THE INFLUENCE OF OXYGEN UPON THE SURVIVAL RESPIRATION OF MUSCLE. By W. M. FLETCHER, M.A., M.B., Fellow of Trinity College, Cambrtidge. (Three Figures in Text.) (From the Physiologial Laboratory, Cambridge.) IN a former paper' I gave an account of the survival respiration of amphibian muscle based upon determinations of the CO. discharge from the excised muscle at short intervals of time during various conditions of rest and activity. This account included some preliminary observations upon the influence of atmospheric oxygen in determininig the course of the survival discharge of CO2, which have been more recently extended by the use of the same methods of estimation. SURVIVAL RESPIRATION IN AN ATMOSPHERE FREE FROM OXYGEN. In the experiments already given under tlis head, the changing rate of CO, discharge fronm a muscle exposed to nitrogen for the whole or part of the survival period, was observed simultaneously with that from a similar 'control' muscle supplied with air. The nitrogen supplied to the chamber containing the excised muscle was prepared by passing air over red-hot copper-filings in a long combustion tube, but this cumbrous method in practice forbad a long series of observations and none were made in continuity beyond the 14th hour from excision. To confirm the results already obtained and in particular to extend them in the direction of the later periods, another method was used. Pure nitrogen, prepared beforehand from urea, was supplied from a reservoir at a rate exactly corresponding with the rate of flow from the aspirators of the estimation apparatus. Between the reservoir and the experiment chamber were placed, first, a tower filled with pumice blocks automatically moistened with an alkaline pyrogallate solution for the removal of last traces of oxygen and CO, and, second, a low-pressure I This Joumnal, xxiii. p. 10. 1898.
RESPIRATION OF MUSCLE. 355 escape valve by which the nitrogen current remained at atmospheric pressure within the experiment chamber. By means of this apparatus it was possible to make continuous observations of the CO2 discharge from a muscle supplied only with nitrogen, and for an indefinitely long time. The results obtained on this plan were in agreement with those already published, and for this reason a fuller account of the apparatus used and of the curves of 002 discharge given by the estimations appears to be unnecessary. These later, as well as the earlier, series of comparisons between the frog's excised muscle exposed only to nitrogen, and the similar ' control' preparation in air, supported the following general account. 1. The CO2 output from the excised muscle during the first five or six hours declines in rate, at first rapidly, then more slowly, from the initial maximum. This output appears to be due, for its largest part, to the escape of CO2 already present as such in the muscle at or just after excision, and is found to be independent of the presence of oxygen. 2. Part of this early output, about a fifth part in the earliest period, and becoming progressively less, can be abolished by the removal of oxygen from the atmosphere, and is probably due to a respiration of the muscle substance continuing that of normal life but disappearing gradually as the changes occur which accoumpany loss of irritability and inaugurate rigor. In the later series of estimations, as in the earlier, two curves of discharge were simultaneously obtained from the muscle in nitrogen,- with a lower rate of output giving a lower curve,-and from the control muscle in air. As before, the initial difference always diminished, giving a convergence of the two curves towards the 6th hour, yet this convergence did not in most of the experiments lead to actual equality of output, as one of the earlier published diagrams shows. The actual meeting of the curves appears dependent on the time relations of the beginning of rigor and the early'diffusion' effect, in particular preparations at particular temperatures. 3. The steadily maintained rate of CO2 output, shown from near the 6th hour onwards, which accompanies the earliest stages of rigor (Zeitstarre) in various parts of the muscle substance, was found to be diminished by kth-ird, in the absence of oxygen. It wa-s found however that shbstituting nitrogen for air in the case of a muscle in which the C0O discharge due to rigor was already well established, did not reduce the discharge to the same degree, and sometimes did not reduce it at all.
356 W. M. FLETCHER. In the later observations these results were extended to the whole period of the development of rigor. The 300/0 reauction of CO2 discharge, due to the absence of oxygen, was found for the later, as previously for the earlier hours. So that the reduction of output already shown in previous diagrams cannot be explained as marking simply a delay in the development of rigor, but must be taken to show that the processes of rigor which result in the discharge of CO2 are partially incomplete throughout the whole survival history of a muscle supplied only with nitrogen. This diminution of the normal survival output is not made good at any stage later, unless it be during bacterial putrefaction. 4. The traces of CO2 which continue to leave a muscle in which natural rigor is complete, or artificial rigor has been produced, are unaffected by the absence of oxygen, and no constant differences were found between the times at which bacterial putrefaction, with its enormous rates of CO2 discharge, showed itself in the cases of the muscles in air and in nitrogen respectively. SURVIVAL RESPIRATION IN AN ATMOSPHERE OF PURE OXYGEN. It has been shown in the previous section that, in respect of one chemical accompanitnent, natural rigor is imperfectly exhibited in a muscle deprived of oxygen. In a similar way it has been found that the development of rigor-in respect again only of the CO2 output Reservaerchambers * Afu1de ckambers * a egaax/ing In waler baleh Fig. 1. Arrangement for the supply of oxygen.
