Departments of Chemical Pathology and Medicine, St. Mary’s Hospital Medical School, London, W. 2
CUSHING’S SYNDROME ASSOCIATED WITH A »CORTICOTROPHIN«-PRODUCING BRONCHIAL NEOPLASM (Results of corticotrophin assays and of pituitary-adrenal function tests)
By J. Landon, V. H. T. James and W. S. Peart
ABSTRACT
A patient is reported who developed bilateral adrenal hyperplasia and Cushing’s syndrome in association with an undifferentiated bronchial carcinoma. »Corticotrophin« was demonstrated by radioimmunoassay in the tumour and its metastases and »melanocyte-stimulating hormone« by bioassay in a metastasis. Treatment with Metyrapone resulted in bio- chemical evidence of improvement but side-effects limited its clinical usefulness. The use of pituitary-adrenal function tests in distinguishing patients with this syndrome from those with Cushing’s disease is discussed.
The association of bilateral adrenal hyperplasia and Cushing’s syndrome with non-pituitary tumours has been reported frequently since the original paper by Brown (1928). Allott & Skelton (1960) reviewed 36 cases from the literature together with 2 of their own and recorded that this syndrome was associated, most commonly, with undifferentiated carcinomata of the bronchus, thymus or pancreas. The finding of elevated plasma levels of ACTH or of an ACTH- like compound in 3 patients (Christy 1961; Bornstein et al. 1961) suggested that the tumours were either synthesizing >ACTH«” or some compound which caused the release of this hormone from the pituitary. Meador et al. (1962) investigated 5 patients with this syndrome and confirmed that the circulating levels of »ACTH« were raised. They also demonstrated the presence of this
* The use of inverted commas is necessary because it is not known whether this compound is identical structurally with pituitary corticotrophin.
compound in the primary tumour and its metastases and found that the pituitary content of corticotrophin was decreased. On the basis of these results they postulated that the bilateral adrenal hyperplasia and increased cortico- steroid secretion was due to production of »ACTH« by the tumour, the elevated steroid levels, in turn, suppressing the endogenous synthesis of ACTH in the pituitary. The finding, by Mattingly et al. (1964), that hypophysectomy did not influence plasma corticosteroid levels in a patient with this syndrome supports this postulate.
Until many more cases have been investigated fully it is impossible to be certain that this is the only mechanism by which the syndrome can arise. Some tumours may secrete a compound with corticotrophin-releasing activity, or which stimulates the pituitary-adrenal axis at the cerebral or hypothalamic level. In the present state of knowledge, however, it would seem essential to demonstrate the presence of »ACTH« in the tumour or its metastases to be sure that the association is not fortuitous (Liddle et al. 1963). The present paper reports a patient in whom this criterion was met. Special attention has been given to the results of >>ACTH« assays and of various pituitary-adrenal function tests. The effect of treatment with Metyrapone is discussed.
CASE REPORT
The patient was a 70 year old retired dental practitioner who had smoked an average of 30 cigarettes and 3 pipes daily for the majority of his adult life. For 6 months prior to his first admission in December 1965 he had experienced numbness of sensation over his penis, scrotum and rectum which impaired control of micturition and de- faecation. During this period he had noted nocturia and polyuria and his friends had commented on his sunburnt appearance. His general health remained excellent until 12 days before his admission; indeed he had played a full round of golf only a fort- night previously. Deterioration, which was dramatic and sudden, followed the ad- ministration of bendrofluazide (tab i daily) because of the development of ankle oedema. Within 24 hours he became confused, polyuria increased and he was unable to stand or walk because of severe generalised weakness.
On admission to his local hospital (under the care of Dr. P. Hughes) he was noted to have generalised weakness and pitting oedema, and to be pigmented and Cushingoid in facial appearance. He had no cutaneous striae or ecchymoses and his blood pressure was 170/80 mm Hg. Preliminary investigations revealed that his plasma potassium was only 1.7 meq./1. After oral and intravenous potassium replacement therapy he was transferred to St. Mary’s Hospital with the suggested diagnosis of Cushing’s syndrome.
When examined, he was confused and euphoric and was noted to have a mild diffuse symmetrical peripheral neuropathy of predominantly sensory type. A clinical diagnosis of Cushing’s syndrome associated with a bronchial carcinoma was made on the basis of his pigmented appearance, the very low plasma potassium (Bagshawe 1960) and the presence of a sensory neuropathy (Denny-Brown 1948).
Preliminary investigations. Haemoglobin 64 %; white cell count 8000/mm3 with 100 eosinophils/mm3; erythrocyte sedimentation rate 117 mm/h; plasma sodium 134 meq./1;
potassium 1.9 meq./1; TCO2 42.0 mM/l; urea 35 mg/100 ml; calcium 9.3 mg/100 ml; inorganic phosphorus 3.9 mg/100 ml; liver function tests (bilirubin, alkaline phos- phatase, cholesterol, SGOT, SGPT, total proteins and protein electrophoresis) were all normal. His initial urinary potassium excretion without potassium supplements was 57 meq./24 h. There was persistent glycosuria and the 2 hour postprandial blood sugar concentration lay in the diabetic range (186 mg/100 ml). Urinary excretion of 5- hydroxyindole acetic acid was normal (6.8 mg/24 h). Radiological investigation of the chest, including tomography, showed the presence of a rounded opacity in the right lower zone and increased shadowing in the right hilar zone. There was no radio- logical evidence of osteoporosis and no vertebral collapse. The pituitary fossa was not enlarged. Retroperitoneal air-insufflation revealed bilateral adrenal enlargement.
