University College, London.)

62.45:577.74.5 THE REPLACEMENT OF DEPLETED ADRENALNE N THE SUPRARENALS. BY G. P. CROWDEN. (From the Department of Physiology and Biochemistry, University College, London.) THE physiological control of the discharge of adrenaline from the suprarenal glands of cats was shown by Elliott(l) to depend on the integrity of the splanchnic nerves. The question whether or not the production of adrenaline in the glands also depends on nervous influences was left undecided, although Elliott tentatively observed that "anabolism seems to proceed without central control" in view of the fact that, following the section of the splanchnic nerves on one side, the denervated gland was invariably found to contain as much, if not more, adrenaline than its fellow. The following research has been carried out in order to determine if nervous control is essential for the production of adrenaline. t was necessary to possess some controllable means of producing, in the first place, a definite degree of exhaustion of the store of adrenaline in the glands from which the subsequent production during recovery could be assessed. Attempts were made to utilize the methods shown by Elliott to produce exhaustion of the glands, namely, the fright caused by morphia or P-tetra hydronaphthylamine hydrochloride, but these methods had to be discarded owing to the variability in the reaction of different animals to these drugs (2). t should be stated, however, that in several recovery experiments following exhaustion by these methods indications were obtained that adrenaline production can take place in the denervated glands. Finally the influence of lowered body temperature was tried and found satisfactory. Several observers have placed on record physiological (3, 4) and histological (5) evidence for the discharge of adrenaline from the glands when the animal is subjected to cold. Preliminary experiments showed that provided the application of external cold is followed by a marked drop in the body temperature of the animal, then in the normally

314 G. P. CROWDEN. innervated gland the adrenaline store is depleted (6). Cold was therefore adopted as a means of producing exhaustion of the glands. Experimental method. The problem has been approached by the direct method of determining the relative total adrenaline content of the glands under controlled conditions for producing exhaustion and for permitting recovery in innervated and denervated glands. Since Elliott(1) proved that the adrenaline content of the two glands remains equal after section of the splanchnic nerves on one side, provided the animal is quiet and undisturbed, it is possible to assess the degree of exhaustion, or of recovery following exhaustion, if the relative quantities of adrenaline can be estimated accurately. The colorimetric method of Folin, Cannon and Denis(7) was employed for this estimation. Preliminary tests showed that very great care must be taken to ensure that in every detail of time, temperature and manipulation, the glands to be compared are treated in the same manner. The work of Baker and Marrian (8) showed that the rate at which the colour develops, after the " uric acid reagent " and saturated solution of sodium carbonate have been added to the extract of the gland, depends on the temperature, and that for a temperature of 2 C. the maximum colour develops in 2 minutes. Estimation of the adrenaline content of glands ( 1-3 g. weight). At the termination of the experimental period the animal was killed quickly by chloroform and coal gas. The glands were immediately removed, weighed and the adrenaline estimated simultaneously in each gland according to the following directions: 1. Place glands with equal quantities of silver sand and 1 c.c. N/1 HC in two 3-inch evaporating dishes, grind thoroughly for three minutes, then add 3 c.c. N/1 HC, grind a few seconds, wash pestle with 5 c.c. distilled water and transfer with further 5 c.c. of water to a small Erlenmeyer flask. 2. Boil 3 minutes over equal gas flames, then add 1 c.c. 1 p.c. sodium acetate and boil j minute; cool quickly under tap. 3. Filter through Biichner, using 7 c.c. H2 for washing. Transfer filtrate to 5 c.c. graduated flask, wash in with 3 c.c. H2. 4. Add 2 c.c. "uric acid reagent," 1 c.c. saturated sodium carbonate and make up to 5 c.c. with water. 5. Place in water bath at 2 C. for 2 minutes and then compare in Dubosc colorimeter for the relative total amounts of adrenaline in the glands. For determination of the actual amounts of adrenaline, a parallel

