THE EQUILIBRIUM BETWEEAT GLYCOGEN AND LACTIC ACID* Otto

THE EQUILIBRIUM BETWEEAT GLYCOGEN AND LACTIC ACID* BP WILDER D. BANCROFT AND GEORGE BANCROFT**

Otto Jfeyerhof, Warburg, A. V. Hill, and others have done considerable work in the last fifteen years on the formation and disappearance of lactic acid in muscles. They find that under anaerobic conditions, that is, in the absence of oxygen, lactic acid is formed from glycogen when the muscle is stimulated. They point out that the amount of lactic acid produced depends on the amount of work done by the muscle so that, in reality, the lactic acid is a measure of the work done. When the muscle is resting after a stimulation, oxygen is absorbed, producing aerobic conditions; part of the lactic acid is burned and part is converted back into glycogen. They assume that this oxidation and synthesis is a coupled reaction; the synthesis cannot take place in the absence of the oxidation which supplies the energy for it. The extent of this coupled reaction is remarkable, for Hill has shown that under suitable conditions the ratio of the amount oxidized to the amount synthesized is one to five or one to six. Such a large ratio is unprecedented, for no coupled reaction of this extent is known in chemistry. In cancer, where there is little accumulation of glycogen in the aerobic phase, this coupled reaction is still assumed to hold; but they believe in this case that the glycogen is broken down nearly as fast as it is formed, so that there is little accumulation. If the resting muscle is stimulated in oxygen and then allowed to rest, there is exactly the same result; part is burned and part of the lactic acid is converted back into glycogen again. There is however the difference that, during the stimulation, less lactic acid is formed in oxygen than in its absence. They assume that this is due to the recovery processes, oxidation and synthesis, going on simultaneously with the stimulation. I t is our purpose ir this paper to show that the formation and disappearance of lactic acid in the muscle can be explained equally as well and in many instances better by the assumption that there is an equilibrium between glycogen and lactic acid in the muscle and that this equilibrium is reached by means of enzymes. The equilibrium point of this reaction is well over on the lactic acid side. As we shall see later, the reaction can be forced back from lactic acid to glycogen by the adsorption of glycogen out of solution on the protein, thereby reducing the amount of free glycogen in solution and causing the formation of more to re-establish the equilibrium. When we say that there is an equilibrium between glycogen and lactic acid we do not mean that * This work is part of the programme now being carried out at Cornell University under a grant from the Heckscher Fund for the Advancement of Research established by August Heckscher at Cornel1 University. Most of the expenses have been defrayed by a grant from the Cancer Research Laboratory of the Graduate School of Medicine of the University of Pennsylvania. * * Holder of Fellowship from the Cancer Research Laboratory of the Graduate School of Medicine of the University of Pennsylvania.

EQUILIBRIUM BETWEEN GLYCOGEN A S D LACTIC ACID

I95

the glycogen necessarily goes directly to lactic acid; on the contrary it probably goes through glucose or some hexose phosphoric ester and may even then go through some other intermediate stage such as methyl glyoxal before it finally reaches lactic acid. The early work of those interested in the changes in frog’s muscles led to a mass of conflicting results, which was not cleared up until the classical work of Fletcher and Hopkins‘ in 1907. They say: “Abundant lactic acid formation is said to accompany the process of natural rigor in a surviving muscle (duBois-Reymond, Ranke, Boehm, Osborne), but this is denied (Blorfle, Heffter); it is said to accompany contraction, and to mark the advance of fatigue (Heidenheim, Ranke, Werther, Marcuse), but this is also denied (Astachewsky, Warren, Monari, Heffter). Indeed it may be said that since Ranke wrote in 1865, no description of the elementary facts of lactic acid formation, despite the fundamental importance of the subject, has been generally accepted.” Fletcher and Hopkins point out that most of the fallacies are due to faulty methods of extraction of the muscle in order to determine the lactic acid content. Thus, many workers procured values for the lactic acid of the resting muscle as high as that of the fatigued, and so believed that there was no change. Also troubles due to bacterial infection were prevalent. Fletcher and Hopkins studied the formation of lactic acid under various conditions. Using induced interrupted current from two Daniel1 cells with a secondary coil (0.5 cm.) they found a maximum production of lactic acid which lay between 0.18 and 0 . 2 5 % with a high value of 0.28% and an average value of 0.21%. These values also confirm the value of 0.229% found by Marcuse.Z Meyerhof, as we shall see, got somewhat higher values than this with slightly less cumbersome methods of analysis. The effect of chemical reagents is to form lactic acid very rapidly. At zo°C four hours exposure to chloroform vapor gives a yield of lactic acid of 0 . 4 3 4 7 ~whereas the control showed 0 . 0 2 7 ~ . Similarly a like amount of lactic acid may be produced by heat rigor in the same time. This is accomplished by heating the muscle a t 4ooC and a maximum of about 0.42% was formed. “We find that an acid maximum is reached on heat rigor effected a t or near 40’ and this maximum is approximately on the same level with that produced in chloroform rigor, or in the slow death by alcohol.’’ The alcohol in four hours gives values slightly over 0.40%. Lactic acid may be formed in another way-by anaerobic rest. The rate of formation in this case is very much slower than by fatigue or by chemical means. I n the course of 20-25 hours a maximum is reached which amounts to about 0.3670 a t zo°C. This is slightly higher than found for the frogs stimulated for a much shorter period ( z hours) and it is less than the maximum reached by chemical means in four hours. J. Phyaiol., 35, zq7 (1907).

* Pflugera Archiv, 39,425 (1886).

196

WILDER D. BANCROFT A S D GEORGE BANCROFT

In 1919-1920 Otto hleyerhof published a series of papers’ on this subject. He fatigues muscles in two ways, namely, by tetanic stimulation and by induction shocks. The results from these two methods were not the same, for he found with single induction shocks values 40-607, higher than by tetanic stimulation. Meyerhof calls attention to the fact that, by using different methods of extraction from Fletcher and thereby shortening the time of manipulation, he obtained analogous maxima but in each case I j to 20% higher. For tetanic stimulation Meyerhof finds a net increase of 0 . 2 0 j 7 ~ , occasionally dropping to a value of 0.1787, as in December, and with a high value of 0.24%. His values for single induction shocks are about 0.35’-& dropping in December to 0.254%. I n each case, however, the corresponding values are 40 to 6 0 7 ~ higher for the single induction shocks than for tetanic stimulation. Incidentally these values are not affected by the means of inducing the stimulation-namely,’ whether applied indirectly to the nerves or directly to the muscle. The rate of formation of lactic acid was studied with respect to single induction shocks and is of interest. Meyerhof carried on a series of experiments with incomplete fatiguing and compared the fatigue maximum reached under similar conditions (frogs from the same source, same time of year, temperature, and method of stimulation). He found in one experiment with indirect stimulation in 2 0 seconds (spark gap 15-17cm.) a lactic acid content of 0.1397~; in another stimulated for 40 seconds and another for 20 seconds he found 0.13% lact,ic acid. By using exhaustive tetanic stimulation a fatigue maximum of 0.237,was obtained and in less than one minute over half of the lactic acid was formed. Metronome stimulation. By exhausting stimulation of fresh fall frogs0.36% 2 minutes 0.118 6

