470 MORRIS : ADRENAL ASCURBK: ACID DEPLETION AND ADRENAL [Vol. 76 The Adrenal Ascorbic Acid Depletion and Adrenal Repair Methods for the Bio-Assay of Adrenocorticotrophic Hormone BY C. J. 0. R. MORRIS (Presented at the meeting of the Biological Methods Group on Tuesday, October 24th., 1950) The methods for the bio-assay of adrenocorticotrophic hormone (ACTH) are discussed. An example of the adrenal repair methods is included in the form of a detailed description of the technique used in the author’s laboratory on male Wistar rats, 35 to 42 days old. Methods that use histological evidence of repair as the criterion of ACTH activity are discussed; these are much more sensitive than those involving adrenal weight. The adrenal ascorbic acid depletion m.ethod is described and discussed, and modifications are mentioned.THE first methods to be used for the bio-assay of the pituitary adrenocorticotrophic hormone (ACTH) were based on increase in size of the adrenals after subcutaneous injection of the hormone. As the technique of hypophysectomy in the rat became more generally practised, it was soon realised that removal of the experimental animal’s endogenous supply of ACTH was essential for reliable assay. This point cannot be too strongly stressed. The use of immature normal animals for ACTH assay by maay workers has led to the publication of much erroneous information. Investigation in our own laboratory has indicated that, even under the most carefully controlled conditions, reliable results cannot be obtained on the intact animal.The point also arises in the assessment of the effect of ACTH on the human subject and must always be borne in mind in such experiments. ,IDRENAL REPAIR METHODS- The technique of the adrenal repair methods for the assay of ACTH on the hypo- physectomised rat is broadly as follows. The animals are hypophysectomised at 5 to 6 weeks old and a period of 7 to 14 days is allowed for the adrenals to atrophy. The test material is then injected, usually twice daily for 3 days, and the animals are killed on the day following the last injection. Adrenals and testes are dissected free of extraneous tissue and weighed. The weight of the testes provides a criterion of completeness of hypophysectomy, although it must be remembered that, in the assay of crude preparations, the presence of gonadotrophic hormones may lead to an increase in weight of the testes.As an example the technique as practised in our laboratory will be described in detail. Nale Wistar rats, 35 to 42 days old, are used. The animals are bred in our own colony, a s it appears that strain and uniform maintenance conditions from birth are essential for repro- ducible results. In particular, adequate nutritional status is most important. Animals of this age and strain should weigh 90 to 110 g, and rats outside this range should not be used. The animals are hypophysectomised by the parapharyngeal approach. They are kept forAugust, 19511 REPAIR METHODS FOR BIO-ASSAY OF ACTH 47 I 10 to 14 days in a ventilated enclosure thermostatically maintained at 25" C. The test material is injected subcutaneously in a volume of 0.5 to 2.0 ml twice dairy for 3 days.Groups of 4 or 5 animals per dose level are used. A similar group may be injected with saline as a control. The animals are killed on the fourth day, and the adrenals are carefully dissected free of extraneous tissue, quickly dried with filter-paper and weighed to 0.1 mg. The testes are also dissected, dried and weighed to 6mg. Under our conditions a combined testis weight of less than 600mg is taken as the criterion of completeness of hypophysectomy. The adrenal weight is expressed as milligrams per 1oOg of body weight a t the time of the * - 1 0 0.2 0.4 0.6 0 8 1-0 Log 10 xdore. p~ Fig. 2. Assay of adrenocorticotrophic hormone by adrenal ascorbic acid depletion method first injection.An arbitrary unit has been taken as equivalent to a 50 per cent. increase in mean adrenal weight over that of the controls. Results for a typicz.1 assay are shown in Fig. 1. It will be seen that there is a satisfactory linear relation between log-dose and biological response. The preparation used was slightly more active than Armour Standard La-1-a ( x 1-18), so that 1 mg of such a preparation gives a response of 0-4 unit. The useful range of the method is between 1 and 4 units (P < 0.05). It will be seen that under carefully controlled conditions the method is at least as accurate as any other and has the advantage of simplicity. It is, however, rather insensitive and requires 4 days, compared with 1 day for the adrenal ascorbic acid depletion method.Various modifications of the adrenal repair method have been