341

J. Physiol. (I943) I02, 34I-356 6I2. OI8

THE OUTPUT OF CORTICAL HORMONE BYTHE MAMMALIAN SUPRARENAL

BY MARTHE VOGT, From the Pharmacological Laboratory,the College of the Pharmaceutical Society, University of London

(Received 19 June 1943)

Substitution therapy in suprarenal insufficiency is based on the beneficialeffects observed, in man or in animals, with extracts of cortical tissue or withsynthetic compounds, and not on any knowledge of the physiological activityof the suprarenal cortex. No data are available on the amount of corticalhormone present in the blood, and information is lacking on the rate of secre-tion by the suprarenal cortex and its possible variations under differentconditions. It is the object of the present paper to supply such information.It was obtained by collecting the blood from the suprarenal veins of differentmammals and assaying its cortical activity. The experiments are restrictedto anaesthetized animals subjected to a severe abdominal operation.

METHODS(1) Assay of cortical hormone

The method used for the assay of cortical hormone is that devised by Selye &Schenker [1938]. For the present purpose it has three essential merits: first,it is more sensitive than most other methods; secondly, it does not requireweeks of observation of the test animals and, cronsequently, deterioration ofthe solutions during the period of assay need not be feared; thirdly, it is notspecific for any particular fraction of the cortical steroids and will, therefore,give a better survey of suprarenal activity than a method which is particularlysensitive to, for example, steroids affecting mostly cell permeability or com-pounds acting mainly on carbohydrate metabolism. Employing this naethod,Kendall [1941] found comparable potencies for corticosterone, compound Eand the 'amorphous fraction', and Zarrow [1942] demonstrated the efficacy ofdesoxycorticosterone acetate, provided it was administered some hours beforethe exposure, thus allowing time for absorption.The principle of the method had been suggested by Hartman, Brownell &

Crosby [1931]. Young suprarenalectomized rats are submitted to stress from22-2

cold environment. Selye & Schenker expose the animals to temperatures of+ 2-4° C. on the day after the operation. The rats are divided into groups andgiven different amounts of cortical extract hypodermically during the firsthours of exposure. The survival time is observed and seen to increase with thequantity of extract administered.

Details of the method, as I have used it, are as follows: The groups weremade up of nine to ten rats of as nearly as possible the same age and weight.As the survival time of the rats increases with weight and decreases, withincertain limits, with the age at which a given weight is attained, the groups hadto be matched carefully with regard to these two factors. Litter mates wereevenly distributed among the groups, and the mean weight of each group wasalways made equal. Although an effect of sex on sensitivity is not certain, thesame ratio of males to females was used throughout the groups.

It is not possible to use rats of different colonies in one assay. Vigorousstrains are more susceptible to cortical hormone than weak ones; the latterrequire very large quantities of extract to protect them. For one colony ofWistar rats, ages from 23 to 30 days and weights between 38 and 60 g. provedworkable, whereas for another strain (Wistar rats, Dr K. H. Coward's colony,reared under strictly constant dietary conditions) suitable figures were 19-23days and 38-45 g. The mean survival time for rats of this last colony wasabout double that of the first.Suprarenalectomy was carried out by the lumbar route from a midline

incision through the skin. Pentobarbitone combined with ether were used asanaesthetics. The rats were given a few drops of 25 % glucose solution soonafter the operation. They took food overnight, usually gained in weight, andwere lively and fit the next morning when the experiment was about to start.For the test, the rats were distributed into individual numbered cages early

in the morning, given their first injection and simultaneously placed in a largerefrigerator. At intervals of an hour and a half further injections were made,their total number being either four or five. The doses were injected as rapidlyas possible, all the cages being removed from and replaced into the refrigeratorsimultaneously. From the 5th or 6th hour after the beginning of exposure,a rapid inspection of the rats was carried out every half-hour, any deathsrecorded, and these observations continued till all or nearly all rats had died.

Calculation of the mean survival time was made for each group. Sometimes,however, when observations had not been continued till all rats in a group haddied, the percentage of deaths observed at the half-hourly intervals wasplotted against time on probability paper and the mean survival time foundgraphically.

Since the rats in each group were not distributed at random, but carefullyselected, so as to have in every group representatives of the same litter, ages,weights, and sexes, the test for 'significance' of the result was based on the

342 M. VOGT

OUTPUT OF CORTICAL HORMONEstandard deviation of the differences in survival times between correspondingpairs of rats in any two groups and not on the standard deviation of thesurvival times themselves. By dividing the standard deviation of the differ-ences by the root of the number of pairs, the standard error E of the meandifference D was obtained and the ratio D/E gave the usual measure for 'signi-ficance', the probability that a result was due to chance being determined bymeans of Fisher's table [1941].

