloride is present, about 30 per cent appears in the Eltrate. After heating in 0.5 N HCl for 4 hours, only about 10 per cent of the hexose content appears as fermentable sugar, though hydrolysis as measured by liberation of phosphate has proceeded to the ex- tent of 30 per cent. Since the hexosephosphate content of the muscles used represents less than 1 per cent of that of the total carbohydrate and only one-tenth of this appears as fermentable reducing sugar, the presence of the est,er has no significant effect upon the data for total carbohydrate.

The optimum time for hydrolysis of glycogen solutions in 0.5 N HCl coincides with the time at which non-fermentable reduction falls to zero; at that time (3 hours) the fermentable reduction is maximum. Similar tests applied to muscle showed after an early rise a decline to a constant value in the reducing non-fermentable fraction. This time (4 hours) has been used for hydrolysis. Be- yond 4 hours heating the fermentable reduction continues to in- crease slightly but at a linear rate. This slow increase is attrib- uted to the hydrolysis of some combined carbohydrate, perhaps similar to that found in blood by Rimington (1929).

When opposite muscles are analyzed by this method, the aver- age difference is 3.6 per cent (Table I). This comparison is a control both on the analytical methods and on the similarity of the initial values in control and asphyxiated muscles.

Preformed glucose WM determined directly on the original a-

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164 Carbohydrate Changes in Muscle

trate freed of Hg by difference in reduction before and after fer- mentation. Glycogen was determined on whole muscles after the usual KOH treatment and precipitation by alcohol, as fer- mentable glucose after hydrolysis.

Estimation of Hexosenaonophosphatexosephosphate was de- termined by a modification of the method of Cori and Cori (1931- 32). In the application of their method to frog muscles difficulty was encountered owing to the high glycogen content, a part of which escapes precipitation with the inorganic Ba phosphates and peptizes these otherwise insoluble salts. Furthermore, we de-

TABLE I

Total Carbohydrate in Groups of Corresponding Resting Muscles from Opposite Legs. Residue + HgC& Extract

Side A Side B

ng. per 100 gm. mg. per 100 gm.

2937 3036 1714 1684 1729 1873 2186 2288 1734 1792 1112 1097 624 597

2158 2181 2311* 2378

-

-.-

. - I Average....................................I

* After 23 hours at pH 6.0.

DitTCWlCC

pm cent 3.3 1.8 8.4 4.7 3.3 1.4 4.5 2.2 2.9

3.6

sired to make hexosephosphate det,erminations on the same HgCL titrates used for sugar determinations and in these solutions the high chloride content prevents the complete precipitation of adenosinetriphosphate. We therefore adopted the following pro- cedure.

From an aliquot of the original solution (Hg-freel) equivalent

1 The HgS precipitate clears the solution completely of glycogen. There is a limit to the amount that a given amount of sulfide will carry down. This condition is easily recognized by the fact that the sulfide is also held in suspension, and can be remedied by addition of more HgCls and again removing the Hg with H&J.

