August, 19511 REISS, HALKERSTON, BA4DRICK AND HALKERSTON 461 Assessment of Adrenocorticotrophic Hormone Activity BY M. REISS, I. D. K. HALKERSTON, F. E. BADRICK AND JEAN M. M. HALKERSTON' (Presented at the meeting oj YJze Biologicnl Methods Group on Tuesday, December 19th, 1950) -4 review of attempts to assess adrenocorticotrophic hormone (ACTH) activity biologically is given. The methods discussed include those based on change of adrenal weight and those u tilising histological changes within the adrenal cortex. Experiences with the Sayers 'adrenal ascorbic acid depletion assay are described, together with an account of pituitary blockade by desoxycorticosterone acetate (DOCA) in an attempt to improve the precision of the method. The use of phosphorus-32 as an index of adrenal function is described.Following a review of indirect assay methods based on changes due to adrenal hormones mobilised by ACTH, attempts to utilise the modification of insulin sensitivity brought about by ACTH are discussed. Some results of clinical adrenal cortex response tests are reported. The possibility of the assay of ACTH by using surviving tissue in place of the assay animal is envisaged. SINCE it was first recognised that extracts made from the pituitary anterior lobe had an adrenocorticotrophic action, there have been numerous attempts a t its qualitative and quantitative assay. The earliest investigators used the weight increase of the adrenal as a basis for the assay of adrenocorticotrophic hormone (ACTH) . Jores and Beck,l for example, used the adrenal weight of normal male mice, which are particularly sensitive ; Moon2 used the adrenal weight of 3-day old rats and later of 21-day old rats3; Bates, Riddle and Miller-4 used the adrenal weight of 2-day old chicks.Results of assays of this type, employing intact as opposed to hypophysectomised animals, should be regarded with the greatest reserve, for many factors, such as changes in environ- mental temperatures or the presence of toxic substances in the extracts used, can influence the weight of the adrenal by mobilising endogenously produced ACTH. CollipJ5 in his first attempts at standardisation, made use of hypophysectomised animals for assays based on adrenal weight change, but experiments published later from his laboratory by Neufelds showed a complete lack of quantitative correlation between dose and response.Investigations were carried out a few years ago in our laboratory on hypophysectomised male Wistar rats of 100-g body weight, using ACTH extracts prepared from cattle pituitaries. The results were disappointing, as can be seen from Table I, in which the individual weights of the right adrenals, removed 7 days after hypophysectomy, are recorded below the weights of the left adrenals removed at autopsy after a 7-day injection period. The very large scatter of the results is indicated by the standard deviation of each response; the dose- response curve in Fig. 1 constructed from the data of Table I only emphasises the unsatisfactory nature of the assay procedure. The causes of the large variation in response are at present being investigated in our laboratory and some are discussed below.Recently, Cortis- Jones, Crooke, Henly, Morris and Morris7 reported a satisfactory linear relationship between the logarithms of the doses injected and the increase in adrenal weights of hypophysectomised rats. They define one ACTH unit, arbitrarily, as an activity causing a 50 per cent. increase in the mean adrenal weight over that of the hypophysectomised control animals. Weight maintenance methods developed by Astwood and Tyslowitzs and by Sayers, White and Longg seemed to yield better results. For a number of years we used as a method of assay the restitution, produced by ACTH injection, of the sudanophobic zone of the adrenal cortex of hypophysectomised rats, onghdly described by Reiss, B a h t , Oestreicher and AronsonlO and later by Simpson, Evans and Li.I1 This assay gave a satisfactory linear relationship between the percentage of animals giving positive response (or better still, the probit of the percentage) and the logarithm462 REISS, HALKERSTON, BADRICK AND HALKERSTON : TA~LE I [Vol.76 INVESTIGATION INTO THE ASSAY OF ADRENOCORTICOTROPHIC HORMONE (ACTH) EXTRACTS WITH 100-g HYPOPHYSECTOMISED MALE WISTAR RATS Mean of individual Number Weight of adrenals, mg percentage weight Dose of of Left