Fig. 1 is a dose-response curve obtained by injecting three groups of tenrats with increasing doses of a cortical extract ('eucortone', Allen and Han-burys). Each rat had five injections containing 0-012 ml. eucortone in group 1,0-03 ml. in group 2, and 0*075 ml. in group 3. In the graph, the logarithms of

hr.18

17

16

15

14

log dose 2-2 24- 26 28 T.oFig. 1. Dose-response curve. Ordinate: mean survival time (hours) of groups of ten rats.

Abscissa: log dose (ml. cortical extract per rat and injection).

these doses are plotted as abscissae against the mean survival times (13.75,15*67 and 17-81 hr.) of the three groups as ordinates. As shown in the figure,the three points lie in a straight line, and indicate that the response is pro-portional to the logarithm of the dose. This result is similar to that obtainedby Biilbring [1937] on the survival times of suprarenalectomized drakes. Acalculation of the standard errors of the differences in survival times as outlinedabove led to the following result: the probability that the mean difference insurvival time between groups is due to chance is 1: 20 for groups 1 and 2, and1:50 for groups 2 and 3. In other words, the differences obtained with thethree doses, though significant, are not sufficiently large to warrant furtherreduction of the differences between the doses. The doses had been so selectedas to increase by the factor 2-5, and the result of the calculation of the errorshows that smaller differences between doses would not have been detected

343

with certainty. This result was confirmed in several other experiments by theobservation that the increase in survival time obtained by doubling the basaldose was not significant, whereas that resulting from trebling it was. In thisrespect the method compares unfavourably with assays on drakes [Biilbring,1937], in which doses differing by 50 % are distinguishable. Its advantage isits high sensitivity; the total dose required per rat is about 1/25th of thatnecessary per drake. In Fig. 1, for instance, the smallest dose used for one ratis 5 x 0-012 or 0-06 ml. extract; the survival time with this dose was 5-75 hr.longer than with saline and the response, therefore, considerably above thres-hold.

If two groups of rats of the same age and average weight are taken from thesame colony and treated with the same dose of cortical extract or injected withthe same volume of saline in two consecutive experiments with, as far as onecan tell, identical environmental conditions, the mean survival time willnevertheless be different. Consequently, in each assay of cortical activity thesurvival time without treatment must be established for a group of rats in-jected with saline only, and the susceptibility to hormone by at least one groupgiven a cortical extract used as 'standard'. If quantitative assay of differencesbetween several unknown samples is desired, two groups of rats given twodifferent doses of 'standard' are required.

(2) Collection of suprarenal bloodCollection of the blood leaving the suprarenals was carried out by Verney's

modification of the method devised by Feldberg & Minz [1934] in their workon the acetylcholine content of the suprarenal effluent. On a heparinized dog,blood is drained from the left suprarenal gland. For this purpose, the branchesof the left lumbar vein are tied, a loose ligature is looped round its entry intothe vena cava, and a cannula introduced into the vessel about 1 cm. lateral tothe suprarenal gland. The cannula is connected by a short rubber tubing toone arm of a T-piece pushed through a small stab wound in the animal's leftflank. The stem of the T-piece is connected by tubing to a cannula introducedinto the left jugular vein. The loose ligature around the caval junction of thevein is tied, and blood may now either be drained from the second arm of theT-piece into a flask, if the tubing leading to the jugular vein is clamped, or beallowed to flow back into the animal, if the second arm of the T-piece isoccluded.

Modifications of this technique had to be applied if mammals other than dogswere used. For the cat, the colon was removed, aorta and cava were tied belowthe renal vessels, the renal pedicles were ligated and a cannula was introducedinto the cava, caudad to the adreno-lumbar veins, which were tied at thelateral edge of the gland. During the collection of suprarenal blood, the cavawas occluded above the suprarenals. The same operation was carried out in

344 M. VOG1:

OUTPUT OF CORTICAL HORMONEthe rabbit, but it was preceded by evisceration. The volume of the bloodsamples required for a test was about 30 ml. In order to obtain this quantityfrom an eviscerated rabbit, blood from another rabbit had to be infusedintravenously while the suprarenal sample was being collected.