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M. Kerly and E. Ronzoni 165

to 2 gm. of muscle the total phosphate compounds were pre- cipitated as the Ba salts with alcohol. 2 cc. of saturated Ba(OH)z, the remaining acid neutralized to phenolphthalein with KOH, 4 volumes of 95 per cent alcohol, and 2 hours standing in the re- frigerator are required for complete precipitation. After separa- tion by centrifugation and removing alcohol by evacuation the precipitate may be dissolved in 5 per cent trichloroacetic acid and the Cori and Cori procedure followed. Under the conditions of our experiments we found that two substances are present with the hexosephosphate, adenylic (or inosinic) acid affecting the phosphate value, and an intermediate carbohydrate which affects the copper reduction values. We have therefore removed the nucleotide by HgAc precipitation (used by Lipmann and Loh- mann (1930) in the purification of hexosephosphate). The method consists in separation of the soluble Ba salts by twice repeated solution in acetic acid and reprecipitation of insoluble salts by neutralizing with KOH. After removal of Ba the free esters are precipitated with HgAc in the presence of 1 per cent acetic acid. The acidity avoids serious loss of hexosephosphate. After freeing the centrifugate of Hg by H2S and of the latter by aeration, the free and total phosphates are determined. The calculated hexose value is compared with the reducing value determined by the Shaffer-Somogyi method (Reagent 50, 1 gm. of, KI).2 Hexose- phosphates prepared from resting muscles have a reducing value with this reagent which is between 68 and 72 per cent of that cal- culated from the phosphate content. The addition of adenylic acid before precipitation has no significant effect on this value and at least 90 per cent of that added can be recovered from the Hg precipitate. The recovery of added hexosephosphate is between 92 and 96 per cent. Even with these precautions solutions from asphyxiated muscles often give a slight pentose test and are there- fore probably contaminated with traces of nucleotide. Reducing values higher than those calculated from P indicate contamina- tion with an intermediate carbohydrate. The values reported

2 The rate at which hexosephosphate reduces alkaline copper sugar reagents is slower than that of glucose. With Reagent 50 the time required for maximum reduction is 35 minutes instead of 20 minutes for glucose. Increasing the alkalinity and salt concentration has the same effect as on glucose reduction.

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166 Carbohydrate Changes in Muscle

in these cases are calculated from the phosphate. Under the con- ditions of our experiments hydrolysis curves of the hexose ester show no indication of the presence of other than hexosemonophos- phate, Embden’s ester.

Lactic acid was estimated after CuSO&-ne precipitation by the

TABLE II

Changes in Muscle during Anaerobiosis for 6 Hours at $5’ The results are expressed in mg. per 100 gm.

I La&k said Total carhohydmta

- 175 174

99 129 - 144 -

91 89 55 81 84

- 80

- 40 41 41 27 40

- 37

-

pH 9.0 19 194 22 196 26 125 19 148

35 44 9 1593 38 46 8 2190 41 38 -3 1927 55 54 -1 2219

1362 231 184 164 182 124 96 134 128

3

31 49 18 2004 1901 46 71 25 3267 3145 33 43 10 2414 2306 22 30 8 1310 1217 46 61 15 2112 1991

163 147

103 109 122 114 108 65 93 89

121 99

109 95

Average. . . . . . . _

pH 7.2 29 120 25 114 10 65 18 99 23 107

Average. . . . . . . . 15

34 106 32 145 33 117 24 116 53 120

72 113 84 92 67

1587 1443 3105 2530 2468 2243

144 112 180 154 194 125 62 119 81 107

pH 6.0 32 72 13 54 17 58 21 48 19 59

Average. . . . . . .

2724 2530 2324

86 133 122

method of Friedemann, Cotonio, and Shaffer (1927) as modified by Friedemann and Kendall (1929), with MnOz in phosphoric acid as oxidant.

The resting values of lactic acid and hexosephosphate of oppo- site muscles are in good agreement, showing variations within the experimental error of the methods.

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M. Kerly and E. Rmaoni 167

The results of a series of experiments are presented in Table II. The pH given is that of the Ringer’s solution at the beginning of the experiment, calculated from the ratio of CO2 to bicarbonate. This pH changes but slightly during the experiments, those at pH 9.0 becoming more acid, those at 7.2 remaining approximately constant, those at pH 6.0 showing a slight increase. These changes are in accord with Lipmann and Meyerhof’s (1930) results. The pH of the Ringer’s solution probably does not represent the true pH of the muscle, since we have found an uneven distribution of lactate between Ringer’s solution and muscle fluid which is in- fluenced by pH. Furthermore, preliminary experiments show an uneven distribution of inorganic phosphate. Hence with the data at present available it is impossible to calculate the pH of the muscle itself.

