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THE HEAT-PRODUCTION OF SURVIVING AMPHIBIAN
MUSCLES, DURING REST, ACTIVITY, AND RIGOR.
By A. V. HILL, Fellow of Trinity College, Cambridge.
(From the (^) Physiological Laboratory, Cambridge.)
IN a recent paper, I showed that it (^) is possible to estimate the heat produced by isolated surviving frogs' muscles, (a) during rest and
(b) during heat- or chloroform-rigor. These experiments lhave led to
a more complete investigation of the energy exchanges of muscles under various conditions. The (^) direction taken by this investigation has been determined largely by ideas derived from Fletcher's work2 on the CO2 (^) liberation, and from the work of Fletcher and (^) Hopkins3 on the lactic acid formation of surviving muscles. A quantitative comparison of the heat (^) produced during the various stages of what is really the dissolution or gradual death of a muscle, with the CO2 set free (^) during these stages, is as Frank5 (^) points out a matter of obvious interest in discussing the internal nature of the muscular machine. A further comparison of^ the heat with the lactic acid formed is of equal importance. It is on lines such as these that we rnay best (^) hope to separate, from^ among the various survival processes, those which exist during normal life and those which occur only during (^) dissolution and death.
The muscle is undouibtedly a clhemical machine: by no stretch of
the imagination can it be supposed to be a heat-engine. Later in the
paper6 is discussed how the developmnents of physical chemistry, making
it possible to^ predict "^ free energy" firom certain constants in a
I (^) A. V. Hill. This (^) Journal, XLIII. (^) p. 280. 1911. (^2) W. M. Fletcher. Ibid. (^) xxiii. p. 10. (^) 1898: xxviii. pp. 354 and 474. 1902. (^3) Fletcher and (^) Hopkins. Ibid. xxxv. (^) p. 247. 1907. (^4) Blix (Scand. Arch. f. Physiol. xii. (^) p. 94. (^) 1902) claims to have (^) established the heat- production of resting muscle. Experience with his instrument (^) makes me, however, very doubtful of the validity of his proof. At any rate he failed (^) entirely to obtain (^) quantitative results.
5 0. Frank. Ergebn. d. Phiysiol. iii. (2), p. 390 &c. 1904. 6 p. 507.
HEAT-PRODUCTION OF MUSCLES
chemical reaction, may help (^) us to develop the theory of muscular contraction upon rational lines. The present investigation is an attempt to follow the disinitegrative processes of a muscle cut off from its circulation from the point of view of the total change of energy in these processes. As regards a comparison of the energy with the chemical products of these reactions I have had (^) many opportunities of discussing the problems involved with Dr Fletcher: to this fact I owe much, both in information and ideas. The (^) heat-production of an isolated muscle under various conditions has however an interest of its own apart from a comparison with the simultaneous formation of chemical substances, in particular of lactic acid and CO2. It can be used as an independent means of (^) investigation, based (^) upon the fact that a rise of temperature in the muscles com- municates itself, with no appreciable delay, to the recording apparatus:
whereas (a) as Fletcher has shown the CO2 once formed takes some
hours to diffuse out of the (^) muscle and (b) the present method of lactic acid estimation is so elaborate that many experiments cannot be done (^) by it. The heat-production is likely, therefore, to be of greater use than the formation of chemical (^) products, in (^) helping us to follow the time-relations of certain muscular processes.
PART I. THE HEAT-PRODUCTION OF RESTING MUSCLES. The method of^ estimation was that of the micro-calorimeter described (^) recently'. Owing however to the (^) extreme smallness of the quantities of heat to be estimated-leading in some cases to a rise of temperature of not more than (^) *01° C. per hour-I have, for reasons given below, adopted somewhat more elaborate precautions to secure equality of (^) temperature outside the two flasks. The (^) differential method is of course based on the supposition that the two flasks are (^) subjected to
identical external temperature conditions, so^ that the physical loss of heat
from one flask is balanced by an equivalent loss of heat from the other.
Naturally I imagined that if the^ two^ flasks^ were standing side by side
on a table, in a cellar of (^) very uniform (^) temperature, such conditions
would be fulfilled.^ For^ all^ ordinary experiments, such as I have
described hitherto, this is in fact the case. But in the estimation of
the very minute quantities of heat given out by resting surviving
muscles in their later (^) stages it was found (^) that there were some-
times certain irregularities which could be accounted for only by the
Op. cit. Throughout this paper calorie denotes gram-calorie.