In the pig and the goat the left renal vein receives most of the blood fromthe left suprarenal; accordingly, the left renal artery was tied, the renal veincannulated, its caval end ligated and the cannula connected to a T-piece, whichallowed the blood to return to the jugular vein in the goat and to the sub-clavian vein in the pig.Ether followed by chloralose intravenously were the anaesthetics used for

all the animals.The blood draining from the suprarenals was collected in centrifuge tubes

cooled in ice water. The red cells were separated off and the plasma was assayedby subcutaneous injection into groups of rats as described above. In mostexperiments the plasma was assayed 14 hr. after collection. Later, however,tests were frequently carried out 40 hr. after obtaining the blood, as the resultsseemed unaffected by that delay. As soon as the red cells had been centrifugedoff, all plasma samples were placed into a refrigerator where they remaineduntil use.

(3) Effect of adrenaline on the assay of cortical hormoneSome of the experiments were carried out on animals in which the splanchnics

were intact, and the blood samples were thus liable to contain adrenaline dueto reflex stimulation of the splanchnics by the operative procedures. It was,therefore, necessary to know whether the presence of adrenaline in the plasmainterfered with the assay of cortical hormone. Groups of rats were treated withsuitable amounts of eucortone and the survival times compared with those ofother groups given the same quantity of eucortone to which adrenaline hadbeen added up to a concentration of 1: 50,000. Similar comparisons were madebetween rats injected with saline and rats given adrenaline solutions 1: 50,000.Adrenaline did not significantly influence the survival times observed in therats. These results are in agreement with earlier observations by Hartman et al.[1931]. They render any correction for the adrenaline content of the plasmaunnecessary.

RESULTS(1) Presence and estimation of cortical hormone in suprarenal venous blood

Blood from the suprarenal veins was assayed for cortical hormone in a largenumber of dogs, several cats, two goats, one rabbit and one pig. A considerablecortical activity was invariably found, whereas power to prolong the life ofsuprarenalectomized rats was lacking in all control plasmas obtained from themain arteries of the body or the right side of the heart. The only exception wasa slight protection afforded to the rats by arterial blood of the cat (see p. 354).

345

The assay was uncomplicated in the dog and the cat as their plasma hadhardly any toxic effect on suprarenalectomized rats. Injections of plasma fromthe other animals, however, proved highly toxic for the rats, and the methodof estimation had to be modified accordingly. An example of two typicalexperiments, one on a dog and one on a goat, will be given first.

hr.

Group l 2 3 4 5Fig. 2. (Exp. 13, dog.) Ordinate: mean survival time (hours). Number of group at the bottom of

each column. The rats received per injection: group 1, 0-2 ml. suprarenal plasma; group 2,0*5 ml. NaCl; group 3, 0*5 ml. plasma from heart and femoral artery; group 4, 0.5 ml. 10 %eucortone in NaCl; group 5, 0.5 ml. 10 % eucortone in heart plasma.

Example of an assay in the dog. In the dog (Exp. 13, Table 1) chosen as an

example, the sample of suprarenal blood was collected while both splanchnicnerves were intact, the blood pressure being over 100 mm. Hg and the flow

through the left suprarenal about 0l2 ml. (0.1 ml. plasma) per min. (This isunusually small and is due to the small size of the dog-5 kg.) Of the plasmaso obtained, four injections of 0-2 ml. were given to a first group of rats; theirmean survival time was 10-7 hr. This time is represented in column 1 (Fig. 2);the other columns are the mean survival times obtained with other solutions

1OF

91-

7'- t

-

M. VOGT346

81

OUTPUT OF CORTICAL HORMONEon the same batch of rats. Thus, a control group treated with 0 9 % NaClsolution survived for 6-5 hr. (column 2). The difference of the two means isstatistically significant. Column 3 represents the effect of blood obtained fromother parts of the vascular system. The rats of that group were given fourinjections of05 ml. of plasma prepared from blood collected from the right sideof the heart and the femoral artery. The survival time hardly differs from thatof the saline-treated group. No detectable amount of hormone, therefore, ispresent in heart blood and arterial blood of this dog, although the quantity ofplasma tested was 24 times as large as that of the suprarenal sample.