adren,al after treatment difference between right ACTH, animals Right adrenal before treatment and left adrenals mg f S.E. of mean 40 8 9 8 8 5 6 8 1 0 8 -z 3 14 -2 3 3-4 3 + 76 & 16.6 + 42 11.3 1 0 1 2 8 6 6 6 1 2 9 -- 8 7 z z 6 8 - 3 7 20 8 + 76 & 15.1 11 10 9 8 7 11 12 7 -3.3 s 3 z 3 - i b 10 8 6 3 7 6 6 6 6 1 0 8 8 S ; ; i 5 6 5 i i + 22 & 8.8 $- 33 & 16.0 Controls .... €4 - 22 6.9 of the dose. If one compares the original adrenal weight method illustrated in Fig. 1 with Fig. 2, showing response curves for standard and unknown ACTH preparations by the histological method, the superiority of the sudanophobic zone restitution method is evident without further calculation. With the histological method 10 to 35 rats per dose and 3 to 4 doses per preparation were required to characterise the response curves, which were found to be very reproducible 420- . # * 22 * 10- * - v) ; +loo- - / - 0 - 0 -10- 0 -30- ’ 0 -20- ,/- I I I -0 310 $0 16.0 20.0 40.0 Total dose, mg Fig.1. Relation between log-d.ose of adrenocorticotrophic hormone and change in weight of adreuals in hypophysectomised rats The dotted lines are one standard deviation from the line of best -fitAugust, 19511 ASSESSMENT .OF ACTH ACTIVITY 463 and in this respect much superior to those we have since obtained with the adrenal ascorbic acid depletion assay of Sayers and Sayers,12 to be discussed later. It is a more time-consuming method, however, requiring the maintenance of hypophysectomised animals for 7 to 11 days before assay and for a 3 to 4-day injection period, so that a considerably higher standard of hypophysectomy technique is involved. Though more consistent, it is less sensitive, requiring 50 to 100 times as much material as the adrenal ascorbic acid depletion method.Dose, mg Fig. 2. Comparison between adrenocorticotrophic hormone of unknown strength and laboratory standard by the histological method Standard - a- Unknown - o ___ Because of the changes observed in the sudanophobic zone, it might have been worth while to follow up more intensively the changes in cholesterol and steroid contents of the adrenals after ACTH treatment, described by Sayers, Sayers, White and Long13 and later by Carryett, Golla and Reiss.14 However, interest in this, and for that matter in any other form of assay, was overshadowed by the introduction of the adrenal ascorbic acid depletion assay by Sayers and Sayers.12 This method makes it possible to obtain results more quickly, with much less material and with comparatively less labour than does, for instance, the sudanophobic zone restitution assay.It has therefore been of great help in the development of methods for preparing ACTH concentrates. It is carried out as a routine method in our laboratory essentially as described by Sayers, Sayers and Woodbury,15 with a few alterations. The use of ether anaesthesia throughout, the choice of the femoral vein as the injection site, a personal preference for the right adrenal as the control and the use of an electrotitrimetric method for ascorbic acid determinations are the only important differences between our technique and that of the originators. Table I1 summarises the results of some of the assays carried out in our department by this method. I t shows the number of rats used, the slopes of the response curves for standard and unknown, the value of 2 (the test for heterogeneity between the two lines), the standard deviation of the assay and the standard error of M (the logarithm of the ratio of potencies) for P = 0.95 and the approximate limits of error calculated from the standard error of M.The first three entries refer to separate one-day (3 + 3) assays comparing the same preparations. The fourth entry gives the evaluation of the combined results of the three assays, which does not show a significant improvement in precision over that of the individual assays. On the other hand the assay carried out on September 27th with only 30 animals gave a result of far greater precision. The irregular nature of the response curves indicated by Table I1 caused much misgiving when we first began to use this method of assay. It appeared that several hundreds of ’ animals would be required to obtain satisfactory limits of error.464 REISS, HALKERSTON, BADRICK AND HALKERSTON (Vol.76 The occurrence every now and then of an assay completely in agreement with results published by Sayers, Sayers and Woodbury,lS