467
HEAT-PRODUCTION OF MUSCLES
The use of whole frogs has, though in an even higher degree, all the advantages, (a) (b) and (c), given above: it has however the dis- advantage that one does not know exactly with what one is dealing: the survival processes in intestine, liver and heart all add their quota to the heat produced by the surviving muscles. The possibility of bacterial decomposition in the gut leading to survival heat-changes can be neglected, for the guts of laboratory frogs are almost always practically empty; in any- case however the muscles provide the^ main^ portion of the active living constituents of an animal: and the exact quantitative and qualitative similarity of the curves of heat-production, for the whole pithed animal and for the legs only, shows that we may safely use the whole animal for the experiments. The experiments were made (a) during the very^ hot^ summer of 1911 and (b) during the months January to May of 1912. The more careful and elaborate experiments are the later ones: some of the earlier experiments however are included here, because although the frogs were in a very different state (owing to the hot weather) the same general type of results^ was^ obtained.^ In^ the^ later^ experiments stirring before each reading (to get the mean temperature of each flask) has been done with thin pieces of^ stick, passing through the^ cotton-wool stopper, and left resting in the flasks throughout the experiment. This I believe to be simpler, less^ liable^ to^ error, and^ more^ efficient^ than^ the bubbling of air previously advocated. With a little stirring a perfectly
constant reading is obtained, so^ that^ further stirring causes^ no^ change
of reading to^ within^ ° C.^ The actual^ galvanometer readings were
made at short intervals during the first few^ hours, and^ at^ longer
intervals later. Curves were drawn through the observed points plotted
on squared paper, and were then corrected^ for^ heat-loss':^ from^ the
curves so corrected the heat-production during the various stages of
survival was read off. It is impossible to give full details of all the experiments quoted. For those however who wish^ to^ see^ the actual
details of a typical experiment, Exp. I, Table I^ and^ Exp. I, Table^ II are
given in^ full. It^ should be^ noted^ that, when the^ contrary is^ not
definitely stated, the^ -Ringer or^ salt^ solution used^ is^ the^ ordinary laboratory solution which^ contains^ a^ certain^ amount of^ dissolved
oxygen, say from^0 2 to^06 c.c.0/0. This is^ important in view of^ the
effects of 02 seen in Table III and described later.
I (^) Cf. the description of (^) method, A. V. (^) Hill, op. cit. The constants of heat-loss of the flasks were very carefully redetermined for these^ experiments, and found^ to^ be^ practically unchanged after nine months' use^ of^ the-flasks.
49
470 A. V. HILL.
TABLE I. Heat-production of resting isolated (^) frogs' Muscles. ExP. (^) I. Feb. 1912. Eight frogs killed, 97 c.c. arms and legs cut off as rapidly as possible, measured and put in flask No. 1 at 13.50 C. in (^) Ringer's solution: total 250 c.c. In control-flask No. 5 285 c.c. water. Coefficient of heat-loss k= (^) -0406 per hour. Stirred with thin sticks before each reading. The whole was very well shaken and stirred before the readings began: the initial reading is therefore a correct' one, and the amount of oxygen2 dissolved in the 250- 97, i.e. 153 c.c. of Ringer, must have been about 1'1 c.c. The time is given in hours from the time of death of the frogs, and the reading in scale divisions: 100 s.d. = .3240 C. Time *3 35 .4 .5 .7 1-0 1P3 1-9 2-5 3 4-6 55 (^) 6- Reading -118 -^113 -^110 - 108 -103 - 97 -90 -76 -74 -70 -58 -49 - Time (^) 8-1 22'7 23-9 26'9 28'3 31'3 33.4 47-7 49 51J1 53'2 74 102 Reading - 36 + 37 + 43 59 68 77 92 128 135 140 143 234 415 At the end of the experiment the muscles were decomposing strongly: that the later large rise of temperature is due to tbis, is seen from the fact that after placing at 102 hours a little (^) chloroform into the flask, the reading fell in 24 hours to 220. Judging by other experiments the chloroform does not (^) immediately check (^) all the (^) processes set up by bac- teria: but^ neglecting the^ first few^ hours^ after the^ introduction of^ chloroform, it is seen that there is practically no heat-production after cessation of the bacterial (^) action. Auto- lytic changes are not stopped by chloroform, so these cannot be responsible for the heat- production in the later stages given in the Table. From the observations given above, and the curve of (^) heat-production corrected for heat-loss, the following numbers are obtained and shown in Fig. 1. The rate of heat- production is calculated in calories per (^) hour, per c.c. of (^) tissue, during the periods named. Time in hours, measured from death of animals. Period beginning at ... *3 (^) *55 '8 1-0 1-5 (^2 3 4 6 ) ending at .... 55 '8 1-0 1-5 2'0 (^3 4 6 8 ) Rate of heat-production (^) *36 '20 '15 (^) *12 '08 (^) '07 '05 '04 (^) -035. Period beginning at ... (^13 23 33 43 50 60 70 80 ) ending at ... (^23 33 43 53 60 70 80 90 ) Rate of heat-production -049 -07 '07 '07 *08 *11 '14 (^) *17 * The rate of heat-production is possibly subject to an error of A '016 cal. (^) per e.c. per hour. The same error, whatever it be, must however affect every estimation alike, and in the same direction. In the following experiments (II-VI) the possible error was 4f '02 cal. per a.c. per hour. Exp. II. Similar Exp.: legs and arms at 130 C. in NaCl .7 (^) e/0. Feb. 1912. (^) Heat- production in gr. cal. per c. c. of tissue per hour, during the (^) pegiods named. Time in (^) hours. Period beginning at ... 0'5 1'0 (^2 3 4 6 9 12 20 ) ending at ... 1'0 2'0 (^3 4 6 9 12 20 30 ) Rate of heat-production '16 '09 '056 '052 '050 '040 '043 '046 (^) '050 ' (^1) If (^) shaking is (^) not carried out carefully the mixture of muscles and Ringer may take some time to (^) settle down to a uniform temperature.