In group 4, the potency of a commercial suprarenal extract ('eucortone',Allen and Hanburys) was tested by four injections of 0 05 ml., diluted 1:10with saline. If we compare the protection thus obtained (column 4) with thatafforded by 0-2 i1. suprarenal plasma, we find only an insignificant difference.We are not justified, however, in concluding from this comparison that 0-2 ml.plasma contain as much active principle as 0 05 ml. eucortone, without firstascertaining that non-specific constituents of the blood are not interfering withthe action of the eucortone. This was done in the following way: a fifth groupof rats was given the same amount of eucortone as group 4, dilution, however(1:10), being made with dog's heart plasma instead of saline. Column 5 showsthe survival time to be equal (within the errors of the method) to tiat ofgroup 4. Direct comparison is thus permissible of the activity of the supra-renal plasma with that of eucortone solution in saline.From the results of Fig. 2 we are able to express the output of cortical

hormone into the blood in terms of the extract 'eucortone'. In order to makethe lowest possible estimate of the glandular activity, we shall assume that allthe hormone is present in the plasma and none has entered, or been adsorbedto, the red cells. One ml. eucortone is the extract of 75 g. gland, but its potencyvaries according to the batch. The sample used in this experiment had onlytwo-thirds of the activity of the most potent batches encountered. For ourcalculations, 1 ml. eucortone will, therefore, be assumed to represent theactivity of 50 g. gland only. It follows that the potency of 1 ml. plasma corre-sponds to that of 12-5 g. gland. In this 5 kg. dog, the output per minute fromone suprarenal (0-1 ml. plasma) was equivalent to the activity contained in1-25 g. gland, and, per kg. body weight, to that contained in 0-25 g. gland.These values are obtained on the assumption that the potencies represented

by columns 1 and 4 or 5 are identical. In fact, column 1 probably representsa somewhat higher value than column 4, and the figures for the yield are likelyto be underestimates.

It was confirmed in many experiments that dog's plasma, taken from anypart of the body, has negligible toxic effects on suprarenalectomized rats. Weneed, therefore, not fear the interference of substances present in the blood whenestimating the content of cortical activity. Cat's plasma was similar to dog's

347

plasma in that respect, but it differed, in some instances, in affording a slightprotection to the rats. The effect was statistically significant but very slight.An example will be given later. Entirely different effects, however, wereobtained with plasma from the cow, the goat, the rabbit and the pig. This isillustrated by Fig. 3 (Exp. 23, Table 1, carried out on a young goat in a similarway as the experiment on the dog represented in Fig. 2).

Group 1 2 3 4 5Fig. 3. (Exp. 23, goat.) Ordinate: mean survival time (hours). Number of group at the bottom

of each column. The rats received per injection: group 1, 0-4 ml. suprarenal plasma; group 2,04 ml. NaCl; group 3, 0.4 ml. plasma from heart and femoral artery; group 4, 0 4 ml. 3.75 %eucortone in heart plasma; group 5, 0-4 ml. suprarenal plasma after splanchnotomy.

In Fig. 3, column 1 represents the survival time obtained with a sample ofsuprarenal plasma, the splanchnics being intact, the blood pressure 68 mm. Hgand the suprarenal flow 4*2 ml. per min. (2.1 ml. plasma). The next columngives the result of injecting saline instead of plasma. The survival time is thesame. The third column illustrates the effect when a mixture of arterial andheart plasma is given in the same quantity (0.4 ml. per injection) as suprarenalplasma: the survival time is much shorter than with saline. The difference

348 M. VOGT

OUTPUT OF CORTICAL HORMONE

between the two results is statistically significant. The fourth column repre-sents the survival time resulting from treatment with 3-75 % eucortone addedto the plasma used for group 3; the survival time is prolonged beyond that ofgroup 3 and reaches the figure observed in groups 1 and 2.The experiment demonstrates a strong toxicity of goat's plasma for supra-

renalectomized rats kept in the cold. In another similar experiment, evenquantities as small as 0*2 ml. per injection reduced the survival time of ratsconsiderably beneath that of saline injected controls. This toxicity is overcomeby adding cortical extract to the plasma. In the example given in the figure,the quantity of eucortone added was just sufficient to raise the survival timeto the level obtained with a first sample of suprarenal plasma, and we are thusenabled to state that this plasma has the same potency as a 3-75 % solutionof eucortone.From a calculation similar to that applied earlier on in the dog, it follows

that the left suprarenal of the goat which gave the results shown in Fig. 3produced, before splanchnotomy, a quantity of hormone corresponding to 6 g.gland per min., which is 0 4 g. gland per min. for each kg. of body weight. Asthe suprarenal weighed 0*55 g., the gland produced per minute 11 times theamount which can be obtained by its extraction.The activity of a second sample of plasma, collected in the same experiment

after section of the left splanchnic, is represented in column 5. The survivaltime is much longer than that in any other group, including the saline-treatedcontrol; this shows that a sufficiently high degree of cortical activity will notonly neutralize the toxicity of goat's blood but render the plasma highlybeneficial for suprarenalectomized rats.