”

0.1687,

7 ’’ 0 . 2 2 6 % We can see that 0.11 8 7 or ~ a good half of the lactic acid was formed in the

first third of the time. Meyerhof gets results for chemical rigor similar to those Fletcher and Hopkins did, although his are slightly higher. He gets values ranging from 0.42y0in the spring to o.jszyO in the fall. These values are considerably higher than those found for electrical stimulation a t the same times of year. The question naturally comes up as to why these values are higher and also why the chloroform causes the formation of lactic acid anyway. This last question is explained by Meyerhof by assuming that the chloroform in some unknown way causes a stimulation in the muscle which is more vigorous than the electrical stimulation and hence causes the formation of more lactic acid. Heat rigor has a similar action to chloroform and Meyerhof finds practically the same values for this as for chloroform.. This is in confirmation of the similar results found by Fletcher and Hopkins. Lacquer* has shown that, if Meyerhof: Pflugers Archiv, 182, 232, 284; 185, I I (1920);188,1 1 5 (1921). *Lacquer: 2.physiol. Chem., 93,60 (1914).

EQUILIBRIUM BETWEEX GLYCOGEN AND LACTIC ACID

197

two percent bicarbonate solution is added to cut-up muscle, a formation of lactic acid takes place which amounts to 0.9% as opposed to 0.4% in the ordinary salt solution. From this he ascribes the increasing acidity as the method which stops the acid formation. This is undoubtedly one of the factors and it, is interesting to note, that neutralization of the acid should increase the yield if the reaction were an enzyme as we postulate, for we are taking away one of the final products. Meyerhof also conducted some experiments on the formation of lactic acid during anaerobic rest; but his values are not easily comparable with those previously given here, because he added NaHC03 and HCX to his solution. As we have seen, the bicarbonate increases materially the yield of the lactic acid and Meyerhof himself seems doubtful whether the HCN does not also have an effect. However, he did find that the acid accumulation was slow and, even with the bicarbonate neutralizing some of the acid formed, he found an end value of 0 . 5 6 7 ~whereas Lacquer found a value for heat rigor and bicarbonate of 0.9%. So we see that this value is cons:derably less than the value procured for heat rigor and this was shown to be the case by Fletcher and Hopkins without the addition of bicarbonate. In any study of the muscle or analysis of the results there are several factors which must be kept in mind in order to get a true insight into the working on the muscle. The first of these is the effect of the time of year. Fletcher and Hopkins (loc. cit., p. 226) say, “The infliction of heat rigor is a convenient method for determining the potentiality of a muscle for acid production at any time. Selecting observations made in other connections and at different times of year, we find that they fall into two groups according to season, thus giving only one for each six months, March ,383 October .54 April ,315 Sovember . S I May , 4 2 0 Av. .36 December . 5 2 Av. . 5 z Whilst we have always found a surprising constancy in the value for the acidity of heat rigor, when duplicate determinations have been made on frogs caught under similar conditions, we have constantly found higher acid maximum for the muscles of autumn frogs than for those of frogs caught in the spring.” Meyerhof also finds differences at different times of year for rigor, chloroform, and the two stimulation maximums. The simplest explanation would seem to be that the glycogen content is different in the different months and indeed it has been shown by Mitchell’ that oysters a t different times of year have markedly different glycogen content. This explanation would be perfectly satisfactory from the point of view of our enzyme theory of equilibrium; but it does not seem to satisfy Meyerhof and his beliefs of the metabolic changes in muscles. He says:‘ “What causes the difference in the lactic acid maxima in the different months? From the experiments of Lacquer on the regulation of the Mitchell: U. S. Bureau of Fisheries, 35, 151 (1916) Meyerhof: Pflugera Archiv., 182, 232 (1920).

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WILDER D. BANCROFT AND GEORGE BANCROFT

lactic acid maximum by H ion, we must conclude that there is a changing sensitiveness in the different months rather than a change in the glycogen supply. This is perfectly plausible. One finds that the anaerobic exhaustion through electrical stimulation is restricted by the addition of acid." Moreover he points out that the rigor maximum is smaller in the winter after the frogs have lain dormant for several months than in the fall. This would seem quite nat'ural as t'he glycogen content should be lower. I t is plain that Meyerhof is trying to explain two different factors by one explanation. These two factors are made clear on the basis of the enzyme theory of lactic acid production, for one would expect less lactic acid to be formed in the spring if there was less glycogen present, even as in the fall one would expect more lactic acid to be produced if there was more glycogen present. I n any equilibrium if the final product is taken out, more of this product will be formed in order to reestablish the equilibrium. The action of the carbonate is to extract some of the lactic acid by neut'ralization and hence cause more to be formed. This last has no effect however on the changes of the amount of lactic acid formed due to the time of year. Another factor which must be taken into consideration is the effect of temperature. Meyerhof found that variations due to temperature affected the maxima formed by all the methods of producing lactic acid. At 0°C the maximum reached by stimulation was singularly small. The difference in the maximum caused by changes from 14' to z z o is much less than from oo and zoo but the change is still considerable, for Meyerhof found an average value a t 14' of 0.17% and a t 22' an average value of 0.21%. The muscle apparently becomes fatigued much more rapidly at the lower temperatures, for he found that a muscle fatigued at g°C was again irritable if heated up to 20°C. This is entirely in keeping with the findings that the muscle will produce more lactic acid a t the higher temperature. There is a limit, however, for Fletcher and Hopkins have shown that in boiling water the muscle will not form lactic acid. This is due to the fact that the enzyme has been destroyed. Cavallo and TVeirs' showed that a muscle exhausted at o°C if heated up to 25' and then cooled down again was able to have a new series of contractions. They believed that the process of heating up and cooling down made the muscle irritable again. Meyerhof was unable to verify this result and he points out that, if one waits long enough after the subsequent cooling down in order to insure that the entire muscle is again a t the lower temperature, and if one takes precautions to provide anaerobic conditions throughout the manipulation, the muscle after cooling is still unirritable. It is not the change in temperature itself which causes the change in irritability but the fact that a t the higher temperature the muscle can produce more lactic acid than at the lower temperature. We have seen that it is possible to produce lactic acid in the muscle by several different methods. The question immediately arises as to where the Cavallo and Weirs: J. Physiol. Pathol., 1, 990 (1899).