2 The solubility of 02 in water at 13.50 is about 3'4 c.c. 0/0: the dilute 02 of the air
is tberefore soluble to the extent of about 0'7 c.c. (^) 0/0.
A. V. HILL.
At the end of the (^) experiment the feet of two of the frogs were still excitable: but there was an obvious smell of (^) decomposition. From the observations a corrected curve of heat- production was made, and the following numbers were obtained and are shown in Fig. 1. H.-P. in calories per c.c. of. tissue per hour, during the periods named. Time in hours, measured from death of animals. Period beginning at .;. 1 2 3 5 8 12 18 26 34 ending at ... 2 3 5 8 12 18 26 34 46 Rate of heat-production *09 *06 *047 *039 '032 '029 030 *031 ' Possible error, 4=^ '007 cal. per c. c. per hour. In Exps. II to IV the (^) possible error (^) was not > ffi02 cal. per c.c. per hour. ExP. II. Whole frogs, unskinned, at 16.50 C. in Ringer. Feb. 1912. Period beginning at ... *3 10 2 3 10 15 20 ending at ... (^) 1.0 2'0 (^3 10 15 20 ) Rate of heat-production '23 '15 (^) '08 '05 '055 065 ' Exp. III. Whole frogs, unskinned, at 12'50 C. in NaCl solution. Feb. 1912. Period (^) beginning at. .5 1 2 3 -4 6 12 20 40-- ending at^ ...^ 1'0^2 3 4 6 12 20 40 Rate of heat-production '10 (^) '07 '055 (^) '037 '034 '030 '021 025 ' EXP. IV. Whole frogs, unskinned, at 210 C. in (^) Ringer. Aug. 1911. Period beginning at 5... 1.0 (^2 4 6 ) ending at ... 1.0 (^) 2'0 4 6 8 10 Rate of (^) heat-production '19 '14 '12 '10 '08 '
From these experinments it is seen that the heat-production of isolated
resting muscles follows a (^) change represented graphically in Fig. 1. The
same relations are shown by the heat-production of pithed frogs. The
heat-production of the normal resting live frog, as was shown in a previous paper', is about '32 cal. per c.c. per hour at 160 C. This value is strikingly the same as values (^) obtained for the initial heat-evolution in Table I exps. I, III, V, Table II exp. II, and Table III (^) exps. II, III and VIII. The (^) production of heat therefoire by the resting surviving muscle starts at a high value which is (^) apparently about the^ same^ as^ that^ for the normal live resting frog. It is impossible, as a matter of fact, to estimate the rate (^) of heat-production immediately after^ death; the^ method requires at least 15 minutes before an observation can be made. If (^) however one exterpolates the curve
back, one finds a value for the initial sturvival evolution of heat which is,
in (^) any case, not less (^) than that shown by normal living frogs; The rate of heat-production falls thereafter for several hours, along a more or less
"exponential" curve, until at 160 C. it reaches a value of about '05 cal.
per gr. of tissue^ per hour, both for frogs' legs or for whole frogs. At I (^) Cf. A. V. Hill. This (^) Journal, xLiii. p. 390. 1911.
HEAT-PRODUCTION OF^ MUSCLES.