by Morris and his co-w~rkers,~ by Prunty16 and by other authors, in that reasonable limits of error were obtained with approximately 30 animals, led us to investigate as fully as possible the causes for the apparently chance variation of our results. Over the question of the suitability or otherwise of the strain of Wistar rats bred in our colony we received considerable help, obtaining, through the courtesy of Dr.Morris, Wistar rats of his own strain. We hope to breed from these animals and compare the results with those obtained on our own rats, which are partly derived from the original Glaxo strain and partly from the strain bred by Dr. Paterson in Porton. That the solution lies solely in the use of a different strain of rats seems unlikely, for it does not explain why our own animals sometimes respond as satisfactorily as those of other workers. Date of assay 2 1.4.50 26.4.50 5.5.50 21.4.50 26.4.50 6.5.50 I- ; ;: : ::} 17.5.50 27.9.50 9.11.50 14.11.60 8.12.60 7.6.50 TABLE I1 SUMMARY OF RESULTS OF SOME ASSAYS OF ACTH BY THE ADRENAL ASCORBIC ACID DEPIXTION METHOD Kumber of rats 44 40 47 131 78 16 30 23 25 26 9 Slupe of log-dose - response curves Standard deviation Standard Approximate limits of 7- of the Error of.M* Standard Unknown X2 assay (P = 0.95) 58.2 63.1 0.059 2 26.7 0.2634 91.3 95.6 0.037 2 33-1 0.2300 144.3 117.4 3.610 f 31.0 0.1355 104.5 85.2 3.210 5 32-3 0.1331 101.0 79.1 2.0 & 24.9 0.1701 149.0 139.0 0.103 & 22.0 0.1510 116.3 114.3 0.0'77 13.7 0,085 1 125.5 142.8 0.049 k 22.6 0.1448 84.25 86.25 0.008 & 25.5 0.2455 116.4 78.1 0.100 * 22.2 0.2028 Log-dose - response curve for 60-g hypophysectomised rats & 34.9 s / b = 0.280 124.6 - -- * M is the logarithm of the ratio of potencies. error, % 2 64 + 56 z 32 - + 31 & 40 & 36.5 19.5 * 34 & 48.5 & 54-45 It would appear more likely that the variation is due to the fact that no other anterior pituitary hormone can be so readily mobilised endogenously as ACTH.The ascorbic acid depletion index shows how even small manipulations on an intact animal, e.g., anaesthesia, injection of test material or even changes in environmental temperature, can bring about a mobilisation of ACTH with a consequent fall in adrenal ascorbic acid concentration. It is obvious that such stresses as fights with other rats and draughts in the animal house can occur in the life of a growing rat, mobilising temporarily increased amounts of endogenous ACTH with its prevailing effect upon the adrenal cortex. This might be the sole explanation for the variation in adrenal weight per unit body weight found among the members of a large rat colony. The point is important, because the dose injected is calculated per 100 g of body weight and may therefore be called upon to influence adrenals of very different weights.Obviously the younger the rat the fewer the occasions for irreversible changes in the adrenal cortex. This consideration, together with the observation that it is easier to induce weight changes in the adrenals of younger than of older rats, led us to use 60-g hypophysectomised rats for assay purposes ; the result of one rather promising experiment is included in Table 11. The precision obtained is comparable with that of most of the assays on older animals, although the scatter of the responses is still unsatisfactory. It would be preferable to use even younger rats, but in a 40-g animal the stress of hypophysectomy is very much greater than in older ones. For this reason we studied the known effect of desoxycorticosterone acetate (DOCA) in blocking the pituitary function of ACTH mobilisa- tion, with a view to using animals pre-treated in this way for assay purposes.August, 19511 ASSESSMENT OF ACTH ACTIVITY 465 In Table I11 is shown the effect of injecting DOCA into 40-g rats on the endogenous production of ACTH by the pituitary, following stress.The stress applied consisted in anaesthesia with ether and the rapid removal of one adrenal. One hour later the animals were killed, the second adrenal was removed and the concentrations of ascorbic acid in the adrenals were compared. TABLE I11 EFFECT OF DESOXYCORTICOSTERONE ACETATE (DOCA) ON THE ENDOGENOUS PRODUCTION OF ACTH BY THE PITUITARY, FOLLOWING STRESS Treatment A r \ Material injected Time before and dose stress, hours 18 18 18 18 No injection - 1 ml of arachis oil 1 mg of DOCA in 1 ml of oil 10 mg of DOCA in 1 ml of oil 10 mg of DOCA in 1 ml of