EQUILIBRIUM BETWEEN GLYCOGEN AND LACTIC ACID

'99

lactic acid comes from. Meyerhof has demonstrated conclusively that, as the lactic acid increases, the carbohydrate decreases in exactly equivalent amounts. This change moreover, concerns chiefly the glycogen as the amount of change of other carbohydrates is small in comparison. He has shown' that during anaerobic rest the carbohydrate decreases as the lactic acid increases. Mg. Glucose per gram Muscle Mg. Glucose Before After Difference Before After

Muscle Wt., grams Glycogen Other Carbohydrates

10.2

10.35

77.

45,2

7.55

4.35

-3.20

19.0

15.5

1.85

1.50

-0.35

-3.55 -3.75

Corrected Lactic Acid

0.20

2.82

4-2.62

In conjunction with the foregoing experiment Meyerhof also determined the resting respiration and he was then able to calculate the amount of carbohydrate burned by this respiration. This amounted to 1.1 mg. If we subtract this value from the amount of carbohydrate decomposed, we have the amount of lactic acid formed. Taking the value 3.75 mg. from the table and subtracting 1 . 1 mg, the amount burned, we have 2.65 mg. which should be converted into lactic acid. This checks very well with the value 2 . 6 2 mg. of lactic acid determined. So we see that, as the carbohydrate decreases, the lactic acid increases in exactly equivalent amounts. The foregoing relation was found in whole muscle and in the following we find the same result in minced muscle in phosphate solution. Time

8h. I 2h. I1 23h. I11 7h I\'

Lactic acid Increase mg./gr.

+

0.272

+ 0.30 4- 1 . 0 5 6

+ 0.885

Glycogen Decrease mg./gr. -0.285

-0.34 -1.00

-0,895

The formation of lactic acid from glucose by enzyme action is not a new theory. In attempting to find out what the intermediate products in the decomposition of glucose to lactic acid were, Embden and his co-workers* carried out some liver perfusion experiments with glyceric aldehyde and dihydroxy-acetone. They showed that both substances increased the lactic acid in the perfused blood but that the glyceric aldehyde was much more effective. Embden advanced the theory that optically active glyceric aldehyde is the intermediary substance formed in the break-down of glucose into d-lactic acid. Keuberg and Rosenthals found that fresh liver tissue would

* Meyerhof:

Pfliigers Archiv, 185, I I (1920). Biochem. Z., 45, 108 (1912). 3 Neuberg and Rosenthal: Biochem. Z., 49, 502 (1913).

200


split methyl glyoxal into a mixture of dl and d-lactic acids. Whether methyl glyoxal or glyceric aldehyde or dihydroxy-acetone is the intermediate product between glucose and lactic acid is outside the scope of this work. The important point is that these authors realize the necessity of the presence of an enzyme in order to get optically-active lactic acid. Dakin and Dudley’ added a solution of phenyl glyoxal t o minced tissue and found a 40% to 60% conversion into 1-mandelic acid. They found that increasing acidity stopped the conversion completely and that, to get this conversion, it was necessary to add sodium bicarbonate to keep the H ion down. Moreover, the enzyme present here was killed if the tissue was boiled, which is just what would be expect#ed. They also found that an enzyme solution prepared from dog’s liver decomposed 4 grams of pure methyl glyoxal into lactic acid completely in I O minutes. They were unable, however, to get only d-lactic acid but got a mixture. Levene and Meyer2 showed that leucocytes and kidney tissue formed lactic acid from methyl glyoxal under aseptic conditions and that a mixture of the dl and d-forms were obtained. They also showed3 that these leucocytes could change glucose into optically active lactic acid. From this they concluded that glucose must break down into lactic acid by way of methyl glyoxal. Meyerhof refers to these authors in a monograph “Chemical Dynamics of Life Phenomena,” 59 (1924) by saying: “They seem to assume that a reversible equilibrium exists between sugar and lactic acid, so that the reaction could go spontaneously either in one or the other direction. This is however, not the case, as will be shown in detail later. The cleavage of sugar into lactic acid is a spontaneous process going to completion. On the other hand the synthesis of sugar from lactic acid requires a supply of energy furnished in the isolated muscle exclusively by oxidation of part of the lactic acid or the corresponding amount of sugar. It can probably be provided also in the other organs only by oxidation.”

As we have seen, the methyl glyoxal apparently breaks down completely into lact,ic acid, yet Dakin has shown conclusively that the equilibrium between methyl glyoxal and lactic acid is r e ~ e r s i b l e . ~An aqueous solution of lactic acid and the enzyme was digested at 37OC. Upon addition of nitrophenyl hydrazine a precipitate of the insoluble methyl glyoxal dinitro phenyl hydrazone was formed, which could be readily separated and analyzed. The fact that the glycogen breaks down completely into lactic acid under certain conditions does not necessarily mean that there can not be an enzyme equilibrium, The conditions necessary to cause all the glycogen to break down is a considerable excess of phosphate which might have the effect of neutralizing the lactic acid formed, thereby displacing the apparent equilibrium. Dakin and Dudley: J. Biol. Chem., 14, I j j (1913). Levene and Meyer: J. Biol. Chem., 14, j51 (1913). Levene and Meyer: J. Biol. Chem., 12, 2 6 j (1913). Dakin: J. Biol. Chem., 14, j j j (1913).

EQUILIBRIUM B E T W E E N GLYCOGEN AND LACTIC ACID

201

Meyerhof has shown that in phosphate solution all the glycogen is decomposed; but that does not mean that all the carbohydrate has decomposed as one is led to believe. Two of his experiments on this might well be quoted here.

G K Time from start of mincing

August

23

h

=

Glycogen other carbohydrate

Carbohydrate Content Total mg. %

G K

20

=

= 31 4

=

9.5

2.23

0.064

39.2

1.12

0.27

G = o o I< = 8 . 6 o . z j

Total

+I.

-1.00

15

8h 1 5 Total

o 898

Lactic Acid Total mg.

G = 1 2 . 4 0.214 K = 1 3 ,j 0 . 2 3 1 G = I< =

0

10.5

4.05

056 corr.

0.010

0.0

0.180 -0.28j

20.0

0.342

+o.