Although results in different experiments vary: the values appear to justify the following conclusions. The pH is without marked effect upon the amount of carbohydrate loss during 5 hours under anaerobic conditions at 25”. But the products of breakdown vary with the pH of the suspension of Ringer’s solution. At an initial pH of 9 the loss of carbohydrate exceeds (one absurdly low value for carbohydrate loss was omitted) on the average 10 per cent of the lactic acid gain. At pH 7.2 hexosephosphate also increases; the sum of this with the smaller lactate formation is 12 per cent less than the carbohydrate loss; while at pH 6.0 the rise of lactate is still less and the hexosephosphate is greater than at higher pH, their sum being again less than the loss of carbohydrate by 8 per cent. Only about 10 per cent of the total carbohydrate present is broken down during the period of anaerobiosis, and from our control experiments an average variat,ion of 3.6 per cent in the determined initial values may be expected. No correction has been made for the 4 t.o 8 per cent loss in hexosephosphate deter- mination nor for the 4 to 5- per cent loss in the lactic acid deter- mination; therefore the discrepancies are probably within experi- mental error, and can hardly be accepted as evidence for the existence of other products of glycogen breakdown. If any methyl- glyoxal were present in the extracts, it would be converted to lac- tic a&d by the treatment with CuS04 and lime and so included in

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168 Carbohydrate Changes in Muscle

the lactic acid determination. That no pyruvic acid is formed is indicated both by a negative nitroprusside reaction and by lack of increase in lactic acid after reduction of the filtrates with Zn (Wendel, 193132).

The rate of the changes taking place was followed in a series of experiments at pH 6.0 (Table III). During the first 3 hours prac- tically no lactic acid is formed, though there is some loss in carbo- bydrat,e, which is nearly accounted for by the hexosephosphate gain.

After a period of 3 hours lactic acid production begins, the in- crease in 20 hours being approximately that produced by a muscle at pH 9. The reason for this would appear t,o be due to the in- creased pH, owing to the liberation of base from the breakdown

TABLE III

Changes in Carbohydrate, Lactic Acid, and Hexosephosphate of Muscle atpH6

Time of anaerobic&

ts.

1 3 54

20

Total Lactic acid + carbohydrate Lactic acid He-phosphste hexosephosphate

mg. per cent glzlcme mg. pa cent n&g. pep cent

glwose mg. per cmt

20 0 10 10 102 8 90 98 155 30 116 146 389 270 100 337 ,

of phosphocreatine, which is complete at this time. This is not reflected in the pH of the Ringer’s solution. The marked ab- sorption of CO2 without a corresponding change in pH of the Ringer’s solution suggests a liberation of nond8usible base within the muscle. The explanation of these changes awaits the de- termination of the true pH of the muscle substance.

Intermediate SwaTa-The glycogen content of muscle should be less than the total fermentable carbohydrate by the amount of any substances not precipitated and determined with glycogen which give rise to fermentable sugar on acid hydrolysis. Table I shows a comparison of these two determinations on opposite rest- ing muscles. The glycogen values are rather less than those for total carbohydrate, as Cori and Cori (1933) found for mammalian muscles, though the differences are probably within the experi- mental error.

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M. Kerly and E. Ronzoni 169

By direct determination the preformed fermentable sugar of the resting muscles averaged 15 mg. per cent. Since the average difference between glycogen and total carbohydrate is of the same order (25 mg. per cent) as the directly observed glucose content, it is obvious that t.here can be at most only traces of other carbo- hydrate in resting muscle.3

During anaerobiosis the fermentable reducing substance in- creases at all react,ions (Table IV), but unless the anaerobiosis proceeds for many hours, the increase is small at normal or high pH. At pH 6, however, the muscle may contain 100 mg. per cent

TABLE IV

Comparison of TotaE Carbohydrate and Glycogen of Muscles from Opposite Legs

The results are expressed in mg. per 166 gm.