this value it may remain constant^ for^ maiiy hours, without^ any very obvious variation, until finally decomposition sets in, followed^ by an increasing rate of^ heat-productiot.^ Before^ proceeding^ to^ discuss^ the bearing of these facts, it may be well to refute certain objections^ that may be made against^ then. In the first place a year's experience with the method has shown^ me that for these experiments the^ apparatus^ is^ working^ somewhere^ near the limit of its power. The greatest liability to error arises from the impossibility of securing exactly similar^ temperatures^ outside^ the two flasks: but it must be remembered that, from several control experi- ments in whicb the final equilibrium difference of^ temperature^ between two flasks (both filled with water)^ has^ been^ estimated,^ it^ is^ possible^ to put a superior limit to the errors which may arise in^ this way: and^ further the errors cannot always be^ positive,^ or^ always^ negative,^ for^ each experiment has been made in a different position upon the^ table. At least 60 experiments altogether have been^ made,^ of which^ the^10 given above are typical: the same results qualitatively and^ quantitatively have been seen in all, so^ there^ is^ no^ possibility of^ any considerable^ error of this type affecting^ the^ general^ result.^ From^ the best of^ these 60 experiments I have calculated below^ an^ average^ value for^ the^ heat-
production of "surviving'" frogs'^ muscles: but^ it^ will^ be^ seen^ from^ the
facts connected with the presence or^ absence^ of^ oxygen, which^ are^ given in Table III, that it^ is^ of^ no^ value,^ obtaining^ very^ exact^ results in^ any
buit the late periods of survival change, because^ thedaumbers^ obtained
depend chiefly upon the^ amount^ of^ oxygen^ present.^ The^ average values given below are as accurate as^ it is^ worth^ our^ while^ to^ make them. The next criticism is as follows. Possibly the^ peculiar conditions
of the experiment have^ rendered^ the^ balancing of the flasks^ against^ one
another no^ longer^ exact.^ The^ difference^ in the^ specific^ heats^ of^ water
and muscle might have^ had^ this effect, were^ not^ this difference^ too
small. It may be^ urged that the^ differential^ method^ applies^ only^ when
exactly similar substances are in^ either flask.^ In these^ experiments a
mixture of solids and^ liquid is^ in^ one^ flask, and^ water^ alone in the^ other.
The solid substance, viz. frogs' muscles, may hinder convection^ currents
and mixing of the fluid, and^ so^ prevent the^ equalisation^ of^ temperature:
this might appreciably change the^ constant^ k^ of heat-loss in the^ flask
considered. The flasks might no^ longer^ be^ properly^ balanced,^ and^ the
mnethod faulty. The one with muscles^ in it^ might always^ lose^ or^ gain
heat slower than the one^ with^ water^ only. As^ a^ matter^ of fact this is
IIEAT-PRODUCIION OF MUSCLES.
analogous in the two cases: the 002 and heat-liberations run parallel, at any rate after the ath hour. As Fletcher' says, the " normal curve
of C02 discharge may be divided into three stages: the first stage
extends over five hours and is irregularly declining. The second stage, the 'plateau,' is many hours longer and shows a very slowly declining rate of discharge ": and again2 "the sudden and final change of direction of the curve, showing a rapidly increasing rate of^ CO,-discharge, is invariably found to be accompanied by a distinctly putrefactive smell." Of these three stages the third and last, that of bacterial invasion, need not be further discussed. It is an accident in survival life which
has only the historical interest of playing a large and^ unrecognised
part in the "muscle respiration"^ studied^ in the earlier work of^ Her^ mann and others.
5 /O^ /S^3
Fig. 2. Course of survival heat-production: mean^ of^23 experiments at average temperature of 15.50 C. Of the survival respiration proper, the second^ stage is that of the long "plateau" of CO2 and heat-production steadily maintained for
many hours, culminating in the mechanical changes of^ rigor. As
Fletcher3 says "^ the^ maintained^ output of^ C02^ marked the^ progress^ of
a continuous chemical change, which only at^ a^ late^ stage induced the
opacity and stiffness of rigor." The C02-output was maintained under
ana3robic conditions, it increased in^ air, and^ might be^ trebled in^ an
atmosphere of oxygen, but^ in^ the^ atmosphere of^ oxygen^ the^ onset^ of
rigor was prevented. The conclusion that the increase of C02 in the presence of abundant oxygen was^ the^ mark of^ the^ oxidative removal of
a product causing rigor if unremoved was confirmed by Fletchner and
' (^) Op. cit. p. 62. (^2) Op. cit. p. 28. This fact is also exactly what I have observed with (^) respect to the evolution of heat. (^3) This (^) Journal, xxviii. (^) p. 476. 1902.
47t
46A. V. HILL.
Hopkins in their work (^) on the lactic acid of muscle. The (^) striking parallelism between the " plateau" of :02-output and the linear rate (^) of
lactic acid and heat-production (under anaerobic conditions) points very
strongly to their^ being due to one and the same process. This will be discussed below.
With regard to the first stage of survival respiration, lasting for
about five hours, during which the CO2 and heat (^) outputs decline (^) rapidly
at first atnd then more slowly to reach their steady constant values, the
interpretation is niore difficult. Fletcher originally suggested the
hypothesis that this stage of the 00,- discharge was due to three factors.