27 2

We see from these figures that all the carbohydrate has not disappeared, even though all the glycogen has. The argument that Meyerhof puts forth, that there can be no enzyme reaction because all the glycogen disappears, is not sound. I n the first place he may have been neutralizing the final product with the excess of phosphate which would displace the apparent equilibrium. Dakin showed that methyl glyoxal apparently went completely to lactic acid but he was able to show conclusively that the equilibrium was reversible. Meyerhof has suggested that in all probability glycogen breaks down into lactic acid perhaps through glucose and probably through an intermediate hexose phosphoric ester. Lacquer' has found that glycogen is a better source of lactic acid than glucose in the separated muscle. According to him the reason for this seems to be that only a glucose which is a derivative of glycogen yields lactic acid immediately whereas fl glucose must first change into the a form. As we have pointed out before, we assume that the formation of lactic acid is due to an equilibrium between glycogen and lactic acid, whose rate is governed by enzymes. I n the decomposition of glycogen there may be an intermediate formation of a glucose and probably a hexose phosphoric ester and lLacquer: Z. physiol. Chem., 116, 169 ( 1 9 2 1 )

202

WILDER

n.

BASCROFT AND GEORGE BANCROFT

perhaps some further intermediate compound as methyl glyoxal before the lactic acid is reached. Furthermore the equilibrium point is well over on the lactic acid side. Glycogen (C6H,OOd”

n

Glucose CsHI’ZO6

M

or Hexose phosphoric ester or both

Methyl Glyoxal CHBCOCHO

n

Lactic Acid CH3CHOHCOOH These might possibly be the steps in the reaction. S o attempt is made in this paper to determine the steps; but it is helpful to have a picture of what may happen in a simplified form in order to understand the processes. Przylecki-and Wojcikl have shown that protein has an extraordinarily high adsorptive power for glycogen, and that, using appropriate concentrations of the protein, which incidentally correspond to those present in the liver, this adsorption may even for 10% glycogen solutions, amount to as much as 90%. With 30 cc. of a 1% glycogen solution and 30 grams of protein, 9970 of the glycogen is adsorbed out of the solution. This adsorption of glycogen is a reversible reaction, for they have shown that by diluting with a sufficient quantity of water the glycogen may be eluted off the protein again. It may be liberated also by the action of various chemicals such as alcohol, and other narcotics. Przylecki worked principally with the enzyme amylase which hydrolyzes glycogen to glucose; he showed that, when the glycogen was adsorbed on the protein, it was so stabilized that the rate of reaction with the enzyme was very slow but in the course of time it was broken down into glucose. If in the muscle the enzyme only reacted with the free glycogen, we should expect during anaerobic rest that the formation of lactic acid would be slow, as the concentration of free glycogen in solution at any time would be small. As the concentration of free glycogen diminished, glycogen adsorbed on the protein would be set free to reestablish that equilibrium, and the free glycogen thus formed would in turn be decomposed to lactic acid until perhaps the increase in hydrogen ions stopped the process. Fletcher and Hopkins,? in order to account for the linear formation of lactic acid, suggested such a possibility as this equilibrium between adsorbed glycogen and free glycogen. They say: “Conceivably the store of precursor in the muscle is partly in insoluble form, partly in solution, the concentration

* Biochem. J., 22, 2

LOC.cit., p. 27j.

1302 (1908).

EQUILIBRIUM BETWEES GLYCOGEN AND LACTIC ACID

203

might be kept, constant by replacement from the insoluble store, and, for a period, conditions would exist for a linear rate of change. The explanation is probably less simple than this, and it is interesting in any case, to observe how in the quiescent unstimulated muscle the survival processes which lead to lactic acid production are so controlled as to lose the exponential character of an isolated chemical reaction.” I n the case of electrical stimulation we have seen that more lactic acid is formed by single induction shocks than by tetanic stimulation. Meyerhof says: “How can one explain that by single induction shocks which are applied sixty times to the minute a much greater amount of lactic acid accumulates in the same time than when twenty-five times the number of irritations were applied tetanically.” He points out that by the use of a tension lever it can be shown that the muscle irritated by single induction shocks performs more work than those stimulated by tetanus. Moreover a muscle which has been exhausted by tetanic fatigue is still irritable to single induction shocks. The lactic acid, according to Meyerhof is a measure of the work done; and since more work was done in the stimulation by the single shocks more lactic acid should be formed. This is, of course, arguing in a circle. We have seen that the glycogen is stabilized by adsorption on the protein, and stimulation causes the glycogen to be liberated from its adsorption thereby increasing the concentration in free solution. As the concentration increases in free solution the rate of formation of lactic acid increases. Indeed, we have seen that where 0.22’3 was formed in seven minutes, over half was formed in the first two minutes when the concentration was the highest. Just how the contraction of the muscle causes the liberation of the glycogen is, at this time, pure conjecture. Bancroft and Richter’ have shown that a man rendered unconscious by an electric shock is in reality in a state of narcosis. Now in narcosis, as they have demonstrated, the proteins are reversibly coagulated, that is if the cause of the coagulation is removed the proteins will return to their peptized state again. I n the case where proteins are irreversibly coagulated the patient dies. Probably the contraction of the muscle coagulates the protein slightly, causing a liberation of some of the adsorbed glycogen. However, this coagulation is not due t o the effect of the electrical current itself, for Meyerhof has shown that it makes no difference whether the current is passed directly to the muscle or directly t,hrough the nerves. The coagulation due to a single twitch is probably slight with a correspondingly small liberation of glycogen. With a single tetanic stimulation the muscle is tetanized, or fully contracted, so that continuous stimulation would have little further effect on the muscle. If a very short period of rest is allowed between tetanic stimulations, the muscle would tend to relax, thus allowing further stimulation to contract it again. It is for this reason essential to allow short periods of rest in tetanic stimulation in order to procure the maximum yield of lactic acid. These periods of rest are only momentary, as the tetanic stimulations are applied well over sixty times to the minute. We know from 1

Bancroft and Richter: J. Phys. Chem., 35,

21j

(1931).