oil Concentration of Number ascorbic acid in of rats right adrenal, 5 343 & 38-1 4 385 & 954 4 337 & 18.4 6 386 & 50.5 4 385 2 50.2 mg per 100 g, & S.D.Mean difference in ascorbic acid concentration between right and left adrenals, mgper lOOg, f S.D. - 73 rf: 23.4 - 63 & 26.2 - 94 f 4.2 - 6 f 10.9 + 62 & 39.9 The response to the stress, in the un-pre-treated control group, is indicated by the considerable drop in concentration of ascorbic acid in the second adrenal. One millilitre of arachis oil or 1 mg of DOCA in 1 ml of arachis oil injected 18 hours before stress did not change this reaction.However, 10mg of DOCA given 18 hours before stress completely prevented depletion of ascorbic acid. It may be pointed out that it is necessary to treat with DOCA at least 18 hours before assay, as the injection is itself a stress: 4 hours after such an injection the concentration of ascorbic acid in the adrenal is still considerably reduced, so that no further decrease can be achieved by a further stress. Only after 18 hours at the earliest is the ascorbic acid concentration sufficiently high to allow the effect of another stress to be recorded efficiently. Table IV shows the reduction of the adrenal ascorbic acid by different doses of ACTH in 40-g male Wistar rats pre-treated with 10 mg of DOCA 18 hours before assay. The scatter of results is still considerable, and we are investigating the possi- bility of improving the technique of pituitary block. TABLE IV REDUCTION OF ASCORBIC ACID IN THE ADRENALS BY DIFFERENT DOSES OF ACTH INJECTED INTO 4O-g RATS PRE-TREATED BY INJECTION OF 10 mg OF DOCA 18 HOURS BEFORE ASSAY Difference between amount and left adrenal, mg per 100 g, f S.D.Dose of ACTH, Number of ascorbic acid in right Log-dose - response curve pg per 100 g of body weight of rats ch axac teris tics 1.0 3 41 & 29.4 Slope ( b ) = 60.3 2.0 4 51 1- 30.7 Standard deviation 4.0 6 76 & 20-0 Index of precision The difficulties encountered in the adrenal ascorbic acid depletion assay will be found in any assay method with rats, owing to the extreme ease in mobilising endogenous ACTH during the animal’s lifetime.It is in our experience particularly true of adrenal repair tests or those based on hypertrophy of the adrenals, when a further complication occurs because the glands are continuously atrophying during the three weeks following hypophysectomy and the weight increases achieved after treatment are at the best a result of the action of the several factors concerned. It might therefore be advantageous instead to investigate the biochemical changes that are the primary basis of the growth phenomenon. Previous investigations by Reissl7 and by Reiss, Druckery and FischP8 on the ovary showed up to 100 per cent. increases in oxygen consumption and glycolysis 1 hour after injection of gonadotrophic hormone, long before any growth could be seen.The adrenal cortexlg also shows similar increases in oxygen consumption and glycolytic processes soon after injection of ACTH. The methods used in such investigations are too cumbersome for application to routine assay procedure, and the (s) = 26.16 (s/b) = 0.433466 REISS, HALKERSTON, BADRICK AND HALKERSTON : [Vol. 76 dose - response relationships are not satisfactory. It is therefore simpler to investigate phosphorylation processes in the adrenal cortex, particularly as Gemzel120 has shown that ACTH increases phosphorylation rate in the adrenal. Changes in the uptake of phosphorus-32, soluble in trichloro-acetic acid, by the adrenal, as an arbitrary index of phosphorylation rate, were investigated in numerous experiments by Reiss and Halkerston.21 The effect of endogenously mobilised ACTH was studied in animals exposed to cold, and increases of several hundred per cent.in uptake of phosphorus-32 by the adrenals were recorded after exposure for 1 hour. This change was not shown by hypophysectomised animals. It was also interesting to note that new-born animals at the age of three and eight days are already able to mclbilise considerable amounts of endogenous ACTH. Table V shows that even the removal of litter mates from the warm environment of the mother and leaving them at room temperature resulted in a considerable increase in the uptake of phosphorous-32 by the adrenals. Removal to a cool room still further enhanced this reaction. These changes in the uptake of phosphorus-32 by the adrenal parallel the