suggested. Collipl used repair of the remaining adrenal in the hypophysectomised unilaterally adrenalectomised rat, the animal serving as its own control. In view of the marked difference in size between the adrenals of a rat, this procedure has little to recommend it. Sayers, White and Long2 used three daily intraperitoneal injections of test material. Comparison of this method with our own of two subcutaneous injections revealed little difference. METHODS MAKING USE OF VISTOLOGICAL EVIDENCE OF REPAIR- In a somewhat different category are the methods that use histological evidence of repair as the criterion of activity. Reiss, Balint, Oestreicher and AronsonS first described a dis- appearance of lipids from the greater part of the adrenal cortex following hypophysectomy.The remaining lipids in the zona glomerulosa become irregular in distribution. Simpson,MORRIS : ADRENAL ASCORBIC ACID DEPLETION AND ADRENAL [Vol. 76 472 Evans and Li4 based an assay method on the ability of ACTH preparations to reverse these changes. A total of 25 pg of a preparation roughly equivalent to Armour Standard injected intraperitoneally, 6.25 pg once daily for 4 days, was sufficient to produce the first detectable signs of repair. The assay is thus much more sensitive than the methods making use of adrenal weight. In our own experience the method is somewhat difficult to carry out, owing to lack of sharpness at the end-point, and in any event, in common with most “all or none” bio-assays, it requires a large number of animals for precision.Here the test material is injected for a period of 14 days, beginning the day after hypophysectomy. The unit is defined as the least quantity of the preparation sufficient to maintain the adrenals at a weight equal to those of normal controls. A total dose of 350pg of a preparation about equal to Armour Standard is adequate. The method is thus roughly ten times as sensitive as the repair test, but requires a large number of animals. It has been described in detail by Sayers, White and Long2 and by Simpson, Evans and Li4; we have ourselves had no experience with it. Another method of this type is the adrenal maintenance method. ADRENAL ASCORBIC ACID DEPLETION METHOD- The method of ACTH assay most generally used at present is the adrenal ascorbic acid depletion method.This is based on the observation of Sayers, Sayers, Liang and Long5 that a single injection of ACTH causes a rapid fall in the ascorbic acid and cholesterol contents of the adrenals. This process is now interpreted as being the first stage in the synthesis of adrenocortical hormones, for it is soon followed by the increase in liver glycogen typical of treatment with certain of these steroids. As this mechanism is set in action by any stress stimulus, it is essential to hypophysectomise the test animal. Long et aL6 showed, however, that the response was diminished and finally disappeared a few days after hypophysectomy, and it is now customary to use the animals the clay after operation.Sayers and Sayers6 and later Sayers, Sayers and Woodbury’ developed the method into an extremely sensitive, rapid and specific bio-assay method for ACTH. We use rats of 90 to 110 g body weight. It appears that in this method the strain is extremely important, and many strains are entirely unsuitable owing to insensitivity and irregularity of response. The literature shows that some of the standard curves quoted have a very low slope, and the common strains of rat appear to fall into two groups in this respect, We have used our own albino Wistar strain and are very fortunate in having a type of rat of extremely high sensitivity. The animals are hypophysectomised by the usual method; as evidence of completeness of hypo- physectomy is difficult to obtain, care should be taken to use only those animals in which the operator is satisfied that removal is complete.The method thus requires a high standard of operative technique. The following day the animal’s left adrenal is removed under anaesthesia, dissected free of extraneous tissue, dried, weighed to 0.1 mg and immediately homogenised in a modified Potter - Elvehjem homcgeniser with the solvent used for extrac- tion. The test material is then injected intravenously in the proportion of 0-2 ml per 100 g of body weight. Sayers, Sayers and Woodbury7 used the tail vein, but we have preferred the jugular vein for this purpose. Exactly one hour after injection, the second adrenal is removed under anaesthesia, dissected, dried, weighed and homogenised as before. The animal is killed and the sella turcica examined under a low power microscope for remnants of the pituitary.Sayers, Sayers and Woodbury7 used