University of Manchester.)

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1 6I : II5 THE LACTIC ACID METABOLISM OF FROG'S MUSCLE POISONED WITH IODOACETIC ACID. I. The lactic acid metabolism of anaerobic iodoacetate muscle. II. The lactic acid metabolism of aerobic iodoacetate muscle. BY COLIN ASHLEY MAWSON. (From the Department of Physiology, the Victoria University of Manchester.) I. THE LACTIC ACID METABOLISM OF ANAEROBIC IODOACETATE MUSCLE. THE work of Lundsgaard [1930] has demonstrated that a muscle poisoned with iodoacetic acid can do a considerable amount of work without any production of lactic acid. The source of chemical energy for both normal and poisoned muscles is to be found in the heat of reaction of the breakdown of creatinephosphoric acid to creatine and phosphoric acid, and in a normal muscle the energy necessary to allow of the resynthesis of creatine phosphoric acid is produced, at any rate in the main, by the breakdown of glycogen to lactic acid. Finally, the status quo ante is restored by the resynthesis of glycogen from four-fifths of the lactic acid produced, the necessary energy being provided by the combustion of the remaining one-fifth of the lactic acid. Such, in brief, is believed to be the course of the main events during and after a muscular contraction. In a poisoned muscle, however, the production of lactic acid is inhibited, and although glycogen breaks down it is converted to hexose mono- and diphosphoric esters [Lundsgaard, 1930] with very little production of energy, so that the restoration of the creatinephosphoric acid broken down during activity is rendered impossible. When, therefore, the store of this essential material becomes exhausted, the muscle ceases to twitch and goes into a peculiar type of rigor. This phenomenon occurs under both aerobic and anaerobic conditions, although an oxygenated muscle will usually survive longer than an anaerobic muscle [Lundsgaard, 1930].

2 202 C. A. MAWSON. It might be that in iodoacetate muscle lactic acid is actually produced, but is subsequently removed at a sufficiently rapid rate to prevent its detection, and previously published evidence does not seem to preclude this possibility. In a form of poisoning closely resembling that of iodoacetate, however, it has been shown [Lipmann, 1927] that lactate is not removed when added to minced anaerobic muscle in the presence of fluoride, but it seemed profitable to carry out a series of experiments to investigate the possibility of removal of lactate by muscle in the presence of iodoacetate. In the first instance, minced frog muscle was incubated for a sufficient time in presence of prussic acid to ensure the production of a considerable amount of lactic acid. Potassium iodoacetate was then added to one portion and incubation continued. No appreciable reduction in the amount of lactic acid present could be obtained by this procedure. An attempt was also made to obtain removal of lactic acid from lactate solution added to freshly minced muscle, but neither incubation of the material with iodoacetate nor leaving the same mixture at room temperature for a considerable period caused any reduction in the amount of added lactate. Experiments on similar lines were then carried out on intact frogs. The animals were pithed and the feet pierced with silver wire electrodes. After giving 100 "make and break" maximal shocks the heart was exposed, the sinus venosus punctured, and 5 c.c. of iodoacetate solution in phosphate-ringer perfused slowly through the frog by injection of the fluid into the aorta. Immediately after this treatment one leg was cut off and worked up for lactic acid, and the other leg allowed to go into rigor. This usually took place after 45 to 100 minutes. The remaining leg was then treated in exactly the same manner as the leg which had previously been cut off. It was considered probable that, if anaerobic reduction of lactic acid did occur at all, the leg which had gone into rigor would have contained little or no lactic acid, but this was not found to be the case. The perfusion of the frog must necessarily have washed out considerable amounts of pre-formed lactic acid, but in general similar amounts would be lost from either leg, and it was believed at first that evidence had been obtained of a reduction in lactic acid content in the "rigor" leg of the order of 25 p.c. Control experiments, however, showed that this apparent reduction was due to the fact that iodoacetate poisoning is a time reaction [Lundsgaard, 1930]. During the mincing of the "cut off" limb lactic acid was produced,