Total carbohydrate Glycogen

2366 2300 1643 1673 1686 1669 1366 1307 1312 1338 986 970

1281 1259 1832 1818

Exoess total carbohydrate Over glycogen

+66 i-79 +86 -7

-26 +16* j-22* +14*

* In these instances the sciatic nerve was cut and the frogs allowed to recover for 1 hour before pithing.

of fermentable reducing substance after 3 hours, and in 20 hours 300 to 400 mg. per cent,. This is in accordance with Lohmann’s (1926) observation that in muscle extract glycogen may be hy- drolyzed to glucose.

The fermentation rat,e of this substance (after removal with HgSOrBaCOa of a large part of the non-fermentable reducing sub-

8 The great differences between total carbohydrate and glycogen values reported by one of us (Kerly, 1931) are accounted for by the non-ferment- able reducing substances precipitated with the sugar by copper-lime. Among the non-fermentable reducing substances in the Cu-lime precipitate are ribosephosphate from hydrolyzed nucleotide and hexosephosphate.

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170 Carbohydrate Changes in Muscle

stances including hexosephosphate4 present in the HgCla filtrate) was compared with the rate of fermentation of ghrcose by a similar sample of yeast, determined by the Warburg technique. Pig. 1 shows that the rate of CO2 production is a little slower than that of glucose solution. But glucose added to the same solution after that initially present had been fermented shows the same slower rate, owing perhaps to the salt concentration of the muscle prepara- tion changing the pH of the phosphate solution from its original

306

206

FIQ. 1. Fermentation rates of muscle sugar

value, 4.6. Besides the rapid CO2 production from glucose there is a continuous slow production of COZ. We conclude, therefore, that the ferment,able substance is in all probability glucose.

The non-fermentable reducing value of the HgCl, extract of resting muscle is approximately constant (Table V). It increases only very slightly during anaerobiosis, except at pH 6.0, when the rise is considerable. Part of this increase is due to a non-ferment-

4 Anaerobically yeast ferments hexosephosphate so slowly that its pres- ence in a solution in which fermentable sugar is being determined introduces but slight error in the glucose value. In oxygen its rate of disappearance is increased.

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M. Kerly and E. Ronaoni 171

able alcohol-soluble polysaccharide and part to hexosephosphate. The presence of intermediate carbohydrate in muscles kept for 20 hours in COZ without 0s was demonstrated by precipitation (before removal of Hg) of the HgClz extract of the muscles with 2 volumes of 95 per cent alcohol and determination of the ferment- able carbohydrate of both fractions after hydrolysis. The fraction of carbohydrate precipitated with alcohol resisted KOH treatment,

TABLE V

The results are eq

pH 9.0

pH 7.2

pH 6.0

Tiie

bra.

5 5 5 5

18 5 5 5

18 3 5 5 5 51

18 20

I

.-

-

eased in mg. per 100 gm.

Fermentable sugar in mhydm- Non-fermentable reduction of lyzed extract unhydrolyzed HgCL extract

Initial Final Increase Initial Final Inmesse

12 13 1 21 31 10 25 43 18 52 45 -7 23 29 6 14 39 25

9 38 21 30 33 3 3 91 88 37 51 14

22 60 38 32 45 7 16 60 44 41 42 1 17 40 23 33 42 9 2 111 109 39 48 9

36 84 48 18 90 62 7 44 37 29 66 37

55 190 135 53 153 100 7 66 59 40 116 76

15 119 104 31 128 97 27 336 309 53 154 101 22 420 398 34 185 151

was completely fermentable after hydrolysis, and was therefore glycogen. However, only a fraction of the total carbohydrate was thus precipitated. The fermentable sugar content of the alcohol-soluble fraction after hydrolysis was 528 mg. per cent as compared with the initial fermentable sugar of this fraction of 240 mg. per cent. Therefore the difference, equivalent to 288 mg. per cent of non-fermentable carbohydrate, not precipitable by alco- hol but hydrolyzable to fermentable sugar, was formed in 26 hours by muscle kept anaerobically at pH 6.