204


Meyerhof’s work that a muscle is still irritable to single induction shocks even though fatigued to tetanic stimulation, so that there are more contractions y i t h the former method, more glycogen liberated, and hence, just as we shohld expect, more lactic acid formed. In this particular case, Meyerhof’s explanation of lactic acid as due to the work done by the muscle is nearly as satisfactory as our explanation. In the case of the quiescent unstimulated muscle under anaerobic conditions we should expect to get a slow formation of lactic acid, as the glycogen in free solution would be decomposed to glucose and hence to lactic acid. As the free glycogen diminishes, more glycogen would be liberated from the protein to keep the concentration constant. As the concentration of free glycogen is small a t any particular time, we should only expect a slow rate of reaction. Meyerhof’s explanation of work done could not hold in this case as no work is done by the muscle in this quiescent state. We can predict what should happen in this case, but do the facts support this prediction? We have seen that a maximum yield of lactic acid in this case is not reached in a few minutes as is the case when the muscle is stimulated electrically but, in the course of 2 3 hours. Moreover, according to Fletcher and Hopkins the rate of formation follows a linear course, which is what we should expect from the fact that the concentration of free glycogen is constant. We do not need to postulate, as Meyerhof does, that the addition of chloroform in some unknown way causes a stimulation of the muscle and in that way produces lactic acid. We have only to bear in mind the fact that the chloroform coagulates the protein thereby liberating the adsorbed glycogen. The increase of the concentration of the free glycogen would increase the rate of formation of lactic acid. Indeed we find that the addition of chloroform causes a formation of lactic acid which reaches a maximum in four hours or less. The effect of the addition of chemical reagents probably liberates more glycogen than stimulation methods do so that there is more lactic acid formed. Heat rigor may be explained on the same basis, as it i p well known that heat will coagulate the proteins. Ether and alcohol apparently have the same effect as chloroform and come under the general head of narcotics. Some other chemicals as arsenate and caffein have slightly different effects. Bancroft and Richter (loc. cit.) have shown that caffein in high enough concentrations acts like a narcotic in coagulating the proteins. According to Przylecki the explanation of the increased formation of lactic acid may not be as simple as this. They say:‘ “The acceleration of the velocity of hydrolysis caused by the addition of narcotics, such as chloroform, ether, or alcohol might on the basis of this research be explained as being due exclusively to elution of polysaccharide. While such an explanation appears to fit very well with experimental findings, it should not be forgotten that in our case we are dealing with a system simpler than that present in our cell, as it contains only one of the components of the latter, namely protein, The possibility remains that, within the cell, enzyme is Przylecki: Biochem. J., 22, 34 (1928)

EQUILIBRIUM B E T W E E N GLYCOGEN AND LACTIC ACID

20s

adsorbed not only on its protein elements, but also upon lipin or nucleoprotein surfaces, from which elution by narcotics may be much greater than from proteins.’, “There can be no doubt that elution of substrate from protein, or change in the state of dispersion of the latter, plays an important part in the acceleration of the velocity of enzyme by hydrolysis of polysaccharides due to the addition of narcotics.” It has been assumed, and qualitatively at any rate the facts seem to bear this out, that the enzyme only affects the free glycogen and not the adsorbed glycogen. However, if there is a combination of enzyme and substrate in the reaction which the enzyme catalyzes, there might also be a combination with some of the adsorbed glycogen. The facts seem to support the belief that the enzyme combines only with the free glycogen, as we have seen in the case of anaerobic rest. ?;evertheless this is not the whole story, for the problem might be further complicated by adsorption of part or all of the enzyme under certain conditions. Thus, with the enzyme 100% adsorbed on the lipin surfaces and the glycogen roo% adsorbed on the protein, t’here would be no reaction. If both are only partly adsorbed the effect of some chemicals might be to liberate only the enzyme, or only the glycogen, or both at once and in each case the resulting effect would be different. There does not seem to be any need in making the problem as complicated as this, for there does not seem to be any evidence as yet that requires us to assume that the enzyme combines with the adsorbed glycogen. On the other hand the enzyme is undoubtedly adsorbed to some extent on the lipin surfaces; but Przylecki has shown that this does not necessarily impair its action on the free glycogen. As Richter and Bancroft have shown, the effect of chemical reagents may be of another class, those which do not coagulate the protein but which are selectively adsorbed on it. These substances, depending on their concentration and the degree of adsorption, would have different influences on the formation of lactic acid. Furthermore substances which would tend to increase the peptization of the protein might increase the degree of adsorption and thereby decrease the tendency towards lactic acid formation. Another essential, but hitherto unconsidered point, is the question why the lactic acid formed in the muscle is dextrorotatory. Evans1 has shown that glucose under the influence of strong alkali yields lactic acid, but the product is inactive. This might be due to racemization of the acid by the strong alkali; but this is improbable, as dilute alkali also yields inactive lactic acid. Moreover, if the glucose passed through an intermediate stage of methyl glyoxal or pyruvic acid, both of which are inactive, the final product would be inactive unless an enzyme were present. It has been pointed out earlier in this paper that, methyl glyoxal in the presence of an enzyme known as glyoxalase yields active lactic acid, and leucocytes and kidney tissue can decompose glucose into optically active lactic acid indicating the presence of Evans: J. Am. Chem. Soc., 47, 3085 (1925).

206

WILDER D. BANCROFT AND GEORGE BAKCROFT

an enzyme. Indeed Meyerhofl has shown that an enzyme can be extracted from muscles which upon purification is many times more active than the muscle itself, and that this enzyme can convert glucose and glycogen into lactic acid. Presumably this is optically active lactic acid although he does not say definitely. Meyerhof has pointed out that the formation of lactic acid is due to several causes, among which the respiration plays an important part in determining the rate. If however, the formation of lactic acid is due, as we believe to enzyme action, increase of the starting products should increase the rate of formation of lactic acid. Stiven2 has shown that increasing the concentration of glycogen with the enzyme extracted from cat's muscle, increases the rate of formation of lactic acid. The following diagram shows this increase. We see from this diagram that as the concentration of glycogen increases the rate of formation of lactic acid increases. This is exactly what we should expect from an enzyme reaction. The concentration of the enzyme was constant in the three cases. What is the fate of the lactic acid during the recovery period in oxygen? So far, we have only considered the anaerobic or working phase. Fletcher and Hopkins found that, with a separated muscle after repeated stimulation FIG.I and recoveries in oxygen over a period From Biochem. J., 22, 872 (1928) of several days, the yield of lactic acid from heat rigor was practically the same as for fresh muscle. It has been known for some time that a muscle which has been fatigued and then allowed to rest in oxygen recovered its irritability again. I n other words the lactic acid, which is a measure of the fatigue, had disappeared. From their experiment Fletcher and Hopkins assume that the lactic acid, all or in part, must be converted back into carbohydrate again in oxygen, for if it was all burned the amount of lactic acid from heat rigor after several days of repeated stimulations and recoveries would be materially reduced. Hill3 found that in the oxidative removal of one gram of lactic acid there is a heat production of about 450 calories. Now the oxidation of one gram of lactic acid leads t o a heat production of about 3700 calories, which is about eight times as large as the quantity observed. Although the measurements 1 Meyerhof: Naturwissenschaften, 14, 196, 756, 117.5 (1926); Biochem. Z., 178, 395, 462 (1926). * Stiven: Biochem. J., 22, 867 (1928). Hill: J. Physiol., 48, x (1914).