3 METABOLISM OF IODOACETATE MUSCLE. because it was worked up immediately after the perfusion, whereas the "rigor" limb gave rise to no lactic acid during the mincing period. The difference in lactic acid content of the two limbs, in fact, disappeared if the "cut off" limb was removed minutes after the end of the perfusion and the "rigor" limb after a further hour's contact with iodoacetate. The conclusion, then, can be drawn that neither in minced muscle, nor in muscle in the intact limlb of the frog, can lactic acid be removed anaerobically in the presence of iodoacetate. EXPERIMENTAL. A series of experiments was carried out in which muscle from four winter frogs was minced in a mincer cooled in ice, and the pulp divided into three portions in a cold dish. One portion was worked up for resting content of lactate, one incubated at 370 C. for 90 minutes with two drops of HCN and 2 c.c. of N/100 potassium iodoacetate solution. The third portion was treated in a similar way, but without addition of iodoacetate. The results (Table I) show that the muscle incubated in presence of iodoacetate produces no appreciable quantity of lactic acid. The percentages of lactic acid are calculated on moist muscle weight. TABLE I. Lactic acid (p.c.) Exp. Resting Incubated with Incubated No. muscle iodoacetate alone * Mean In the next series of experiments the muscle was minced and incubated at 370 C. for 2j hours. It was then divided into three portions, one of which was incubated for a further 3 hours with addition of two drops of HCN. The second portion was incubated for a similar period with 2 c.c. of N/100 potassium iodoacetate and a similar amount of HCN, and the third portion was treated in the same way, but incubated overnight. The results given in Table II show that no appreciable reduction of lactic acid took place in the presence of iodoacetate. Lithium lactate solution was then added to freshly minced muscle, as it was thought possible that the enzyme system might have been disturbed by previous incubation of the pulp. It is known, for instance

4 A. MAWSON. TABLE II. Lactic acid (p.c.) Incubated Incubated Exp. Incubated 3 hr. with overnight with No. 3 hr. iodoacetate iodoacetate * * A 0* [Lohmann, 1926], that incubation of muscle extract for 15 minutes at 370 C. will destroy the glycolytic activity of the extract. The minced muscle was divided into four portions. One was estimated as resting muscle. The next was treated with 1 c.c. of lactate solution and also given resting muscle treatment, the difference between the two giving the amount of added lactate. The same amount of lactate was added to the third portion, which was incubated for 4 hours with iodoacetate. The last portion was incubated alone for 4 hours. Toluene was used as a preservative in each case. In column 4 the added lactate has been subtracted from the observed lactate. TABLE III. Lactic acid (p.c.) Incubated 4 hr. +Lactate and iodoacetate Exp. Resting (less added Incubated No. Resting + Lactate lactate) 4 hr * In column 4 it is shown that the difference between the observed lactate and added lactate is a close approximation to the value for the resting muscle, as would be expected from the results given in Table I had no disappearance of lactate taken place. If any lactate had disappeared, subtraction of "added" from "observed" lactate would have given negative values in column 4. In the experiments on intact frogs the perfusing Ringer solution contained N/2000 potassium iodoacetate (1 part in 10,000). The amount of lactic acid found respectively in "cut off " and "rigor" limbs is given in Table IV. The amounts are expressed as percentages of the dry weight of the muscles as the perfusion gave rise to an cedematous condition in some cases.

5 METABOLISM OF IODOACETATE MUSCLE. 205 TABLE IV. Lactic acid (p.c.) _ Exp. "Cut off" "Rigor" No. limb limb 59 0* l621 0*474 67A B Mean 0* The difference of p.c. appeared significant, but some doubt arose when a control experiment was done using phosphate-ringer solution without addition of iodoacetate. This gave p.c. lactic acid in the limb immediately severed and only p.c. in the limb left for an hour, but a more important result was obtained by perfusing four frogs with Ringer containing N/100 iodoacetate and estimating lactic acid in their muscles, removed and minced in a warm room without precautions after different periods of rest. TABLE V. Time of severance after perfusion (min.) Lactic acid on dry weight (p.c.) The "injury" production of lactic acid is clearly likely to be much greater in the "cut off " limb than in the "rigor" limb, where it will be negligible. This point was proved by experiment on frogs perfused with N/300 iodoacetate Ringer solution. TABLE VI. Lactic acid (p.c.) Exp. No. Cut off Left 17 min. 56A B Mean The mean difference of p.c. is clearly of the same order as that of p.c. in Table IV. The point was finally settled by doing experiments in which, in each experiment, two frogs were used. One was treated as before, one leg being cut off immediately after perfusion, and the other when rigor had set in. The other frog was allowed to remain minutes after perfusion before the " cut off " leg was removed. Two typical results are given in Table VII.