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172 Carbohydrate Changes in Muscle

Lohmann (1926) and Barbour (1929-30) have both shown that a trisaccharide can be formed from glycogen by muscle extract. It is possible that this same compound is formed here, though we have not found so constant a relationship between the reducing value before and after hydrolysis. Barbour states that the tri- saccharide has 30 per cent of the reduction value of glucose, de- termined with a Shaffer-Hartmann reagent ;6 Lohmann using Bertrand’s reagent found somewhat lower values.

Several preparations of the Ba salt of this compound have been prepared by the method of Barbour, precipitation with alcohol of the Ba salt after removing hexosephosphate with HgSOd- BaC03. The Ba-free solution from this precipitate is reducing but non-fermentable. On hydrolysis the reducing value increases between 3.9 and 7 times the initial value. We conclude that a variable mixture of reducing carbohydrates is formed which yields on hydrolysis fermentable sugar. Lohmann states that he found dextrin present in some of his preparations, depending upon the treatment of the muscle.

A method is described by which total carbohydrate, soluble carbohydrate, hexosephosphate, and lactic acid have been esti- mated on the same muscle. This procedure is thought to give a more accurate picture of the carbohydrate changes in muscle.

The effect of pH on the anaerobic breakdown of muscle carbo- hydrate has been investigated. At an alkaline reaction lactic acid increase approximately balances carbohydrate decrease, and there is only a small increase in hexosemonophosphate. As the reaction is made progressively more acid, lactic acid formation becomes less, while hexosemonophosphate formation increases, the sum of the two about balancing the loss in total carbohydrate.

During anaerobiosis at an alkaline or neutral reaction the forma- tion of lower carbohydrates from glycogen is very small. At pH 6.0 considerable quantities both of glucose and of intermediary carbohydrate are formed. The latter is alcohol-soluble, has an

6 Heating for 35 minutes is required for the maximum reduction of Shaffer-Somogyi Reagent 50 (1 gm. of I). This may be decreaeed by using more alkaline solutions.

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M. Kerly and E. Ronzoni 173

alcohol-insoluble Ba salt, and is reducing but is non-fermentable. On hydrolysis it forms fermentable sugar.

BIBLIOGRAPHY

Bsrbour, A. D., J. Biol. Chem., 85, 29 (1929-30). Cori, C. F., and Cori, G. T., J. Biol. Chem., 199, 323 (1933). Cori, G. T., and Cori, C. F., J. Biol. ?T’hem., 94, 561 (193132). Eggleton, G. P., and Eggleton, P., J. Physiol., 88, 193 (1929-30). Friedemann, T. E., Cotonio, M., and Shaffer, P. A., J. Biol. Chem., 73,335

(1927). Friedemann, T. E., and Kendall, A. I., J. Biol. Chem., 82, 23 (1929). Kerly, M., Biochem. J., 26, 671 (1931). Lipmann, F., and Lohmann, K., Biochem. Z., 222, 389 (1930). Lipmann, F., and Meyerhof, O., Biochem. Z., 227,84 (1930). Lohmann, K., Biochem. Z., 178, 444 (1926). Meyerhof, O., Arch. ges. Physiol., 185, 11 (1920). Rimington, C., Biochem. J., 23, 430 (1929). Shaffer, P. A., and Somogyi, M., J. Biol. Chem., 199,695 (1933). Wendel, W. B., J. BioZ. Chem., 94, 717 (1931-32). West, E. S., Scharles, F. H., and Peterson, V. L., J. BioZ. Chem., 62, 137

(1929).

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Margaret Kerly and Ethel RonzoniMUSCLE

ISOLATED ANAEROBIC FROGCARBOHYDRATE CHANGES IN

THE EFFECT OF pH ON

1933, 103:161-173.J. Biol. Chem. 

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