"The bulk of the (^) discharge is due to destructive processes (^) going on
within the nmuscle independently of the surrounding mediumii": that is
to say the events (^) causing the later CO2 " (^) plateau " (^) were supposed to be present also during the first stage, in addition to (^) the other processes yielding the extra CO2. In (^) the next place he believed that "part of
the earliest dischargre is of 00, already existing as such within the
muscle, and which (^) escapes with the rest by diffusion outwards." (^) Since such diffusion can cause no appreciable heat (^) changes, and since the same fall is observed in the (^) heat-production as in the C02-output, this factor cannot, as Fletclier believed, explain the (^) general initial form of the curve, and it is probably of no (^) importance. Finally he suggested that "the (^) remaining part of the discharge is due to a process in (^) which oxygen is absorbed fronm the air and (^) CO2 produced, and which is to be taken as an imperfect continiuation of part of the normal (^) process of (^) respiration during somatic life." (^) And in (^) a later paper' he showed that the muscle
could avail itself from the earliest periods onwards of a supply of oxygen
in the (^) atmosphere, producing a much larger yield of CO2. It is this last factor, in a inodified form, which (^) I believe to be one of the chief causes of the (^) phenomena observed. It should be noted that (^) these hypotheses, and others which were considered (^) and rejected, were put forward before the (^) dalys when the brilliant work of Fletcher and Hopkins on the lactic acid formation of (^) surviving muscles had made so many factors clcar. It will be shown below that in all (^) probability the long continued "plateaus" of CO,- and of heat-production are due to one (^) and the same factor, viz. the chemical (^) breakdowns leading in the absence of oxygen to the liberation (^) of lactic acid. We know however that these (^) breakdowns do
not occur in the presence of sufficient oxygen2, and Dr Fletcher agrees
(^1) This Journal, xxviii. p. 358. 1902. (^2) Fletcher and Hopkins. This Journal, xxxv. (^) p. 282. 1907.
A. V. HILL.
Let tls now turn to the further experimental evidence at hand. It was noticeable in the first^ place that the^ rate of^ heat-production in the initial stages was much more (^) variable, from (^) experiment to (^) experiment, than that occurring in the later stages: even though the percentage error of observation is much greater in these later (^) stages. This suggested that some arbitrary unknown factor was at work, such as the amount of oxygen dissolved in the^ water^ in^ which^ the frogs' muscles lay. The apparatus is not suitable for experiments on the evolution of heat (^) by muscles hanging in oxygen gas: in^ fact^ I^ doubt if any such apparatus can at present be made. It is necessary to have the muscles (^) lying in salt solution in the (^) flasks, in order both to (^) calculate or balance correctly the loss of heat by the flask, and also to get an exact reading of the mean temperature inside. The (^) experiments were therefore made as follows, and in all cases except Exp. I upon whole frogs unskinned. Ringer's
soluition, or water, was boiled and cooled, so as to boil off the oxygen;
and then one experiment was made with frogs in the oxygen-free water, and another (^) experiment with (^) frogs in the same (^) water after shaking some time with oxygen. Water at 160 C. does not dissolve much oxygen, only about 3-2 c.c. 0/0, but^ quite enough to show the characteristic effects given in Table III. It will be noticed that the initial evolution
of heat is very largely increased by the presence of oxygen in the
water. Further the experiments are the most accurate hitherto (^) made, and one^ can^ be quite confident of the^ genuineness of the phenomena seen. Exp. I in the Table shows the same effect of (^) oxygen, but was done in a^ different way.
TABLE III. Effects of oxygen on the heat-production of surviving muscles. Exp. I. This experiment is of a different kind from the rest in the Table. Five frogs killed, skinned, and left^ in^ oxygen 14 hours. Then their heat-production estimated immediately in the calorimeter at 160 C. Time in hours from death. H.-P. in cal. per c.c. of^ skinned animal per hour. Period beginning at 14 15 16 17 18 20 22 ending at^15 16 17 18 20 22 Rate of H.-P. ... *19 *10 *08 -06 -05 -04 - There is, for three hours, a much higher rate of H. -P. than occurs (^) normally in (^) muscles 14 to 17 hours after death. Exps. II, III, IV and V. Made on the same (^) day with the same (^) batch of (^) frogs. The possible error was -fi *o1 cal. per c. c. in every (^) reading in each (^) Exp. Exps. II and III. In Ringer's solution saturated with oxygen, at 15.60 C. and 160 C. respectively. Period (^) beginning at *3 (^6 1 2 4 6 10 20 30 ) ending at^ *^6 1 0 2 4 6 10 20 30 40 Rate of H.-P., Exp. II '40 *19 *10 *065 (^) -045 037 -035 *032 -037 - Rate of H.-P., (^) Exp. III *60 *3 *20 *08 (^06) *049 *049 *048 -