pentobarbital, but we have had most unsatisfactory experience with this anaesthetic and now use ether exclusively. Some American workers have had a similar experience. This effect also appears to be related to the strain of animal. Analysis of the adrenals for ascorbic acid can be carried out by any of the common methods. Sayers, Sayers and Woodbury7 used the Roe-Kuether methods but we have used a modified indophenol decolorisation m e t h ~ d . ~ In this method the decolorisation of a buffered 2 :6-dichlorophenol- indophenol solution is measured photo-electrically exactly 30 seconds after addition of the test solution. It is at least as precise as the Roe - Kuether method and is probably more reliable.It is in routine use by Armour Laboratories, who have probably carried out more assays by the Sayers method than any other group. The adrenal ascorbic acid value is expressed in pg per 100mg of adrenal tissue. Typical results for an assay of a sample of unknown potency against Armour Standard La-1-a are shown in Fig. 2. These data give a log-potency ratio of 0.07, a standard error of the mean of 0.066 and a combined standard deviation of 19.7. At the usual significance level of 1 in 20 this The choice of anaesthetic is of importance. It is also much quicker.August, 19511 REPAIR METHODS FOR BIO-ASSAY OF ACTH 473 corresponds to an accuracy of 3.16 per cent. for the use of 28 animals. This is well within the limits of precision quoted by Sayers, Sayers and W~odbury.~ By suitable choice of strain of animal and meticulous experimental technique this precision is always possible, despite reports to the contrary that have appeared in the literature.However, 2 to 3 per cent. of the animals give completely anomalous values, which can easily be discerned. This effect can also be found in the adrenal repair assay. The adrenal ascorbic acid depletion assay is thus about 2000 times as sensitive as the repair assay, while precision and specificity are probably comparable. The form?r is also very much quicker, but requires more labour and the most meticulous attention to detail. The high sensitivity may in some ways be a drawback, as the extremely dilute test solutions may show the instability characteristic of ACTH in neutral solutions.In routine work we make up our solutions for injection in 0.02 N acetic acid, which is tolerated well on intravenous injection and prevents adsorption and other inactivation effects. Some modifications of the Sayers, Sayers and Woodbury7 technique have been suggested. Munson, Barry and KochlO do not perform the unilateral adrenalectomy before injection but compare the mean total adrenal ascorbic acid in an injected group with the mean of a control group. Sayers, Sayers and Woodbury7 made a statistical comparison of both methods by using the same experimental data and right-adrenal ascorbic acid values as Munson responses so that identical statistical treatment could be applied to both. They concluded that, to attain the same precision, twice as many animals were required for the Munson method as for the difference method.The Munson method decreases the number of analyses to be performed. It is used in routine work by Amour Laboratories. Stoerk, Porter and Silberll have developed a modified Sayers assay in which normal rats are used, the pituitary being blocked by prior administration of adrenal cortical extract. No details are available of this method, but it is understood that this group is now using the usual Sayers assay. This paper is an attempt to give an account of the chief methods for the assay of ACTH. Only two seem to be of practical value, the adrenal ascorbic acid depletion method and adrenal repair method. The choice between these must depend on the user’s special requirements. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Collip, J. B., J . Mount S i n a i Hosp., 1934, 1, 28. Sayers, G., White, A., and Long, C. N. H., J . Biol. Chem., 1943, 149, 425. Reiss, M., B a h t , J., Oestreicher, F., and Aronson, V., Endokrinologie, 1936, 18, 1. Simpson, M. E., Evans, H. M., and Li, C. H., Endocrinology, 1943, 33, 261. Sayers, G., Sayers, M., Liang, T. V., and Long, C. N. H., Ibid., 1945, 37, 96. Sayers, M., and Sayers, G., Fed. Proc., 1946, 5, 200. Sayers, G., Sayers, M., and Woodbury, L. A., Endocrinology, 1948, 42, 379. Roe, J. H., and Kuether, C. A., J . Biol. Chem., 1943, 147, 399. Mindlin, R. L., and Butler, A. M., Ibid., 1938, 122, 673. Munson, P. L., Barry, A. G., and Koch, F. C., quoted in Endocrinology, 1948, 42, 379. Stoerk, H. C., Porter, C. C., and Silber, R. H., quoted in J . Amer. Chem. SOC., 1950, 72, 1040. ENDOCRINE UNIT THE LONDON HOSPITAL WHITECHAPEL ROAD LONDON, E.l