207

were not very accurate it is obvious that all the lactic acid is not burned, but that some is returned to its precursor, glycogen, under the influence and with the energy of the oxidation, either (a) of a small part of the lactic acid itself, or (b) some other body. I n 1922’ a more careful study of the liberation of heat showed that there are three stages in which heat is given off in the working muscle, which Hill characterizes as the initial anaerobic heat, the delayed anaerobic heat, and the oxidative heat. These amount to 285, 8 j and 340 calories respectively. If we suppose that of one gram of lactic acid z grams are oxidized in the recovery and (I - x) grams restored to its previous state as glycogen, the muscle will have returned to normal except for the z grams oxidized. The heat of combustion of glycogen according to Stohmann is 4 1 9 1 cal./gr. and according to Emery and Benedict 4 2 2 7 cal/g. If we take the mean of these two quantities, namely cal./g., we see that the heat of combustion of 0.9 g. of glycogen which corresponds to I gram of lactic acid is 3788 calories. Hence the total energy available to cover all breakdowns in the complete cycle is 3788 X cal. Equating this to (285 8j 340 = 7 1 0 cal.,), we find x = 710/3788 or 0.188. Thus of one gram of lactic acid passing through the whole cycle of contraction and recovery 0.188 gr. are oxidized and the remainder viz. 0.812 grams are restored to its previous state as glycogen. We can see, therefore, that one-fifth to one-sixth of the lactic acid is burned and the remainder is converted back into glycogen. These myothermic measurements of Hill’s do not show what the lactic acid is converted into, as there was no chemical analysis of the changes. Meyerhof2 however, in a study of the carbohydrate exchange of frog’s muscles demonstrated that, as the lactic acid disappears during the recovery period, so the carbohydrate increases in exactly the extent as calculated from the difference of the lactic acid disappearance and the oxygen consumption (recovery consumption minus resting consumption). Again the change concerns the glycogen chiefly, and there is a synthesis of glycogen from lactic acid. The glycogen content a t the end of the recovery period is the same as before the stimulation, minus the carbohydrate disappearance equivalent to the oxygen consumption. T o quote one experiment of Meyerhof’s where he found the following balance in mg./g. of muscle.

+ +

Before recovery

After recovery

3 37

4 75

2 01

I

Glycogen Other Carbohydrate Lactic acid

66

5.38

6.41

2.56

0.44

Difference +I -0

38 35

+ I .03 -2.12

I n oxygen experiments carried on a t the same time, he found 1.20 mg of excess oxygen were used in the recovery period which would burn I . I 2 mg. of 1

Hill: J. Physiol., 56, 367

(1922).

* Meyerhof: Pflugers Archiv, 182, 284

(1920).

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lactic acid, so we see that 2 . 1 2 - 1 . 1 2 or 1.00mg of lactic acid should have been converted back into glycogen. This checks very reasonably with the determination that I .03 mg of glycogen were formed. Meyerhof has also shown that, if the muscles are not minced too finely, there is apparently a formation of glycogen in the aerobic phase which indicates that there is no necessary relation between structure of the muscle and the formation of glycogen. When the muscle is minced very finely, the negative results are probably due to a diffusion of phosphate away from the enzyme, thus preventing the esterification of the glucose to the intermediate glucose phosphoric ester. This is in accord with his findings for the necessity of phosphate for the decomposition of glycogen to lactic acid. Let us now consider Meyerhof’s explanation for the recovery period in the muscle. I n “Chemical Dynamics of Life Phenomena,” j j (1924)~he says: “In the oxidative phase one molecule of sugar or the corresponding amount of lactic acid is burned. The rest of the lactic acid is reconverted with phosphate to the ester (glucose phosphoric ester) and again becomes glycogen. Here we have a coupled reaction similar to the alcoholic fermentation.” In short he assumes that the oxidation and synthesis is a coupled reaction, where the synthesis cannot take place without the oxidation to supply the energy for it. From a purely chemical point this reaction should not require much energy, as glucose stoichiometrically is exactly two molecules of lactic acid, and glycogen is a straight polymer of glucose with a splitting out of water. If this is in reality a coupled reaction, one should be able to take lactic acid, oxidize it in the presence of protein, and form glycogen. It apparently does not require the muscular structure as Meyerhof found a synthesis of glycogen with minced muscle. Lactic acid is oxidized readily in the presence of ferrous salts by hydrogen peroxide but there is no formation of glycogen under these conditions even in the presence of protein which would naturally adsorb any glycogen that was formed. Indeed from our hypothesis of an equilibrium between glycogen and lactic acid, catalyzed by enzymes, we should not expect to form any glycogen under these conditions. I t is our belief that the oxidation of lactic acid is not coupled with the synthesis of glycogen, but occurs simultaneously in the presence of oxygen. We have stated that we assume that glycogen and lactic acid are in equilibrium, the reaction being catalyzed by enzymes. When the muscle is stimulated, considerable quantities of glycogen relatively are liberated from adsorption on the protein, and this free glycogen changes over to lactic acid due to the fact that the equilibrium point between glycogen and lactic acid is well over on the lactic acid side. When the muscle comes to rest, on the other hand, in the presence of oxygen, the concentration of adsorbed glycogen has been considerably reduced from what it was before the stimulation. To re-establish this former state more glycogen must be adsorbed out of solution. We know from the mass law that, when two substances are in equilibrium, extraction of one substance will cause a formation of that substance from the other in order to re-establish the equilibrium. In other words, as the