478
HEAT-PRODUCTION OF MUSCLES. 479
Exps. IV and V. (^) In 02-free Ringer's solution at 15.90 C. and 15.80 C. Period (^) beginning at... *3 *6 10 2 3 5 10 20 30 40 ending at^ ...^ *6^ 1-0^ 2-0^3 5 10 20 30 40 Rate of H.-P., Exp. IV *12 *08 05 *042 037 -035 *038 033 035 043 Rate of H.-P., (^) Exp. V *15 *13 07 -053 *046 *038 *036 035 *041 * Exps. VI and VII. Frogs kept (^) (alive) in oxygen two hours before experiment began. Exp. VI in water^ saturated^ with^02 at^ 16.50^ C.: Exp.^ VII^ in^ 02-free^ water at^ 16.50^ C. Period beginning at ... 1-0 2 3 5 10 15 20 25 ending at (^) ... 2 0 3 5 10 15 20 25 30 Rate of H.-P., Exp. VI *12 *10 *08 *065 -05 -043 -045 * Rate of H.-P., (^) Exp. VII -057 -044 *032 *032 *031 (^) *031 -034 (^) * Exps. (^) VII and IX. (^) Exp. VIII in distilled water saturated with 02 at (^) 14.20 C. Exp. IX. in 02-free distilled water. Period (^) beginning at ... .5 1-0 2 4 10 20 ending at^ ...^ 1.0^2 0 4 10 20 Rate of H.-P., Exp. VIII *20 -075 -03 *015 *021 * Rate of H.-P., Exp. IX^ ... *12 *05^ *02 *016^ *016 * Here from Exp. I, we see that immediately after removal from 02 there is a much higher rate of H.-P. than (^) normally occurs in muscles 14 hours after death: it is^ only after removal^ from the 02 that^ the second
stage comes on, although in the absence of 02 it would have been
present ten hours previously. The oxygen in fact, which is known to prevent the liberation of lactic acid', and the onset of rigor2, and nearly to treble the rate of^ C02-output, keeps up the respiratory
oxidative activity of the surviving muscle to a high level, and even
after removal from the oxygen, the rate of H.-P. only falls slowly, i.e. in three or four houirs, to the normal rate for the second stage
of survival. With regard to the rest of Table III, it will be noticed
that the presence of oxygen increases^ very largely the heat-
production during the first few hours after death: but that in the later (^) stages there is little or no noticeable difference. (^) By the time the second stage comes on, the oxygen is^ practically exhausted however, as is shown by the following calculation. In the oxidation of carbohydrate 1 c.c. of 02 produces 5-4 calories: 150 c.c. of water at 160 C. holds some 48 c.c. of 02 after shaking with 02: this quantity of 02 is equivalent therefore to 26 calories. With 100 c.c. of frogs' muscle in 150 c.c. of water this yields an extra heat-production of *26 calorie per gram of muscle: in Table III, Exps. II to V, just these quantities of material were used, and a short calculation shows that in the period *3 hour to 5 0 hours the muscles in the oxygenated Ringer produced just about -30 calorie per c.c. of muscle, more than the muscles in 02-free Ringer. The equivalence between this -30 and the *26 is striking. (^1) Fletcher and Hopkins. Op. cit. p. 284.
2 Fletcher. This Journal, xxviii. p. 479. 1902.
HEAT-PRODUCTION OF MUSCLES.
bicarbonate of the tissues to form sodium lactate and (^) CO2: so that CO is liberated at a rate proportional to the production of lactic acid. According to this scheme we have, NaHCO3 + HL -.^ NaL + H20 + C02, so that one molecule of lactic acid liberates one molecule of CO2. Now according to Fletcher and Hopkins1, there is a possible production of
lactic acid up to about *27 grm. 0/0 (36 0/0 Zn-lactate) in frogs' muscles
duringr the months of March, April and May. The muscles in the experiments which I have carried ouit at 160 C. have usually, been inexcitable in 36 hours after death: so that there must have been a
lactic-acid production of about -000075 gr. per gr. of muscle per houir.
This is equivalent in the above reaction to an amount *018 c.c. of CO per grm. of muscle per hour. In F 1 e t c her's original paper the weights of the frogs' legs used were not recorded. In order therefore to obtain a (^) comparison I have assumed an average weight, viz. 12 gr. for each
pair of legs. This nuimber cannot be far wrong, and with it the
following results were obtained.
TABLE IV. After Fletcher. CO2-liberation in c.c. per gr. of (^) frogs' legs per hour, during the periods named. Exp. I. Fletcher, This Journal, xxiii.^ P. 98. 1898. At 150 C. Period (^) beginning at... 5 11 4 7 9-2 11 27 ending at^ ... 1.0^ 1-7 5-2^ 7-6^ 10-3 23 27- Rate of CO2 liberation -043 030 *022 *028 *027 (^) -020 *017! Exp. 54. Fletcher, Ibid. p. 99. In (^) nitrogen at (^) 15-17° C. Period (^) beginning at ... .5 1P8 3-8 53 11. ending at ... *8 2-2^ 4.3^ 5-8^ ^11- Rate of (^) C02-liberation -037 (^022) *018 *013 -
It is seen that the production of CO2 in the later^ stages is^ strikingly
equivalent to the amount^ of lactic acid that must have been formed.