i4 far as can be told from the small number of experiments carried out withplasmas from cows, pigs, rabbits and goats, their toxicity for suprarenalec-tomized rats is of the same order. We have just seen that the addition of3-75 % eucortone to a sample of goat's heart plasma neutralized its toxicproperties so that the mixture acted like saline. In another experiment, thesame amount of cow's plasma was made innocuous by addition of 3 %eucortone. Rats treated with this solution survived for 11 hr. and so dida control group injected with saline; a third group, however, which was given3 % eucortone diluted with saline instead of with plasma lived for 13-7 hr.(The difference between 11 and 13*7 hr. is statistically significant.) In a firsttest with pig's plasma, the addition of 8 % eucortone made the plasma justharmless, and in another test 10 % were far in excess for the purpose of'neutralizing' its toxic properties; a similar result was obtained in the rabbit.

It follows from these observations that, in assaying the cortical activity ofplasma with rabbits, pigs, goats and cows, it is not sufficient to compare thepotency of the sample with that of solutions of a cortical preparation in saline.Comparison has to be made with solutions of the known extract in plasma

349

350 M. VOGTobtained from any part of the body other than the suprarenals. Rats injectedwith that same plasma without the addition of cortical extract take the placeof the saline-treated controls in other experiments.

TABLE 1. Output of cortical hormone by the suprarenal gland of one side, per min. per kg. bodyweight. The output is expressed by the amount of suprarenal tissue ('g. gland') which yields,on extraction, the same activity

Output per mi.'g. gland' in terms of

No. of Animal per min. 'content' ofexp. used Conditions per kg. own gland10 Dog Splanchnics intact 02613 ,, ,, 0-315 ,, ,, 0*5329 ,, Splanchnics intact, eviscerated 0 75 17 times14 ,, Left splanchnic cut >04617 ,, ,, 0-3818 ,, ,, c. 05 c. 9 times20 ,, ,, 0-38 4 ,,34 ,, Left splanchnic cut, eviscerated >0-41 >4-5 ,,37 ,, ,, ,, 0 95 13 ,,39 ,, ,, ,, 2-0 29 ,,42 ,, ,, ,, -062 1046 ,, ,, ,, c.05 c. 1157 ,, Splanchnics cut, eviscerted 039 5-751 Cat* ct, ev s10* >-rte ,,55 ,, Splanchnics intact, eviscerated 0.75* 526 Pig Splanchnics intact >0 41 =5 ,,23 Goat ,, 040 1133 ,, Left splanchnic cut, eviscerated >048 > 1665 Rabbit* Splanchnics intact, eviscerated 2.0* 13

* Unlike the other figures, those for the cat and the rabbit represent the output not of one, butof both suprarenals.

The results obtained in these two experiments are no exceptions, as may beseen from Table 1, which summarizes all the experiments in which the activityof suprarenal plasma was compared with that of 'eucortone'. The amount ofhormone released from one suprarenal gland, per min. per kg. body weight,varies in different experiments but is of the same order in the five mammalstested. The yield ranges between 0.26 and 2-0 'g. gland'. In the last column,the suprarenal output is compared with the hormone 'content' of the animal'sown suprarenal, 'content', however, signifying the activity which can beextracted from the gland as distinct from its real content, which is unknown.In all instances in which it was possible to make this calculation, the minuteoutput was many times higher than the 'content' of the secreting gland. Inhalf of the experiments, the amount given off per minute was at least 10 timesas high as the 'content' of the animal's gland, and in one instance it was nearly30 times as high.

This considerable rate of hormone production also becomes apparent, ifcomparison is made between the concentration detected in plasma preparedfrom suprarenal blood, and the quantity obtainable by extraction of 1 g. glandtissue. This calculation has been carried out for experiments on dogs (Table 2).

OUTPUT OF CORTICAL HORMONE

TABLE 2

1 ml. suprarenal 1 ml. suprarenalplasma equivalent plasma equivalent

No. of exp. to 'g. gland' No. of exp. to 'g. gland'10 2-1 37 10 013 12*5 39 10015 5-0 42 6-517 3-8 46 8*320 2*2 52 6-229 10*0 57 4-8

It is obvious that 1 ml. of plasma always contained much more hormonethan can be obtained by extraction of 1 g. of glandular tissue. In one-third ofthe experiments the ratio was 10: 1 or greater.