ascorbic acid depletion known to occur under similar conditions.TABLE 1' EFFECT OF ENVIRONMENT ON ADRENAL UPTAKE OF PHOSPHORUS New-born rats, litter mates, injected with 6 pc of phosphorus-32 intraperitoneally and killed 60 to 80 minutes later. Trichloro-acetic acid extracts of adrenals measured in a liquid counter. Impulses per Litter number Rat number Treatment 100 mg of adrenal 1 (3 days old) 1 Left with mother 550 2 Kept 1 hour at room temperature 850 3 Kept 1 hoiir in cold room 1165 2 (8 days old) 1 Left with mother 465 2 Left with mother 455 3 Kept 1 hour at room temperature 660 4 Kept 1 hour in cold room 830 6 Kept 1 hoiir in cold room 1940 Recently we have taken advantage of the fact that during routine use of the ascorbic acid depletion assay it is possible to introduce by intravenous injection, after the removal of the control adrenal, a dose of phosphorus-32 together with the ACTH test solution.An aliquot of the metaphosphoric acid filtrate from the left adrenal contains a greater amount of phosphorus-32 than that of control animals, and Table VI shows that promising dose - response relationships are obtained between the percentage of the phosphorus-32 taken up by the adrenal and the dose of ACTH injected. TABLE VI DOSE - RESPONSE RELATIONSHIP BETWEEN PHOSPHORUS-32 TAKEN U P BY . ADRENALS AND DOSE OF ACTH INJECTED Standard preparation of ACTH Unknown preparation of ACTH A A f \ f Percentage of dose Percentage of dose Dose in pg per Number of taken up by Dose in pg per Number of 32P taken up by 100 g of body weight of rats left adrenal 100 g of body weight of rats left adrenal 0-5 5 0.049 0.75 4 0.061 0-25 6 0.03 7 0.375 5 0.052 1.0 5 0.054 0-5 6 0.04 1 0-25 2 0.03 1 1.0 0.5 0.25 4 5 7 0.058 0.0475 0.0435 Such changes in oxygen consumption and uptake of phosphorus-32 are by no means confined to the adrenal cortex.The turnover rate for phosphorus-32 is increased in most growing tissue, particularly tumours. Similarly, changes in the ascorbic acid concentration of the adrenal need not necessarily be connected with the endocrine function of the adrenal cortex; one should therefore be somewhat reluctant to use this index as a basis for the biologicalAugust, 19511 ASSESSMENT O F ACTH ACTIVITY 467 assay of preparations having adrenocorticotrophic action. The use of the adrenal ascorbic acid depletion assay before the mechanism of the depletion is known reminds one of previous attempts to standardise the hormonal activity of ‘pituitary posterior lobe extracts, prepared for their clinical pressor, oxytocic and antidiuretic activities, by their action on the melano- phores of frogs.Indirect tests, based on the quantitative action of adrenocortical hormones mobilised by exogenous doses of ACTH, might therefore be more satisfactory from the point of view of the physiologist and the clinician. Several workers have shown that animals may become more sensitive to certain injurious factors after hypophysectomy and have indicated the possibility of increasing their resistance by treatment with ACTH or adrenocortical hormones. Tyslowitz and Astwood,22 for instance, reported the effect of ACTH on the resistance of hypophysectomised rats to low environmental temperatures and later reportedB the increased resistance of these animals to low environmental pressures after treatment with ACTH.Li, Simpson and EvansM described the action of ACTH in increasing resistance to cold, starvation and anoxia, while Reiss, MacLeod and Golla? showed that rats treated with ACTH exhibit increased resistance to secondary shock symptoms (rapid fall in oxygen con- sumption and body temperature, rise of blood haematocrit value and fall in blood sodium) induced by intraperitoneal injections of hypertonic glucose solution. Recently, Buttle26 reported on the increased resistance of guinea-pigs to histamine after ACTH administration, and Pruntyl6 has based an assay on the antagonistic effect of ACTH to wound-healing in mice.All of these actions can, of course, be used as a basis for quantitative bio-assay as soon as standardised conditions can be worked out. Recently we have investigated the effect of ACTH in increasing the resistance of mice to insulin. Table VII shows the results of two such experiments where groups of about TABLE VII EFFECT OF ACTH ON SENSITIVITY OF MICE TO INSULIN Dose of ACTH, given 1 hour before injection mg per animal Percentage of mice reacting 