Where, from^ above, we^ should^ expect *018 c.c,^ of^ CO, per gr. per hour
we find 013 to *020. On^ the other^ hand,^ if^ we^ suppose the^ CO2^ ,in^ the
later stages to originate from oxidative^ processes, then^ supposing the
oxidation to be that of carbohydrate the CO2 formed must be calculable
directly from the heat-production. From the^ average heat-productiou
at 15.50 C.^ calculated^ above2,^ assuming that^ the^ oxidation^ of^ 1.gr.
carbohydrate gives 4000 calories and therefore that 1 calorie^ -185^ c.a.
Of C02, we^ can^ obtain the^ following table.^ The^ CO2^ is^ reckoned^ per
C.C.3 of animal per hour, at^ 15.50 C.
481
1 Op. cit. p. 266.^2 P. 474. 3 At nGrmal temp. and pressure.
A. V. HILL.
Period (^) beginning at ... 3 *5 10 2 3 5 10 20 30 40 endingat ... 5 1-0 2-0 3 5 10 20 30 40 50 Rate of C02-production 057 *026 *020 *0144 *012 -0093 *0083 *0085 *0091 * It is seen that the numbers obtained in the later stages are decidedly smaller than those observed by Fletcher: this is even more definitely the case, because undoubtedly the lactic-acid formation liberates a considerable quantity of heat (see the next part of^ this^ paper, on^ the heat-formation in rigor) and this must be subtracted from the observed heat-production if^ we are to reckon^ how much heat^ is^ due^ to^ the supposed oxidative processes. Probably we ought to reduce the quantity of (^) heat to at least a half (^) by this (^) subtraction, so that the above values
of the CO2 calculated from the heat are also to be reduced by a half
This makes the numbers so small that under^ no^ conditions^ can^ they be
supposed to correspond to Fletcher's observations. The CO2 in fact,
in the "^ plateau "^ stage, is not due mainly to oxidative processes, for there is too little heat to correspond to such oxidations.
The equivalence observed between the lactic acid and CO2 formations,
in conjunction with many other facts, gives us very good grounds for
believing that the chief part of^ the CO2 comes from the reaction^ between
lactic acid and the sodium bicarbonate of the tissuies. On the other
hand, during the first hour or two, there is a very close coincidence between Fletcber's CO2 observations and the values calculated from
the heat, on the supposition that both heat and CO2 are due to the
oxidation of carbohydrate. This further strengthens the view that the
first stage is characterised by still surviving oxidative processes. In further confirmation of these conclusions is the fact that Fletcher found' that in the first stage of the CO output about a fifth part of it
can be abolished by the remo:val of oxygen from the atmosphere, and
this "is^ probably due^ to a^ respiration of^ the muscle substance con- tinuing that of normal life, but disappearing gradually as the changes occur which^ accompany loss^ of^ irritability and inaugurate rigor." Even
without a contemporary supply of oxygen from outside the earlier
processes in^ survival life^ are^ oxidations: the later are, as Fletcher suggested, the same as lead finally to the phenomena of rigor. Further evidence for this view^ is given in the next part of
this papelr In any case one thing is certain: the known liberation of
lactic acid in survival processes not^ only may^ but^ must drive off CO
from its combination with sodium: and the amount of CO2 so driven
off must in any case be a large fraction of^ the^ total CO2-evolution. In
(^1) This Journal, xxviii. p. 355. (^) 1902.
482
A. V. HILL.
of work the methods are of very doubtful validity: certainly however
they established the bare^ fact^ that there is^ an^ evolution of heat duiring
the onset of rigor. Fick and Dybkowsky first plunged a thermometer into a collection of^ muscles, which they then warmed very slowly on a water-bath. As the temperature rose the muscles became stiff, and after awhile were at a higher temperature than the bath. This proved that there is a liberation of heat; but the absence of any heat- insulating apparatus made them lose about 97 0/0 of the heat produced, so that their. estimations are many times too small. Later they used a therinopile, one set of junctions of which wvas covered with a fresh
muscle, the other with one already stiff. This they warmed in an
incubator, and during the moments when the muscle began to go stiff always found a positive deflection of the galvanometer. According to
their observations, the heat-production occurred only during the actual
contraction and^ stiffening at the onset of^ rigor: this^ is not in accordance with my experiments, which have shown that the gradual production of lactic acid leading finally to rigor, is responsible for the evolution of heat, and I^ believe^ the^ explanation of^ the difference^ to^ be that^ it was impossible by their method to warm the muscles uniformly
throughout. The^ movements^ of^ the muscle^ during shortening brouight
the thermopile junctions into closer contact with warmer parts of the
muscle, and hence the only observation made was made during the
shortening. The numbers they obtained by the first method for the
rise of^ temperature were:^ (a) frogs'^ muscles: not greater than .030 C.,
(b) rabbits' muscles: .23°C., results which are very much too small.