6 206 C. A. MA WSON. TABLE VII. Iodoacetic Time of Lactic acid (p.c.) Exp. acid con- No. of restr No. centration frog (min.) Cut off Rigor Difference 54 N/ N/300 IA A * It is therefore clear that, if the muscle is allowed to remain until it is thoroughly poisoned, no evidence of reduction of lactic acid content in presence of iodoacetate can be obtained by perfusion of the intact frog. II. THE LACTIC ACID METABOLISM OF AEROBIC IODOACETATE MUSCLE. Since the earliest work by Lunds gaard [1930] on the metabolism of muscle poisoned with iodoacetic acid it has become increasingly clear that the alteration in the chemical mechanism of the contractile process is not to be looked for in the earlier stages of the contraction-relaxation cycle of the stimulated muscle. Myothermic work by Hartree [1931] and Fischer [1931] demonstrated that not only was the appearance of the heat-production curve the same in poisoned as in normal muscle, but that the relationship between tension developed and heat produced was also identical in the two cases. The identity of the processes during the early stages of contraction of normal and poisoned muscles was shown to be possible when Lehnartz [1931] demonstrated the truth of the contention that most of the lactic acid was produced after the conclusion of the mechanical response. The mechanical response itself was investigated by Henriques and Lundsgaard [1931], who found that the latent period, period of contraction, and course of the contraction curve as well as the tension-length developed, were quite normal in iodoacetic acid muscle. From these results it is clear that, in the earlier stages of a response to stimulation, a muscle poisoned with iodoacetic acid behaves as if it were a normal muscle. If, therefore, we are to find the link in the chain of events which is displaced by the action of the poison we must investigate the later stages of the response, and in this respect observation of the respiration of poisoned muscle assumes particular interest. Me ye rho f and Boyland [1931] showed that the respiratory quotient of poisoned muscle was in the neighbourhood of 0-7, which would seem to indicate that the fuel being utilized was fat rather than carbohydrate. Addition of lactate to the muscle restored the respiratory quotient to a value of 0-95, which

7 METABOLISM OF IODOACETATE MUSCLE. 207 is that observed for normal muscle, but the most striking feature of the observations was the threefold increase in respiration of the poisoned muscles on addition of the lactate ion. Addition of lactate to normal muscle causes an increase in respiration [Meyerhof, Lohmann and Meier, 1925], but the increase in oxygen uptake of poisoned muscle exceeds this by 50 p.c. Fig. 1. Exp (Control for Exp. 128.) Sartorious in oxygenated Ringer solution. Ten single twitches per minute. A. Normal contractions. B. After soaking for 30 min. in 1/20,000 iodoacetate. At X rigor began after 113 twitches. 13I Fig. 2. Exp Sartorius in oxygenated lactate-ringer solution. Ten single twitches per minute. A. Twitches in presence of lactate. B. First 100 twitches after 30 min. in 1/20,000 iodoacetate. C. Twitches from No. 500 to No D. Twitches from No to No Rigor began at X. The question then arose as to the manner in which the muscle was using up its increased oxygen consumption. It seemed likely that the added lactate was being utilized as fuel for the provision of energy for an endothermic reaction, and it was possible that the most essential reaction in muscular metabolism-i.e. the resynthesis of creatine phosphoric acid, was being provided for in this way. It is well known that this resynthesis does not normally take place in muscle poisoned with iodoacetic acid [Lundsgaard, 1930]. If the combustion of added lactate in

8 208 C. A. MA WSON. oxygenated poisoned muscle did indeed result in the resynthesis of, perhaps, some part of the creatine phosphoric acid broken down, it would be -expected that a poisoned muscle which received added lactate and oxygen would show, powers of contraction superior in duration to those of a similar muscle supplied with oxygen alone. This was, in fact, found to be the case. A solution of sodium d-lactate, prepared from cat muscle, was made up in phosphate-ringer solution to a Fig. 3. Exp Sartorius in oxygenated lactate-ringer solution. Six single twitches per minute. A. First 100 twitches after soaking in 1/20,000 iodoacetate. B. After 300 further twitches. a. Twitches in absence of oxygen after 15 min. anaerobic rest. At X rigor began, 25 twitches after resumption of stimulation. Fig. 4. Exp (Control for Exp. 157.) Sartorius in oxygenated Ringer solution. Six single twitches per minute. A. Normal contractions. B. After soaking for 30 min. in 1/20,000 iodoacetate. At X rigor began after 50 twitches. concentration of about 0 04 p.c. lactic acid. A pair of sartorii of Rana temporaria, which had been poisoned by soaking for half an hour in sodium iodoacetate-ringer solution (1/20,000 CH2ICOONa), gave about 100 twitches in oxygen before rigor commenced (Fig. 1), but a sartorius which had been soaked for half an hour in lactate-ringer before addition of sufficient iodoacetate to give the same concentration of poison would, after half an hour's soaking in the iodoacetate, give as much as 1400 twitches before signs of rigor began to appear (Fig. 2). In cases where