209

glycogen is taken out of solution by the protein, more glycogen will be formed from lactic acid in order to keep the equilibrium constant. I n this way three quarters of the lactic acid which disappears is reconverted into glycogen. The other one quarter is burned by the oxidation which occurs a t the same time. It is not necessary that there should be a great amount of synthesis at any one time for, as glycogen appears, it is extracted from solution by adsorption until the amount of adsorbed glycogen has reached the normal resting value. Bayliss in “The Nature of Enzyme Action,” 56 (1925) says: “It might be thought that a synthesis of a small degree could not be of much practical importance. This would be an error, as the following considerations will show. Let us take the case of amylase where the hydrolysis progresses almost to completion, and let us suppose that no more than one percent of starch is formed when the enzyme acts as maltose or dextrin. Since the product is an insoluble body, the equilibrium will exist only for a moment, so that more starch will be formed in order to replace that thrown out of the system by precipitation. As the rate of this reaction is slow, as shown above, the amount of starch per unit time will not be great, although by no means negligible. The process, it will be noted, is analgous to that of the precipitation of chloride as silver salt. It is most likely as Croft Hill’ points out, that the storage of starch in the plant and that of glycogen in the animal are to be explained on these lines.” I n such an enzyme equilibrium where the glycogen is adsorbed out of solution on the protein, thereby displacing the equilibrium and causing the formation of more glycogen, the rate of recovery to the normal state must necessarily be slow, as there is only a small amount of synthesis at any one time. Meyerhof has shown that the recovery period is slow, requiring fifteen to twenty-three hours before the muscle returns to its normal resting state. This could be prophesied from our theory but would not necessarily be so if the synthesis were coupled with the oxidation of lactic acid. If the glycogen and lactic acid are in an equilibrium whose rate is catalyzed by enzymes, we should expect, all other things being equal, that increase in the amount of glycogen should increase the rate of reaction and the amount of the final product lactic acid. Thus ten milligrams of glycogen with the enzyme should be converted to lactic acid more rapidly than one milligram and with a larger final yield of lactic acid. This was apparently the case in the experiments of Stiven which have been cited. It should be borne in mind, however, what is meant by all other things being equal. The enzyme concentration must be constant throughout the reaction. Moreover the final products must not affect or poison the enzyme. Dakin found that the enzymic conversion of phenyl glyoxal to 1-mandelic acid was stopped completely by increasing acidity. I n other words, the acid poisoned the enzyme. We know that the addition of bicarbonate increases very materially the lactic acid production in the muscle. This, however, may be a combination of two factors: one, reduction of the acidity; and two, extraction of the final product J. Chem. SOC., 73, 634 (1898); J. Phydol., 28, Proc. XXVI.

WILDER D. BANCROET AND GEORGE BANCROFT

210

by neutralization. It is possible therefore, that, in any particular case, the increasing acidity might stop the reaction a t a definite value of lactic acid. When the muscle is stimulated and then allowed to recover in the presence of oxygen, part of the lactic acid is burned and part is converted back into glycogen. This means that the store of glycogen is becoming less and less, providing there is no replenishment from outside. This replenishment probably comes in the normal animal from the glucose of the blood. There is no provision in Meyerhof's theory for the formation of glycogen from glucose except by the way of lactic acid which seems improbable. Mitchell' has shown that the glycogen content of oysters may be very materially increased by feeding the oysters glucose in the presence of air. It seems distinctly unlikely that this glucose is first broken down into lactic acid before being converted into glycogen. From our enzyme theory it would be perfectly possible for the glucose to go either directly or, after esterification with phosphate, to glycogen, for the two substances must be in equilibrium. The glycogen would be formed as fast as it was adsorbed out of solution by the protein, otherwise the equilibrium would be displaced. The formation of glycogen from lactic acid, as we have pointed out, depends upon the extraction of glycogen from solution on the protein, thereby disturbing the equilibrium and causing the further formation of glycogen i r order to re-establish it. Under these conditions it should be possible to add lactic acid to the enzyme in the presence of a suitable protein adsorbent and perhaps phosphate and form glycogen. Preylecki (loc. cit.) has shown that glycogen is strongly adsorbed on such proteins as are present in liver, as well as on coagulated egg white. Using 15 cc. of a 0.2% solution of d-lactic acid, I O cc. of M/z KH2P04, and I O CC. of enzyme solution and approximately 30 grams of egg white we attempted to show the formation of glycogen. That we were not able to establish the formation positively may be due to the present methods of analysis of glycogen. The enzyme solution was prepared according to the direction of Meyerhofz in which the rabbit was killed by a blow on the head, the hind legs rapidly skinned, the muscles cut out and placed in a glass evaporating dish surrounded by carbon dioxide snow. The muscles were then cut into thin slices and extracted with 50 cc. of isotonic KC1 solution a t 0°C. The muscle residue was separated from the solution by centrifuging. The clear solution resulting from this procedure after two hours digestion with 0.8% glycogen solution showed a strong qualitative test for lactic acid. The qualitative test was that recommended by Fletcher and Hopkins on the formation of a cherry red color with thiophene after oxidation with CuS04and H2S04. It is interesting to note that in the same period when egg-white was added to the mixture, there was only a faint test for lactic acid. This is entirely in accord with the findings of Prsylecki who showed that glycogen was hydrolyzed only very 1

LOC.cit.

* Naturwiasenschaften, 14, 196, 756 (1926).

EQUILIBRIUM BETWEEN GLYCOGEN AXD LACTIC ACID

211

slowly by amylase in the presence of protein. Indeed it could have been prophesied from the theory that this would be the case when the glycogen was stabilized by adsorption on the protein. Evans1 pointed out that all the glycogen is not precipitated when using Pfluger’s method of analyzing for glycogen which necessitates the use of as much as 90 cc of solution in the precipitation of glycogen by alcohol after treatment with KOH. This will amount to a large percentage error when only small amounts of glycogen are present. In a very recent paper Kerly* says that this is probably due to the solubility of glycogen in aqueous alcohol, but there may also be some peptization by the KOH decomposition products of the protein. Holmes and HolmesS undertook some experiments to show that glycogen could be recovered from such a large volume of solution and They, however, they found that the glycogen could be recovered IOO%. added solid glycogen to water and then precipitated it with alcohol, which according to Kerly does not resemble the true conditions, as she points out that I 7 to 18 percent of the glycogen is in solution; but that it dissolves very slowly, taking a week to come to equilibrium. Under these conditions Holmes and Holmes were not precipitating glycogen from solution, and their good results may be attributed to this fact. Inasmuch as we were working with egg albumin, enzyme, lactic acid, and perhaps glycogen, it seemed that the procedure might be simplified to precipitation of the protein, with a subsequent careful washing in order to removeall the glycogen. The protein was precipitated with mercuric chloride and with thorium nitrate in alkaline solution. Careful washing caused some of the protein to become peptized and pass over into the filtrate. If these last traces are not removed, reducing substances caused by the subsequent HCl hydrolysis affect the results materially, making them too high. The more serious difficulty, however, is that the last traces of glycogen cannot be removed from adsorption on the protein. Where small amounts of glycogen are present this amounts to a large percentage error, and makes the method impracticable for quantitative work. This question of adsorption has not been considered carefully heretofore in the methods for determining glycogen. It is obvious that the usual means of testing the efficaciousness of a method by adding known amounts of glycogen to tissue already containing glycogen and showing that one can recover 1 0 0of~ the ~ added glycogen is no criterion for that method. There is the same error of loss due to adsorption both in the portion to which glycogen was added and also in the blank. This error is of course not so serious when one is merely comparing the relative amounts of glycogen in two samples, but it is necessarily of prime importance in our case where we have no glycogen in the blank and wish to show a formation of glycogen. We are now working on a method of analysis with which we hope to surmount these difficulties. ‘Biochem. J., 19, 1115 (1925).