Schiffer, working more carefully on^ rnuch^ the^ same^ lines, found^ for
the stiffening of fish-muscles a rise of temperature of 1' C., which he
compared with^ the rise^ of^ temperature of^ 0.50 C.^ in^ clotting blood.
With the fish he found that the heat was produced before the stiffening
began: with the^ mammal that there^ was^ a^ contintious evolution of heat
during the^ whole^ period until. stifftness^ camne on.^ In^ opposition to^ Fick
and Dybkowsky he concluded that the heat-production is^ not due to
the change in^ the condition of aggregation of the proteins, accompanying
rigor, but to the chemical processes. preceding it. This conclusion, as
a Miatter of fact, is^ exactly- the^ same^ as^ the^ one^ to^ which^ we^ are^ led^ by
.the experimeintal. evidence. given below: we know more definitely now
what those chemical^ processes are. Burridge and Scottl have pointed out^ that in^ heat-rigor at 370C. two stages of contraction occur, one^ half^ of^ the^ total^ shortening occurring rather^ rapidly at^ first, and (^1) Proc. Physiol. Soc. Feb. (^) 17, 1912. Thia (^) Journal, xmIV. (^) p. iii. 1912.
484
HEAT-PRODUCTION OF MUSCLES.
the other half more (^) slowly and beginning only after the first stage isi almost complete. At 420 C. (^) thes9 stages are fused together^ and^ only^ one^ contraction^ is^ seen.^ This^ I^ have been able to^ confirm.^ At lower^ temperatures^ su6h^ as 360 C. the^ onset^ of^ heat-rigor^ is^ very slow and regular: the shortening may not be complete for many minutes. On the other hand at higher temperatures such as 420 C. and over, the shortening ensues and the rigor is complete almost immediately on raising the temperature of the muscle. Thus at 360 C., the shortening does not occur suddenly after several minutes exposure^ to^ the temperature, but continues from the very beginning. The same seems to be true of the evolution of heat, both in heat- and chloroform-rigor. The gradual^ shortening^ is^ presumably^ 4ue^ to the action of the chemical substances (lactic acid especially) which, at^ lower temperatures, are liberated gradually in the muscle: and the heat-production^ arises^ from the^ reactiori liberating these bodies.
The method I have adopted for estimating the heat produced^ in heat-rigor is again that of the micro-calorimeter. Hot ivater is poured into one flask with its temperature so^ adjusted that^ when^ a^ measured quantity of-cold^ muscles^ is^ thrown^ into^ it,^ the^ final^ temperature^ shall be approximately the same as that of the other^ hot^ water^ in^ the^ ordinary control-flask. The miiuscles are prepared and their.^ temperature taken,^ Water^ at about 360 C. is^ poured into^ the control^ flask,^ hot^ water^ adjusted^ to^ the calculated temperature is poured into the^ experimental flask, and then when all is ready the muscles^ are^ thrown^ suddenly^ into the hot^ water: the experimental flask is then stirred vigorously for about^ two^ mitlutes, until the whole of^ its^ contents^ are^ at^ one^ uniform^ temperature
throughout. Readings of the rise of temperature are^ then^ made^ with
the thermocouple anid galvanometer as before. The temperature is
usually adjusted to be the very lowest possible temperature which^ will cause the complete development of^ rigor within about^ one^ hour;^ for thereby the processes leading to rigor occut slowly, and^ no^ great
error is^ caused^ by the^ impossibility of^ making readings^ in the^ first
tbree or -four minutes.. It is in fact absolitely impossible to obtain reliable readings within^ the^ first few^ minutes:^ the^ muscles,^ however, well stirred, require some little time in order to settle down uniformly -to the same- temperature as^ the^ water^ and the walls of the flask.' As
mixing goes on, the readings decrease rapidly at^ first, owing to^ cooling
of the hot water by the^ cold^ museles:^ when^ however^ there^ is a^ uniform
temperature throughout the flask the readings increase slowly, because
of the gradual liberation^ of^ heat^ by the^ muscles.^ I^ have^ generally
aimed at securing a temperature- of about^ 35.50^ C.^ in the^ experimental
flask, in which^ case^ rigor is^ usually complete in from^40 to^50 minutes.
If-the temperatiure is^ a^ degree^ or^ so^ higher^ there^ is^ a^ considerable^ error
due to the unknown quantity of^ heat liberated^ in^ the first few^ .miw,es;
485