RESPIRATION OF MUSCLE. 357 marking it,-becomes exaggerated in a muscle surrounded by an atmosphere of oxygen. Crossed pairs of legs, prepared as in previous experiments from the frog, were arranged in simple chambers put side by side in a waterbath. One chamber containing the muscle preparation for 'control' was supplied in the ordinary way with air freed from CO2, drawn throuah it at constant rate and at atmospheric pressure by an aspirator. Through the other was drawn at the same rate by a similar aspirator a current of pure oxygen, supplied from a reservoir to which, falling from the aspirator, constantly flowed a bulk of water very nearly equal in volume per unit of time to the bulk of gas removed. The arrangement is shown in Fig. 1. Air is dissolved in the drops which fall from the aspirator, but the amount of contamination suffered by the oxygen store through the (liffusion of air from the quietly incoming water at the bottom of the reservoir was found to be negligible. The temperature of the oxygen reservoir was kept as constant as possible by means of thick non-conducting jackets packed with sawdust. With this precaution it was fouind in practice that a constant rate of flow (120 c.c. per hour) couild be maintained at atmospheric pressure through the experiment chamnber, and its constancy easily checked at any moment by timning the rate of dropping from the aspirator siphon. The hydrodynamic system is such that constancy of rate of flow marks a position of unstable equilibrium, and the necessary balancing must be effected by addition or subtraction of water at the regulating tap shown in the figure. Two typical experiments may be described. Exp. I. Two freshly caught frogs were killed at 10.30 a.m. and bled. A '"crossed' pair of the skinned legs was arranged to hang free in each of the experimental chambers. The chambers were plunged side by side in water maintained throughout at 20O5'-21' C. The volume of each chamber was 30 c,c. A current of air free from 00 was started through each at 10.55 a.m. Pure oxygen was substituted for air in the case of one chamber at 11.45 a.m. and both currents were maintained at the same rate (120 c.c. per hour) throughout the whole experiment. The number of CO2 estimations made, the period of each, and its result are shown graphically in Fig. 2-the diagram being constructed on the same plan and scale as those in the previous paper already referred to. The first CO2reading began at 11.20 a.m., when the preparations, both in air, showed identical outputs of *13 c.c. CO2 per half-hour. It may be taken PH. XXVIII. 23
358 W. M. FLETCHER. for granted that the ouitput of each during the preceding quarter of an hour had been at the rate of *17-*20 c.c. per hour..30 in air in oxygen 265 beginning of oxygen 'L3 1 ~ L Hours I 2 3 4 5 6 7 8 9 10 11 12' 2223 24 25 26 after excision. Fig. 2. Survival discharge from 'crossed' pairs of legs, one in air, the other in oxygen. The second reading, begun at 12 noon, a quarter of an hour after oxygen alone had been sent to one preparation, showed an increased rate for that preparation, and a normally falling rate for the 'control' in air. The rate of output from the former continued to increase from this point, while the latter entered upon the normal 'plateau' of CO2 production. Eleven hours after excision the muscle in oxygen was found to be discharging CO2 at more than three times the normal rate. After the 22nd hour its rate of discharge began to sink as the completion of the rigor period approached, while that of the muscle in air gave a positive indication of early bacterial putrefaction by rising. At the 26th hour the chambers were opened. The 'control' muscle in air gave a distinct smell of putrefaction, while the muscle in oxygen gave none. Exp. II. With similar arrangements, 'crossed' legs, from a large frog, excised at 5.8 p.m., were placed in chambers maintained at 19-20 0. throughout and the air current started through each at 5.20 p.m. At-the 16th hour oxygen was sent to one as before. The results are given in Fig. 3. Here the preparations showed normal and nearly identical rates of 002 output up to the 16th hour, when pure oxygen was supplied to one. Immediately the rate in this case rose until it was nearly double that of the control in air. In this experiment bacterial putrefaction began in both preparations at nearly the same time (after the 24th hour) but is not shown in the figure. From these and other similar experiments it is clear that the supply of oxygen to an excised muscle enormously increases the output of CO2 which accompanies the early stages of rigor, and this is true whether
RESPIRATION OF MUSCLE. the extra supply of oxygen is brought to the muscle before the beginning of the rigor stages or later in their development. I in air 30 - in ox)gen oxygen i,,25 to one 0 359 W '. ' after Fig. 3. Hiours 1 2 15 16 17 18 excision Oxygen supplied to one muscle preparation after the 16th hour. It may be noted in passing that bacterial putrefaction, as demonstrated by a rise of 002 discharge, was delayed in the case of the muscle exposed to the oxygen current, in four out of five experiments in which the observations were carried on long enough to reach that stage. SUMMARY. 1. A former account of the survival respiration of Amphibian muscle in an atmosphere of nitrogen has been confirmed and extended, by the use of a more convenient method. In particular it has beer shown that the introductory processes of rigor which are marked b.y a steady output of 002 are partially incomplete in the absence of oxygen. The rate of 002 output characteristic of the gradual development of rigor in air is diminished by about 30 p/pian atmosphere of nitrogen. 2. It has been found that the normal rate of co2 discharge during the rigor periods for a muscle in air is always largely increased in an atmosphere of oxygen, the increase ranging from 80-300n/. 23-2