* Biochem. J., 24, 67 (1930). a

Biochem. J., 20, 1196(1926).

212


It is interesting to note that one of the characteristics of cancer is a disruption of the ordinary metabolic changes in the muscle. The rate of formation of lactic acid is greatly increased and there seems to be little conversion of lactic acid back into glycogen. It is necessary for Meyerhof to postulate that the glycogen is broken down as fast as it is formed, for he claims that the glycogen is formed by the coupled reaction between oxidation and synthesis. This is absurd. Warburg and Xegelein’ have shown that the rate of formation of lactic acid in cancer is very little reduced by the presence of oxygen. On the other hand, in the normal tissue there is very little or no lactic acid formation in the presence of oxygen. This indicates that the formation of glycogen, which reduces the concentration of lactic acid, is reduced to a minimum in cancer tissue. The increased rate of formation of lactic acid may be due either to an increased amount of available enzyme, or to less adsorption of glycogen on the protein. If some of the enzyme is ordinarily adsorbed on the lipin surfaces, as Przylecki suggests, this might be liberated by the selective adsorption of some foreign substance in cancer which would give an increased concentration of free enzyme. On the other hand, less adsorption of glycogen due either to less protein or to some substance being selectively adsorbed on it might account for the increased rate. With less adsorption of glycogen, according to our theory, there would be no reason for much formation of glycogen from lactic acid, and apparently there isn’t. Due to the lack of facilities we have not been able to test whether there is less adsorption of glycogen by cancer tissue than by normal tissue. Warburg also points out that carcinomas form lactic acid from methyl glyoxal a t the same rate as from glucose, which is very surprising if methyl glyoxal is an intermediate compound between glucose and lactic acid. There should be a time factor unless the reaction between glucose and methyl glyoxal is instantaneous. Moreover he points out that h e r tissue breaks down glucose a t only one-tenth the rate of cancer tissue but breaks down methyl glyoxal at the same rate. He concludes from this that there is no difference between cancer and fresh tissue with respect to methyl glyoxal We conclude from this that either there is some mistake in the determination of the rates of lactic acid formation from glucose and methyl glyoxal, or, in carcinoma, glucose does not break down to lactic acid through the intermediate stage of methyl glyoxal. Warburg also calls attention to the similarity between embryonic and cancer tissue in the rapid growth, increased rate of glycolysis and the small amount of glycogen present. This fact led Harrison and &fellanby* to the belief that the increased rate of glycolysis might be due to a lack of some substance ‘the growth regulator’ which was absent from the embryo and from the cancer tissue. Inasmuch as cancer seldom attacks the pancreas, they believed that this organ must contain considerable quantities of this hypothetical substance. They found 1 Warburg and Negelein: Biochem. Z., 152, 309 (1924). ‘Harrison and Mellsnby: Biochem. J., 24, 141 (1920).

EQUILIBRIUM BETWEEN GLYCOGEN AR'D LACTIC ACID

213

that an extract of the pancreas did inhibit glycolysis in cancer tissue from 1 5 to IOO per cent both in the anaerobic and aerobic phase. They found, moreover, that cancer tissue could not form lactic acid from hexose monophosphate or diphosphate, whereas the normal tissue forms lactic acid from these substances readily. They came to the conclusion that in cancer tissue glucose does not go through the form of a phosphate ester before going to lactic acid, and that in this respect the cancer tissue was different from normal tissue. We know, however, that cancer forms lactic acid from glycogen rapidly. If Harrison and Mellanby are correct, this must mean that in cancer glycogen is not esterified with phosphate during the course of glycogenolysis. There seems to be ample evidence to show the formation of an intermediate phosphoric ester in the normal tissue. Furthermore we have seen from Warburg's work that glucose apparently breaks down into lactic acid without going through the stage of methyl glyoxal. On the basis of these facts we must conclude that glycogen and glucose are converted into lactic acid by different ways in cancer and in normal tissue. This difference in itself might possibly account for the difference in rate of glycolysis. The problem as outlined here of why and how glycogenolysis takes a different path in cancer than in normal tissue should be of considerable importance in the study of cancer. It is one of the problems which must be solved in the future. The following general conclusions are made: The theories of Meyerhof, Warburg, and Hill are not the only exI. planation for the chemical changes during fatigue and recovery in the muscle. 2. The amount and the rate of lactic acid formation may be readily explained on the assumption of an equilibrium between glycogen and lactic acid, whose rate is catalyzed by enzymes.

3. The slow linear formation of lactic acid during anaerobic rest is easily understood and could be predicted on the basis of an enzyme theory where the glycogen is stabilized by adsorption on the protein. 4. The formation of lactic acid as a result of chloroform and heat rigor is obvious when we realize that the effect of these agents is to liberate the glycogen from adsorption on the protein. 5 . A suggestion has been made which should account for the effect of various other chemical reagents as caffein, arsenate, oxalate, etc.

6 . An adequate explanation is put forward by means of this theory for the formation of dextrorotatory lactic acid in the muscle, a phenomenon which has been more or less ignored in the Meyerhof theory.

;. I t has been shown that, despite the fact that the equilibrium point is Fell over on the lactic acid side, it is quite possible for the lactic acid to be converted back into glycogen again, due to the extraction of glycogen from solution by adsorption on the protein.

214

WILDER D. BANCROFT AND GEORGE BAXCROFT

8. Some of the errors in the methods of analysis of glycogen have been pointed out which have prevented up to this time the demonstration of the formation of glycogen from lactic acid by means of the enzymes and the adsorption of the glycogen on the protein. 9. Glycogen apparently takes a different path in cancer in the formation of lactic acid from that which it takes in the normal muscle. IO. Several possibilities are suggested, any one or all of which might account for the increased rate of glycolysis in cancer.

We are indebted to Dr. Ellice McDonald of the Cancer Research Laboratories of the Graduate School of Medicine of the University of Pennsylvania who suggested this work and gave us valuable aid in supplying references. Cornell UniLerszty.

THE EQUILIBRIUM BETWEEAT GLYCOGEN AND LACTIC ACID* Otto

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