2 hours after injection of of insulin, Number of mice insulin (a) 0-2 units of insulin injected subcutaneously- nil (controls) 20 0.1 19 0.4 20 1.0 20 (b) 0.045 units of insulin injected subcutaneousIy- nil (controls) 30 0.1 18 1.0 22 65 32 25 20 93 39 4.5 20 mice were injected with ACTH 1 hour before insulin administration.In the first experi- ment the occurrence of insulin coma or death 2 hours after 0.2 units of insulin was taken as a criterion of insulin action, while in the second experiment the criterion was the percentage of animals showing at least paralysis of the hind limbs 2 hours after a lower insulin dose. In both experiments the degree of protection increased with dose. The results appear encouraging, particularly since it should be possible for all laboratories with well-established facilities for mouse insulin tests to apply them to the bio-assay for ACTH. The method could doubtless be considerably improved in precision and sensitivity by pre-treatment of the animals with DOCA, which increases the insulin sensitivity considerably by blockage of the pituitary anterior lobe.A discussion of the assessment of ACTH activity would not be complete without reference to the response of human subjects to ACTH administration. Specific response tests for adrenocortical and adrenocorticotrophic efficiency worked out by Forsham, Thorn , Prunty and Hills,27 Pincus28 and other authors are becoming more and more a routine procedure in pathological laboratories. It is at once apparent when reviewing the different indirect methods of assay that different adrenocortical products are concerned in such effects as resistance to cold, low atmospheric pressure, starvation, insulin and secondary shock .symptoms. This becomes still more obvious with the responses of different human subjects to a single injection of ACTH ;468 REISS, HALKERSTON, BADRICK.AND HALKERSTON [Vol. 76 even if the response tests are restricted to three secondary actions, vix., decrease in circulating eosinophils, increase in uric acid - creatinine ratio and increase in 17-ketosteroid excretion after ACTH administration, very different reaction types can be found among human subjects. In a large proportion of the tests the results show an eosinophil count decreased by at least TABLE VIII RESPONSE OF OVER 400 PATIENTS TO INJECTION OF DOSES OF ACTH AND TO STIMULATIOK OF ENDOGENOUS ACTH BY INJECTION OF GLUCOSE AND BY ELECTRIC SHOCK TREATMENT Response measured by A 7- 7 Circulating Uric acid - 17-Ketosteroid Treatment eosinophils, creatinine ratio, excretion, 0 ’ % % /O Glucose injection .. .. .. 41 34 9 Low ACTH dose, 15 to 26 mg . . 76 58 18 High ACTH dose, 50 mg . . .. 95 62 86 Electric shock . . .. .. .. 100 85 35 30 per cent., an increase in uric acid - creatinine ratio by at least 25 per cent. and an hourly 17-ketosteroid excretion increased by at least 20 per cent. In other tests, however, only the eosinophil count falls, while the other two indices do not alter. Other cases show an un- changed eosinophil count, one or both of the other components showing significant increases. The fact that an increase in the ACTH dose produces a full response, when the low dose failed to do so, suggests that different independent mechanisms are concerned, some of which are TABLE IX INDIVIDUAL RESPONSES OF PATIENTS TO INJECTIOle OF DOSES OF ACTH -4ND TO STIMULATION OF ENDOGENOUS ACTH BY ADMINISTRATION OF GLUCOSE AND BY ELECTRIC SHOCK TREATMENT Patient 1 2 3 4 5 6 7 8 9 10 11 Patient 1 2 3 4 5 6 7 8 9 10 11 Age 25 42 26 28 30 32 42 40 26 40 46 Age 25 42 26 28 30 32 42 40 26 40 46 Response to small dose of ACTH (15 to 26 mg) A I 7 Uric acid - 17-Keto- creatinine steroid Eosinophils ratio excretion + 0 0 0 + 0 0 0 + + -t + + + + + -t + 0 + + + + + 0 + + 0 0 + 0 0 Response to glucose (150 g administered orally) I A 7 Uric acid - 17-Keto- creatinine steroid Eosinophils ratio excretion + 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + 0 0 + 0 + 0 + + 0 + Response to higher dose of ACTH (50 mg) r > Uric acid - 17-Keto- creatinine steroid Eosinophils ratio excretion + 0 0 + + 0 + -+ + I - + 0 + 4- 0 Response to electric shock treatment Eosinophils 1- + + + + + + + + + + Uric acid - creatinine ratio + + + 0 + + + 0 + + 0 3 17-Keto- steroid excretion 4- + 0 0 0 0 0 0 0 0 +August, 19511 ASSESSMENT OF ACTH ACTIVITY 469 disturbed under pathological conditions.The threshold becomes increased