9 METABOLISM OF IODOACETAI'E MUSCLE. 209 rigor developed at all in presence of lactate it was always much smaller in amount than in the poisoned muscle to which lactate had not previouisly been added. The fact that this apparent protection of the muscle from the action of the poison is due to the oxidative removal of the added lactate, and not to interaction between the lactate and the iodoacetate as such, can be demonstrated by cutting off the oxygen supply (Fig. 3). The effect is not immediately visible owing to the amount of oxygen present in solution in the Ringer, but in a remarkably short time the muscle develops typical symptoms of iodoacetic acid poisoning, very soon ceases to twitch, and goes into rigor. An attempt was made to obtain quantitative evidence of the disappearance of lactic acid from the solution during the activity of poisoned muscle, and for this purpose an apparatus was used which was suitable for the estimation of quantities of lactic acid of the order of 0.1 mg. [Mawson and Ritchie, 1932]. After the muscle had been at rest in oxygenated lactate-ringer solution for half an hour a sample was withdrawn, suitably diluted, and the lactic acid estimated. Iodoacetate was added, the muscle left for a further half hour, and stimulation commenced. After some time another sample of the solution was removed and the lactate estimated again. Some typical results are given in Table VIII. TABLE VIII. Weight of lactic acid (mg.) Exp. W TL, A TL No. (g.) (kg.-cm.) Before After Loss Loss * * * * W = moist weight of muscle (g.). L =length of muscle (cm.). T =tension developed (kg.) during experiment. The figures given are probably not sufficiently reliable to allow any conclusion to be drawn from the value arrived at for the relation (T) between the energy output of the muscle and the lactic acid consumed, but they are sufficient to demonstrate the removal of lactic acid by the poisoned muscle during a period of aerobic activity. Several experiments were carried out in which creatine was added to the lactate-ringer solution used in the other experiments, but it did not seem PH. LXXV. 14

10 210 C. A. MAWSON. to have any appreciable influence on the results obtained, and no disappearance of creatine from the solution could be detected on stimulation of the muscle. It was shown by Lipmann and Meyerhof [1930] that the breakdown of creatinephosphoric acid was accelerated by raising the concentration of carbon dioxide in the muscle, so it would be anticipated that addition of C02 to the oxygen supply of a poisoned muscle in presence of lactate would tend to prevent the utilization of the lactate for the resynthesis of creatinephosphoric acid. It was found that the addition of up to 4.0 p.c. C02 (giving ph 6.8) to the oxygen supply had only a slightly deleterious effect, but any considerable increase of C02 tension above this value seriously hastened the onset of rigor, and resulted in a series of twitches similar to those given by a poisoned muscle working in the absence of added lactate. A curious feature of these phenomena which is difficult to explain is that, once symptoms of poisoning have developed, the muscle cannot be saved. If, for instance, a muscle is first of all poisoned, and lactate added subsequently, although it will twitch for a somewhat longer period than if no lactate were added, the postponement of rigor and non-irritability is very limited, and this is also true, though to a less extent, if poison and lactate are added simultaneously. This may be due, at any rate in the former case, to the fact that even when the muscle is at rest its metabolic processes are slowly proceeding, and consequently the initially poisoned muscle is losing creatine phosphoric acid, without resynthesis, during its period of soaking. This point of view is strengthened by the fact that if a poisoned muscle, twitching vigorously in presence of lactate, is deprived for a few minutes of its oxygen supply, resumption of the passage of oxygen does not postpone for very long the development of marked symptoms of iodoacetate poisoning which would not, had the oxygen supply been continuous, have appeared until after several hundred further twitches. It was also noticed that at temperatures below 100 C. poisoned muscles would continue to twitch for very much longer than they would, say, at 150 C. The initial part of the tracing of the muscle at low temperature was not greatly different from a trace taken at 150 C., but after about 100 twitches a very prolonged series of small, irregular twitches commenced which might continue to the number of three or four hundred. Rigor developed at about the hundredth twitch, as at , but it was very small in amount and did not increase nearly as rapidly as at the higher temperature.