ADRENALNE REPLACEMENT. colorimetric estimation was simultaneously carried out with a standard solution of uric acid. The accuracy of this method was checked with a standard solution of pure adrenaline hydrochloride (British Drug Houses, Ltd.). The error due to the presence of blood was found to be negligible. Exhaustion of adrenaline store by cold. Experiments were carried out to ascertain the conditions under which cold depletes the adrenaline store in the glands and to provide data for a curve of progressive exhaustion. The animals (male cats) were kept in a warm animal house, and after each animal had settled down to cage life operative section of the splanchnic nerves and the nerves to the semilunar ganglion on the left side was performed. The animals were then allowed to recover undisturbed for a few days at a room temperature of 65 F. in different cages separated by sacking and on a uniform diet of meat and milk. Under these conditions at the end of a day or two the denervated and innervated glands were almost equally loaded with adrenaline. This fact, already observed by Elliott(l), was confirmed by the experiments shown in Table. TABLE. Relative content of adrenaline in suprarenal glands. Condition Relative content of glands A Right Left Normal animal 1 18,, 1 1,, 1 14,,9 1 97 Left splanchnics cut 12 days 1 18 Left splanchnics cut, animal killed after operation 1 98-5 Left splanchnics cut by ligature 1 97.5 315; This and subsequent tables do not give the absolute amounts of adrenaline present in the right and left glands, but show their relative contents in terms of the content of the gland on the side taken as normal which is expressed as 1 p.c. There is a slight variation in the relative equality of content of the glands, but for the purposes of this investigation it is sufficiently accurate to assume that the glands start with an equal content of adrenaline. On the day of a cold experiment the animal was not fed. After taking its deep rectal temperature it was transferred to a room at C. The fur and skin up to the neck were wetted and the animal was then left undisturbed except when its rectal temperature was taken. After the required interval the animal was killed quickly and the adrenaline

316 G. P. CROWDEN. content of the glands compared. Table gives the results of this series of experiments. TABLE L. Exhaustion of suprarenal glands by cold. Total Average fall in intensity of Relative content Duration of tempera- Degree internal of glands extemal cold ture (deep hr. cold. r_ rectal) internal Degree hr. De- A- n- Exp. hr. min. C. cold per hr. nervated nervated 1 4 35 5x3 19 4-1 1 71-2 4 15 4x5 14 3-29 1 65-5 3 1 3 12-9-5 6-3 1 85-5 4 6 1 5-13 2-1 1 82-5 5 4 6-12-5 2-2 1 62-5 6 8 1-5 -5 Nil 1 11-7 7 3 1-4 2- -26 1 115- As regards "degree hours internal cold," a degree hour (1 hr.) may be defined as a difference in temperature from the initial normal temperature of 1 C. persisting for a period of 1 hour. f the rectal temperature of an animal were found to remain 2 C. below its normal temperature for a period of 1 hour the cold stimulus to which it had been subjected would be, according to this definition, 2 hours internal cold. A continuous record of body temperature was not made, but readings of the deep rectal temperature were taken at intervals of approximately 1 hour while the animals were exposed to cold. From the data thus obtained the temperature charts shown in Figs. 1 and 2 were constructed. The area between the temperature curve and the normal temperature base line in Figs. 1 and 2 gives the number of degree hours internal cold. The intensity of the cold stimulus is given in terms of degree hours per hour in Table. t will be observed that, although the number of hours exposure to external cold may be considerable, unless there is a resulting adequate cooling there is not a depletion of the adrenaline store of the innervated gland as compared with the denervated gland, the content of which is taken as 1. n animal 6 which had been fed in the morning in error, shivering caused the body temperature to rise slightly and apparently give the total chilling a small negative value in spite of the ultimate fall in temperature of.5 C. n animal 7 the temperature curve is also irregular, the final result being a slight fall representing 2 hours internal cold at an intensity of.26 hours per hour. n the normal animal there must be a balance which is converted to a depletion by conditions which cause an excess of discharge of adrenaline over production. On the other hand, it is possible that a weak stimulus,

ADRENALNE REPLACEMENT. 317 of a kind which if intensified would lead to such depletion of adrenaline, may in itself produce a preliminary production equal to or in excess of 34 w tr 36 Q- c) F 38 a 4L m 1 _ nternal i Cold _J 19'hrs. M J External ot4 4hr35m4 2 4 6 TME N 34 F 36 : UJ 38 4 2 26 28 n-ernaz Cold, 14 hrs j a: _ 3 Ex.CoZd 4hrs.15rn w 2 4. 32 34 ' a _ 36 a Lu a J,f5nternal 1, L ~~~~~~cold,, 38 ( 13 hrs,es. CodT6,'r. loin.~ 4 ' 2 4 6 Fig. 1. Temperature charts of animals 1, 2, 3, 4, 5, during exhaustion of suprarenal glands by cold. n all cases male cats were used. A degree hour, 1 hr., = a difference in temperature of 1 C. from normal persisting for a period of 1 hour. The degree of internal cold is expressed in these units. Temperature = deep rectal temperature. discharge and so account for the fact that in experiments Nos. 6 and 7 there was no depletion of the adrenaline store of the innervated glands. Cramer's observations(5) of the histological changes in the suprarenals of the mouse when subjected to cold support this view. On plotting the degree of exhaustion of the innervated gland against the duration of the external cold stimulus, no curve of progressive exhaustion is obtained owing to the fact that animals 6 and 7 showed no depletion in spite of considerable extemal cold. A similar result is obtained when the degree of exhaustion is plotted against the total fall in body temperature. The rapid drop in temperature of 12 C. in 1l hours in the case of animal 3 did not result in a proportionate degree of