(2) The effect of blood flow and blood pressure on the output of cortical hormoneIn the interpretation of results bearing on the question whether certain

conditions modify the output of cortical hormone, the limited accuracy of themethod has to be taken into account. Changes can, as a rule, only be ascer-tained if they are large, e.g. threefold increases or reduction to one-third of thebasal value. The effect of a particular factor can be tested by comparing theyield in several experiments differing from each other with regard to that factor.Such information, based on the foregoing experiments, will be described here.Experiments in which the output is measured in consecutive samples obtainedfrom the same animal under varying conditions will bereported in another paper.

(i) Blood flow. The weight of the suprarenals varied between 0 4 and 1l3 g.,their blood flow, however, between 0X2 and 8X2 c.c./min. The figures apply to onegland only, except in the case of the cat and the rabbit. In spite of this widerange of blood flows, no correlation was found between yield ofhormone and flowthrough the gland. In fact, when the blood flow changed considerably during thesame experiment, the concentration of cortical activity varied inversely withthe flow, the hormone production per minute remaining comparatively un-changed. An example was given in Fig. 3. Between the collection of the first(column 1) and the second (column 5) sample of suprarenal plasma, the bloodflow through the gland had fallen to one-third of its original value. The fallwas partly due to the fact that the left splanchnics had been severed after thecollection of the first sample. The considerable increase in hormone con-centration is apparent in the figure. It may well be an expression of anunchanged rate of hormone production and, therefore, of the independence ofhormone output on blood flow. This argument is, of course, only valid if sectionof the splanchnic in itself does not alter the cortical activity. Evidence fdrthat assumption will be published later.

(ii) Arterial blood pressure. During the collection of suprarenal blood samplesin different experiments, the mean ar,terial pressure varied from 25 to 132 mm.Hg. There was no correlation between blood pressure and amount of hormone

351

secreted per unit of time. In order to obtain a high concentration of activeprinciple, it is, therefore, best to work at a low arterial pressure and a corre-spondingly small blood flow.

(3) Fate of the cortical hormone in the organismThe fact that no cortical hormone could be detected in arterial blood, or in

blood from the right side of the heart, indicates that the body disposes at greatspeed of the released hormone. The gastro-intestinal tract, spleen and liver,are not essential for this disappearance, since the result is the same in theeviscerated animal. Even in blood samples which were obtained from theheart several hours after evisceration, cortical activity was absent. If the liverplayed a predominant role in the inactivation of the hormone, its exclusionfrom the circulation would lead to accumulation of cortical substance in thegeneral circulation. As no such accumulation was detected, and the hormoneis stable in blood, it must be either very rapidly inactivated by the tissues orvery speedily excreted by the kidney. In order to decide between the twoalternatives, the following experiment was devised: A female cat (2.4 kg.) wasanaesthetized with chloralose, and 25 ml. blood were drained from one of itscarotids into a tube containing a little heparin (sample 1). The blood losswas partially replaced by infusing intravenously 15 ml. heparinized bloodtaken from another chloralose cat which had been bled completely and thendiscarded. The animal was now eviscerated and both its ovaries and kidneyswere excluded from the circulation. Two and a half hours later, 10 mg. heparinwere given intravenously, a record of the blood pressure was started and asecond sample of 25 ml. blood collected from the carotid. The blood pressurefell from 85 to 6 mm. towards the end of the haemorrhage, but recovered com-pletely when the blood loss was replaced as soon as the sample had beenobtained. The aorta was tied distal to the renal arteries, the cava cannulatedat the level of the renal veins, the lumbar veins tied at the lateral edge of thesuprarenals, the cava ligated above the suprarenals, and the suprarenaleffluent drained from the cannulated cava. Twenty-five ml. blood (sample 3)were collected during the next 28 min. and the blood pressure was kept at anaverage of 62 mm. Hg by slow intravenous infusion of blood (diluted withsaline towards the end of the experiment).The three blood samples were assayed for cortical hormone (Fig. 4, columns

1-3), and their activity was compared with that of saline (column 5) and of a4 % eucortone solution (column 4). The survival times observed with the twocarotid blood samples are identical. Therefore, in the general circulation of theeviscerated, nephrectomized cat, no accumulation of cortical hormone hadtaken place.

The suprarenal sample, on the other hand, collected in a period of 28 min.,contained a considerable amount of hormone (column 3). Its potency was the

352 M. VOGT

OUTPUT OF CORTICAL HORMONEsame as that of a 4 % solution of eucortone. Assuming that 125 ml. are anadequate estimate of the blood volume of the eviscerated nephrectomized cat,we can infer from that figure and the blood flow through the glands (25 ml. in

hr.'AI4 U

13k

121-

II -

101-

9k

8k

U

Group 1 2 3 4 5Fig. 4. (Exp. 55, cat.) Ordinate: mean survival time (hours). Number of group at the bottom

of each column. The rats received at each injection: group 1, 03 ml. carotid plasma beforeevisceration; group 2, 03 ml. carotid plasma 2* hr. after evisceration and nephrectomy;group 3, 03 ml. suprarenal plasma; group 4, 0-3 ml. 4 % eucortone; group 5, 0.3 ml. 0*9 %NaCl.