11 METABOLISM OF IODOACETATE MUSCLE. 211 It is not easy to relate the phenomena described to the metabolic processes of normal muscle, but it has been observed that part of the resynthesis of creatinephosphoric acid in normal muscle has to await the aerobic processes following the twitch [Hill, 1932]. It may be that this mechanism is, in poisoned aerobic muscle, called upon to bear the whole burden of resynthesis of creatinephosphoric acid. A poisoned muscle, even in the presence of lactate, although it will remain active much longer than if no lactate were present, becomes non-irritable long before a normal muscle would show serious signs of fatigue, and this may well be due to the fact that the synthetic system kept in action by combustion of lactate is insufficient to keep up the store of creatinephosphoric acid to its normal level, with the result that constant wastage occurs. It must not be forgotten, too, that poisoned muscle is continuously losing glycogen, and abnormal amounts of hexose phosphoric esters are accumulating. It is, however, clear that the reason for the rapid deterioration of poisoned muscle, even in oxygen, is the actual absence of lactic acid, and that the non-production of lactic acid is not merely a symptom of the conditions. The results also tend to strengthen the point of view that lactic acid is an intermediate step in the oxidation of glycogen to carbon dioxide and water, rather than the theory that lactic acid lies off the main path of carbohydrate oxidation. The point of action of the iodoacetate is evidently not at the end of the chain of events, and as we know that the beginning of the chain is also unaffected by the poison the broken link may be sought in a more limited field. The suggestion of Meyerhof [1926] that the appearance of hexose phosphoric esters is due to abnormal stabilization of a labile hexose monophosphoric ester is very attractive, but would seem to be difficult, from its nature, of verification. I wish to acknowledge a private communication from Dr E. Lundsgaard, who informs me that he has recently carried out experiments on the addition of lactate to oxygenated iodoacetate muscle with results similar to those reported in this paper. 14-2

12 212 C. A. MAWSON. SUMMARY. PART I. No anaerobic disappearance of lactic acid takes place in a muscle poisoned with iodoacetic acid. PART II. 1. A muscle poisoned with iodoacetate in the presence of oxygen and lactate will continue to contract long after a similar muscle, without added lactate, has become non-irritable and has gone into rigor. 2. Lactate disappears during such a prolonged series of twitches, and it is suggested that it is used, at least in part, as fuel to provide for the resynthesis of creatinephosphoric acid. 3. Addition of more than 4 p.c. of C02 to the oxygen supply tends to prevent the prolongation of activity due to added lactate. 4. The disappearance of added creatine during the activity of aerobic iodoacetate muscle could not be demonstrated. In conclusion I wish to acknowledge a grant from the Medical Research Council and to express my gratitude to Mr A. D. Ritchie, M.A., for his invaluable advice and encouragement, and to Prof. H. S. Raper, F.R.S., for his assistance in the revision of this paper. REFERENCES. Fischer, E. (1931). Pflueger8 Arch. 226, 500. Hartree, W. (1931). J. Physiol. 72, 1. Henriques, V. and Lundsgaard, E. (1931). Biochem. Z. 2386, 219. Hill, A. V. (1932). Phy8iol. Rev. 12, 56. Lehnartz, E. (1931). Hoppe-Seyl. Z. 197, 55. Lipmann, F. (1927). Biochem. Z. 191, 442. Lipmann, F. and Meyerhof, 0. (1930). Ibid. 227, 84. Lohmann, K. (1926). Ibid. 178, 444. Lundsgaard, E. (1930). Ibid. 227, 51. Mawson, C. A. and Ritchie, A. D. (1932). Biochem. J. 26 (in the Press). Meyerhof, 0. (1926). Biochem. Z. 178, 462. Meyerhof, 0. and Boyland, E. (1931). Ibid. 237, 406. Meyerhof, O., Lohmann, K. and Meier, R. (1925). Ibid. 157, 459.