and, therefore, the particular response is absent at low dose levels. In Table VIII are shown the results of response tests performed on over 400 patients29 each of whom received in turn 25 mg of ACTH, 50 to 100 mg of ACTH, then, as mobilisers of endogenous ACTH, 150 g of glucose by injection, and finally electric shock treatment. From this table the general pattern of the response would appear to be an enhanced reaction with increase in the strength of stimulus.That this is only true for the low and high ACTH dose is shown by the individual responses set out in Table IX. Patients 1 to 3, for example, react according to the over-all picture in Table VIII, while patients 4, 5, 6 and 8 show a complete response to even the low ACTH dose, though not to the greater stimulus of electric shock. In other patients it would appear that different adrenal cortex responses are evoked by the different stimuli. Many of the results are difficult to explain and the whole picture remains obscure, but it seems difficult to avoid the conclusion that there are different adrenocorticotrophic components. A review of all the bio-assays and possibilities of bio-assay for ACTH emphasises the shortcomings of purely biological methods. It would seem that it will not be possible to overcome all the inherent difficulties of assays based on living animals with their individual demands for adaptation.This demand is a prominent feature of the whole life of the animal before being taken into the assay experiment and is concomitant with the mobilisation of adrenal cortex hormones. In other words, it would be a great advantage if one could dispense with the assay animal altogether and investigate the influence of ACTH and different ACTH fractions on adrenal cortex tissue in vitro. As a step in this direction we are trying to determine the amount of “cortin-like” substances (i.e., neutral lipoid-soluble reducing substances) produced by adrenal cortex tissue in vitro and to find how this production can be influenced by ACTH added to the medium.The results obtained so far have proved encouraging and we shall report more fully when the experimental conditions have been stabilised. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. REFERENCES Jores, A., and Beck, H., 2. ges. ex$. Med., 1936, 97, 622. Moon, H. D.. Proc. SOC. Exp. Biol. Med., 1937, 35, 649. Bates, R. W., Riddle, O., and Miller, R. A., Endocvinology, 1940, 27, 781. Collip, J. B., J . Mount Sinai Hosp., 1934, 1, 28. Neufeld, A. H., Proc. Soc. Exp. Biol. Med., 1943, 54, 91. Cortis-Jones, B., Crooke, A. C., Henly, A. A., Morris, P., and Morris, C. J. 0. R., Biochem. J., Astwood, E. B., and Tyslowitz, R., Fed. Proc., 1942, 1, 4. 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J . Physiol., 1941, 133, 472. -- , Ibid., 1942, 136, 22. Li, C. H., Simpson, M. E., and Evans, H. M., Science, 1942, 96, 450. Reiss, M., MacLeod, L. D., and Golla, Y . M. L., J . EndocrinoZ., 1943, 3, 292. Buttle, G. A. H., “Proceedings of the Meeting of the Society of Endocrinology,” Oct. 25th. 1960. Forsham, P. H., Thorn, G. W., Prunty, F. T. G., and Hills, A. G., J . Clin. Endocrinol., 1948,8, 16. Pincus, G., Psychosom. Med., 1949, 2, 74. Hemphill, R. E., and Reiss, M., “Proceedings of International Congress of Psychiatry,” Pans, -, Ibid., 1940, 43, 42. 1950, 46, 173. 200. in the Press. 1950, in the Press. BIOCHEMICAL AND ENDOCRINOLOGICAL RESEARCH DEPARTMENT BRISTOL MENTAL HOSPITALS FISHPONDS, BRISTOL470 MORRIS : ADRENAL ASCURBK: ACID DEPLETION AND ADRENAL DISCUSSION [Vol. 76 DR. W. PERRY said that variations introduced by stress could be reduced by animal training. He thought i t might be possible to use non-hypophysectomised animals for assay purposes on a basis of the modification of their immunity responses. DR. L. J. HARRIS enquired whether the electrometric; method used by the authors for the estimation of ascorbic acid was a photo-electric or a potentiometric method and, if the latter, the nature of the electrode used. DRS. HALKERSTON and h r s s said, in reply, that they had not tried any of the possibilities suggested by Dr. Perry, but they did not think that any advantage could be gained by their use. The method used or ascorbic acid estimation was potentiometric, with clean platinum electrodes and a small potential applied across them for a dead-stop end-point. The oxidising agent was 2 :6-dichlorophenolindophenol