353

28 min.), that the suprarenals produced in 2 hr. enough hormone to raise theconcentration in the general circulation to the figure found in the suprarenaleffluent, provided inactivation had not taken place in the body. As no accumu-lation whatever occurred, it follows that the substance is destroyed in thetissues and that renal excretion can only play a negligible role in its disposal.This result confirms, for the natural hormone, an observation by Pfiffner,Swingle & Vars [1934], who found only very small amounts of hormone in theurine of a dog injected subcutaneously with a massive dose of cortical extract.A comparison of the survival time of the saline-treated group (column 5)

with those obtained by injection of the two carotid blood samples (columns 1and 2), shows the slight protection occasionally observed with arterial bloodfrom the cat and mentioned on p. 345. The differences be'tween the survivaltimes, although significant, are so small that there is considerable doubtwhether they are due to traces of cortical hormone or to some other con-stituent of the plasma. As in this cat the ovaries had been tied off at the begin-ning of the operation, it is unlikely that progesterone was responsible for theprotection. Progesterone could be ruled out entirely by another experiment,in which the same phenomenon occurred in a male cat.

DISCUSSION

The foregoing experiments have shown that there is invariably present, inthe venous blood leaving the suprarenals, an amount of cortical hormonedetectable by biological assay. Its quantity is surprisingly high. The averageyield obtained per min. per kg. body weight, by the suprarenal of one side,is equivalent to the amount which can be extracted, by methods used commer-cially, from 0-6 g. glandular tissue. For a dog of 10 kg. this would represent adaily production by both glands of the equivalent of 17,300 g. suprarenal tissuecontained in 230 ml. of a commercial extract.The discrepancy between the amount of hormone obtainable by chemical

treatment of glandular tissue and the quantity present in the venous blooddrained from the gland can be interpreted in several ways; either the methodsof extraction are very inadequate, or the steroids are not stored in the glandin their potent form but changed into the active principle (or principles) ontheir release into the circulation. It is not possible at the moment to decidebetween the two alternatives. If storage of hormone in the cortical tissue isindeed negligible, this fact would distinguish the suprarenal cortex from mostother endocrine glands, and particularly from the suprarenal medulla.Another characteristic feature. of cortical activity is the apparently con-

tinuous release of the hormone into the circulation; this again is in strikingcontrast with the rhythmic function of many endocrine glands, the action ofwhich is initiated either by nervous or by chemical stimuli. This observationof permanent activity is, however, so far restricted to the special conditions

354 M. VOGT

OUTPUT OF CORTICAL HORMONE(anaesthesia, severe abdominal operation) under which the present experi-ments were performed. Whether it also holds under normal circumstancescannot be decided before methods have been employed which allow thecollection of suprarenal venous blood in the surviving animal.

Comparison of the quantity of cortical hormone secreted by the glands of amedium-sized dog, with the quantities employed for maintaining life in supra-renalectomized animals, discloses another great discrepancy. The figures givenby different authors concerning the hormone requirements of suprarenalec-tomized animals are very variable; some of the differences are obviously due toinequalities in the potency of various extracts; the values, however, arealways much smaller than the daily output of hormone into the circulationwhich, according to the foregoing experiments, corresponds to 230 ml. of acommercial extract for an animal of 10 kg. Eversole, Gaunt & Kendall [1942]state that the maintenance dose for a dog is 1 ml. of cortical extract (75 g.gland), and Cleghorn, Fowler, Wenzel & Clarke [1941] give a similar figure(1x8 ml.) for a dog kept on a diet containing 1 % NaCl. The latter authors addthat 7-15 times those amounts are required in the state of collapse; even27 ml. extract, however, are little compared to 230 ml. According to Nowak[1938], 5 ml. extract daily (approximately 2 ml./kg.) are adequate for a cat,and rats are reported to need between.1 (Eversole et al. 1942] and 4X5 ml./kg.[Hartman et al. 1931]. It would, therefore, appear that the doses of hormoneindispensable to life are far smaller than those available in the animal pos-sessing its suprarenals and required for optimal function of its organs. Someevidence to the effect that the amounts of extracts hitherto used are notadequate for the full restitution of the functions of the body can also beobtained from the treatment of cases of Addison's disease and from functionaltests in suprarenalectomized animals. It is well known that human patients,despite the administration of desoxycorticosterone acetate or water-solublecortical preparations, have a low efficiency of the muscular and circulatorysystems. Observations on cortin-treated suprarenalectomized cats and dogs[Hartman, Lewis, Thatcher & Street, 1942] reveal an abnormally high globulincontent of the plasma in spite of a clinically satisfactory condition. Further-more, in suprarenalectomized rats, the cortin requirements for normal workperformance far exceed the maintenance dose; even with very large amountsrestitution of function is not complete [Ingle, 1936]. The possibility has, ofcourse, to be borne in mind that the extracts used are not only given in toosmall a quantity, but might be qualitatively different from the natural hormone.