318 G. P. CROWDEN. v 36-6 Q 38 nternal Cold O hr PP3 m 39 External ol6hrlo m-ns. 2 4 6 8 Time in hours 36r 38 7 ntern,al Cold Z hr External Cold 7hr 3min. 4 2 4 6 8 Time in hours Fig. 2. Temperature charts of animals Nos. 6, 7. 12% 7 6 @ 8% - 4f ~~ ~ ~ ~ ~ 1 Cs 6% o @ 4% 2% 4hr 8ohr. 12hr 16hr: 2hr. 24hr 28hr. titernal cold in degree hours Fig. 3. Chart showing progressive exhaustion of adrenaline store of innervated glands by internal cold. The numbered points indicate the content of the innervated glands of animals 1-7 expressed as a percentage of the content of the denervated glands. The four unnumbered points indicate the relative content of the glands of control animals which were not subjected to cold.

ADRENALNE REPLACEMENT. 319 exhaustion of the adrenaline store. When, however, the degree of exhaustion is plotted against the number of degree hours internal cold with which the bodies of the animals had attempted to cope, these aberrant points fall approximately into line (Fig. 3), because the figures expressing degree hours internal cold take into account the actual loss of heat regulation reached. t is evident therefore that it is internal cold, probably within certain limiting intensities, i.e. the lowering of the temperature of the body, which acts as the stimulus producing an excess of discharge of adrenaline over production. n Fig. 3 the slope of the dotted line roughly indicates the average increasing exhaustion of the glands as the internal cold stimulus increases. The general result is clear, namely, that with an internal cold stimulus of 12 or more degree hours, the body temperature not falling too rapidly or too low, there will be a depletion of the adrenaline store of the normally innervated gland representing some 2 or 3 p.c. of the content of the denervated gland. This enables the recovery production of adrenaline to be assessed. Recovery of innervated glands after exhaustion. The following experiments were devised for the investigation of the subsequent recovery production of adrenaline in glands which were allowed to remain normally innervated after exhaustion by cold. The experimental animals, which had had their left suprarenals denervated some days previously, were subjected to the effect of cold such as had produced exhaustion in the experiments described above. After being exposed to cold the animals were returned to the warm animal house, rubbed down with warm towels, placed near a hot water bottle and given warm milk to drink. Under these conditions they rapidly regained their normal temperature and TABLE. Recovery of innervated glands after exhaustion. Experiment No.......... 8 9 1 Duration of external cold 6 hr. 55 min. 7 hr. 1 min. 6 hr. Fall in body temperature (deep rectal) 12.1 C. 4.6 C. 3.8 C. Degree hours internal cold 33.5 13.5 13 Average intensity of internal cold. 4-8 1-88 2'16 Deg. hr. per hr. Estimated relative content of glands after exhaustion: Denervated 1 1 1 nnervated 32 73 74 nnervated recovery period 18 hr. 25 min. 69 hr. 71 hr. Relative content of glands after recovery: Denervated 1 1 1 nnervated 79 19.5 97.5 Note on recovery ncomplete Complete Almost complete

32 G. P. CROWDEN. behaviour. Then after varying periods of time the animals were killed quickly and the relative content of adrenaline in their glands estimated. Table gives the results of the innervated recovery experiments. t will be observed that 18 hours under the conditions of the experiment No. 8 were insufficient for the innervated gland to replenish its normal store of adrenaline. n the subsequent experiments the recovery period was increased to approximately 3 days in order to give ample opportunity for full recovery. t is probable that animal 9 had replenished its adrenaline store before 69 hours had elapsed. Recovery of denervated glands after exhaustion. n the case of the denervated recovery experiments animals in which the left glands had been denervated some days previously were exposed to cold, then quickly put under an anesthetic and the nerves to the right gland cut. They were then returned to the warm animal house and treated as in the case of the innervated recovery experiments. A period of recovery of approximately 3 days was allowed, after which the routine estimation of the relative content of adrenaline in the glands was carried out. The data for these experiments are given in Table V. TABLE V. Recovery of denervated glands after exhaustion. Experiment No....... 11 12 13 Duration of external cold 5 hr. 35 mi. 7 hr. 1 min. 6 hr. Fall in body temperature (deep 4.5 C. 7.8 C. 9.8 C. rectal) Degree hours internal cold 12-5 23 21-5 Average intensity of internal 2-24 3-48 3*58 cold. Deg. hr. per hr. Estimated relative content of glands after exhaustion: Denervated left 1 1 1 nnervated right 75 54 58 Denervated recovery period 71 hr. 68 hr. 3 min. 69 hr. 15 min. Relative content of glands after recovery: Denervated left 1 1 1 Denervated right 1 18-5 93 Note on recovery Complete Complete Almost complete Experiment No. 9 in Table and 12 in Table V and similarly 1 and 13 are two pairs of parallel experiments in which the animals in each pair were subjected to identical cold conditions at the same time. There is considerable variation in the degree of resistance offered by different animals to the influence of external cold, and therefore in order to make the test as conclusive as possible the animals selected for the denervated recovery experiments (Nos. 12 and 13) were the ones which had developed the greatest degree of chilling. The temperature charts of these four

ADRENALNE REPLACEMENT. 321 animals and the graphical representation of the exhaustion and recovery of their glands are shown in Figs. 4 and 5. On comparing the figures c3 c' ~ 341 9 361 38 _ xterna -nternal 13-5hrs. Cold g Cok'1,7)7hrJ1m. 4 u a 3r 321 341 14 r 2 4 6 8 35-1 37 - nternal Coldl EH 39 13hrs, External Cold 6hr. 41. 2-4 6 7 29-3 - 32-34 13 ~ PS 36 38 4U' External Cold 2 4 ternal Cold 23 hrs a 7hr 1m.O 6 8 E- 36 /nternal, Exl;ernaz ColdZ 6hrv. 4 2 4 6 7 Fig. 4. Temperature charts of animals Nos. 9, 1, 12, 13, during exhaustion of innervated glands by cold. n Nos. 9 and 1 the innervated glands were allowed to recover after exhaustion, n Nos. 12 and 13 the innervated glands were denervated after exhaustion and then allowed to recover. Exp. Nos. 9 and 12, and similarly Nos. 1 and 13, were carried out under identical conditions. for the relative content of adrenaline in the glands after innervated and denervated recovery in these four experiments, it is evident that there is no significant difference in the final results, namely, that in each case replacement of the depleted adrenaline store of the glands has occurred. t is therefore held that these experiments prove that the production of adrenaline can take place after denervation of the suprarenal glands.

322 G. P. CROWDEN. 2 vd 171 87% 6% 2% k9,12 xge 12t TME OF DENERVATON _ 9 External Cold 7hr _O.i. Ai J 8 1 6 24 32 4 48 56 64 72 8 Time in hours 1% 1,13 cd 8% \ 1 ir.ated Grl_t j9ecoveryv.. 7,d _ - - 13 6% g 4% T ME OF DENERVATON V 2% Fig. 5. Excterno Co 6hr&. 1 8 24 3 4 4 8 1 6 24 32 4 48 56 64 72 8 Time in hours Chart showing exhaustion of adrenaline store of innervated glands of animals 9, 1, 12, 13, and subsequent recovery production of adrenaline. No. 9. Exhaustion by 13.5 hr. internal cold in 7 hr. 1 min. followed by recovery for 69 hr. No. 12. Exhaustion by 23 hr. internal cold in 7 hr. 1 min. followed by denervation and then recovery for 68 hr. 3 mi. No. 1. 71 hr. Exhaustion by 13 hr. internal cold in 6 hr. followed by recovery for No. 13. Exhaustion by 21.5 hr. internal cold in 6 hr. followed by denervation and then recovery for 69 hr. 15 mi.

ADRENALNE REPLACEMENT. 323 SUMMARY. 1. The application of external cold is a controllable method of producing exhaustion of the adrenaline store of the suprarenal glands of cats, provided a marked fall in body temperature occurs and persists over a sufficient period of time. The effective stimulus is estimated in terms of degree hours internal cold produced. 2. The replacement of adrenaline in innervated and in denervated glands following partial exhaustion of the glands by cold has been investigated and the conclusion is drawn that the replacement of adrenaline may proceed without nervous control. My thanks are due to Dr G. V. Anrep for drawing my attention to this problem, to Prof. T. R. Elliott and Prof. C. Lovatt Evans for their interest in the work, to Dr M. G. Pearson for valuable assistance with the experiments and to the British Medical Association and the Medical Research Council for grants which enabled the work to be carried out at University College, London. REFERENCES. 1. Elliott. This Journ. 44. p. 374. 1912. 2. Crowden and Pearson. Proc. Physiol. Soc. June 23, 1928. bid. 65. 3. Hartman, McCordock and Loder. Amer. Journ. Physiol. 64. p. 1. 1923. Hartman and Hartman. bid. 65. p. 612. 1923. 4. Cannon and Querido. Proc. Nat. Acad. Sci. 1. p. 245. 1924. 5. Cramer. Vth Rep. mp. Cancer Res. Fund. 1919. 6. Crowden and Pearson. Proc. Physiol. Soc. May 19, 1928. This Journ. 65. 7. Folin, Cannon and Denis. Journ. Biol. Chem. 13. p. 477. 1912-13. 8. BakerandMarrian. Biochem. Jourin. 21. p. 15. 1927.