SUMMARY1. Experience is reported with Selye & Schenker's method for the assay of

cortical hormone (treatment with cortical extract of young, suprarenalec-tomized rats exposed to low temperature). The response (mean survival time)

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was found to be proportional to the logarithm of the dose. The accuracy of thetest is inferior to that of the assay on drakes; its sensitivity, however, is about25 times higher.

2. With the aid of this assay, estimation was made of the cortical hormonein the venous effluent of the suprarenal gland of the dog, the cat, the goat, therabbit and the pig. Considerable cortical activity was invariably found in thevenous blood collected from the suprarenals. It was absent in arterial bloodand in blood taken from the right side of the heart.

3. The potency of suprarenal blood was compared with that of a commercialextract ('eucortone', Allen and Hanburys), of which 1 ml. is equivalent to75 g. gland. The potency of the blood may then be expressed by the weight ofsuprarenal tissue which has to be extracted in order to obtain a solution of thesame activity. The average output of one suprarenal gland, per min. per kg.body weight, was equivalent to 0-6 g. of suprarenal tissue. Its range laybetween 0-26 and 2-0 g. tissue. The order of magnitude was the same in thefive species used. The potency of 1 ml. suprarenal plasma was at least severaltimes, occasionally 10-12 times, as high as the activity obtained by extractionof 1 g. gland.

4. The figure of 0'6 g. suprarenal tissue per min. per kg. amounts to a dailyoutput, by both suprarenals of a 10 kg. dog, of 17,300 g. gland contained in230 ml. of a commercial extract. This figure is probably an underestimate. Itfollows that either our present methods of extraction are inadequate, or thesuprarenal cortex does not store its principle (or principles) in the active form.

5. As far as can be detected by the available methods, the minute-output bythe suprarenal cortex is not affected by changes in blood pressure or blood flow.

6. Cortical hormone is rapidly inactivated in the tissues. Liver, spleen andgastro-intestinal tract are not essential for this process. Neither does theexcretion by the kidney play an important part in the disposal of corticalhormone.

It is a pleasure to thank Dr K. H. Coward, Prof. J. H. Gaddum and, particularly, Dr D. E. Lea, fortheir advice in the statistical treatment of the results. My thanks are equally due to Dr F. J. Dyerfor his help in some of the assays.

REFERENCESBiilbring, E. [1937]. J. Physiol. 89, 64.Cleghorn, R. A., Fowler, J. L. A., Wenzel, J. S. & Clarke, A. P. W. [1941]. Endocrinology, 29, 535.Eversole, W. J., Gaunt, R. & Kendall, E. C. [1942]. Amer. J. Physiol. 135, 378.Feldberg, W. & Minz, B. [1934]. PflUg. Arch. ges. Physiol. 233, 657.Fisher, R. A. [1941]. Statistical Methods for Research Workers, p. 167. London: Oliver and Boyd.Hartman, F. A., Brownell, K. A. & Crosby, A. A. [1931]. Amer. J. Physiol. 98, 674.Hartman, F. A., Lewis, L. A., Thatcher, J. S. & Street, H. R. [1942]. Endocrinology, 31, 287.Ingle, D. J. [1936]. Amer. J. Physiol. 116, 622.Kendall, E. C. [1941]. J. Amer. Med. Ass. 116, 2394.Nowak, S. J. G. [1938]. Arch. int. Pharmacodyn. 60, 129.Pfiffner, J. J., Swingle, W. W. & Vars, H. M. [1934]. J. biol. Chem. 104, 701.Selye, H. & Schenker, V. [1938]. Proc. Soc. exp. Biol., N.Y., 39, 518.Zarrow, M. [1942]. Proc. Soc. exp. Biol., N.Y., 50, 135.

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