The results of the preliminary studies of adrenocortical function are given in Table 1. The markedly elevated plasma and urinary steroid values and the high cortisol pro- duction rate confirmed the diagnosis of Cushing’s syndrome, and the raised circulating level of »ACTH« suggested that this was associated with bilateral adrenal hyper- plasia. The plasma level of aldosterone was within normal limits.
Subsequent course. Therapy with 12 g daily of effervescent potassium chloride by mouth resulted in a marked clinical improvement associated with a rise of the plasma potassium to between 3.0 and 3.7 meq./1. The patient became less confused and euphoric and was able to stand and walk without help.
On 28. 12. 65 an exploratory thoracotomy (Mr. L. Bromley) confirmed the presence of a bronchial neoplasm in the right lower lobe together with extensive lymphatic involvement. The largest posterior hilar gland, approximately 4 cm in diameter, was resected and was examined histologically and assayed for corticotrophic and melano- cyte stimulating activity. Following thoracotomy plasma 11-hydroxycorticoid (11- OHCS) levels lay in the range 17 to 30.0 ug/100 ml (see Fig. 1) and were lower than in the pre-operative period. It seems unlikely that this was due to removal of a single metastasis since more than 90 % of tumour tissue had been left in situ. Unfortunately
| Patient's values | Normal range | Method employed | |
|---|---|---|---|
| Urinary 17-KS (mg/24 h) | 21.6-26.4 | 5-19 | James & Caie (1964) |
| Urinary 17-OHCS (mg/24 h) | 80.7-109.4 | 8-20 | James & Caie (1964) |
| Plasma 11-Hydroxycorticoids | |||
| (ug/100 ml) | 44.6-55.3 | 5-24 | Mattingly (1962) |
| Plasma cortisol (ug/100 ml) | 53.9 | 5-20 | James & Fraser (1966) |
| Plasma corticosterone | |||
| (ug/100 ml) | 2.6 | 0-2 | James & Fraser (1966) |
| Plasma aldosterone | |||
| (mug/100 ml) | 9 | 1-17 | James & Fraser (1966) |
| Cortisol production rate | |||
| (mg/24 h) | 245 | 9-31 | James & Caie (1964) |
| Plasma »ACTH« (uug/ml) | 125 | less than | Yalow et al. (1964) |
50
Metyrapone.
30/ day
(56) . (60)
4’5 g / day
30
Plasma 11-OHCS (pg / 100ml)
www
wwwwww
20
Normal
10
Range
wwwww
wwwwww
0
120
Urinary 17-OHCS
(mg/ 24 hrs)
80
40
wwwww
www
Normal Range
0
1
5
10
15
20
25
30
10
15
12
January.
Febuary
May. 1966.
urinary 17-hydroxycorticosteroid (17-OHCS) determinations were not performed at this time.
Subsequent treatment was confined to an attempt to ameliorate the effects of adrenocortical hyperfunction medically as the neoplasm was inoperable and the patient’s poor physical condition would have rendered bilateral adrenalectomy hazardous. On the 5. 1. 66 he was started on the 118-hydroxylation inhibitor Metyrapone. During the initial 6 hour infusion at a rate of 500 mg/h, plasma 11-OHCS fell from 17.0 to 3.8 ug/100 ml. Treatment was continued orally with doses of 500 mg 4 hourly later in- creasing to 750 mg 4 hourly. It was not possible to give the drug less frequently be- cause plasma 11-OHCS began to rise after 4 hours (see Fig. 2). During the administra- tion of Metyrapone plasma 11-OHCS values were maintained within the normal range, glycosuria ceased and the plasma potassium increased from 3.2 to 4.0 meq./1. Urinary 17-OHCS levels were markedly elevated and were similar to the values obtained immediately after his admission. The drug was discontinued after 14 days because the patient developed anorexia, diarrhoea, oliguria and hypotension together with a pyrexia of 38.9º C. These symptoms rapidly improved after stopping Metyrapone and urinary 17-OHCS values fell to within normal limits for the first time since his ad- mission. His plasma 11-OHCS remained in the upper range of normal after an initial rebound (Fig. 1). During this apparent remission plasma »ACTH« levels were below the sensitivity of the assay in our hands (less than 50 uug/ml). However, an in- fusion of dexamethasone failed to lower his plasma 11-OHCS suggesting that the bronchial neoplasm was continuing to secrete »ACTH«. Three weeks after discon- tinuing Metyrapone, urinary and plasma steroid values began to increase slightly. The patient was discarged from hospital and remained symptom-free for 3 months apart from continuing parasthesiae and progressive pigmentation. He was readmitted for a short period in May 1966 at which time his plasma 11-OHCS had increased to more
18
16
14
PLASMA II-OHCS ( p9/100ml.)
12
IO
8
6
4
2
0
O
2
3
4
5
6
HOURS.
than 55 ug/100 ml and his urinary 17-OHCS to more than 110 mg/24 h (Fig. 1). He died 2 weeks after returning home.
Pituitary-adrenal function tests. There was no circadian plasma 11-OHCS rhythm (10 a. m .: 44.6 ug/100 ml; 10 p. m .: 42.7 ug/100 ml). The oral administration of 0.5 mg q. d. s. of dexamethasone for 2 days then 2 mg q. d. s. for a further 2 days (Liddle 1960) caused no suppression of urinary steroid secretion. The intravenous infusion of dexamethasone caused no significant change in plasma 11-OHCS values either during the initial period of investigation or when the patient was in an apparent remission (Table 2). No firm conclusion can be drawn regarding the presence, or absence, of a pituitary-adrenal response to Metyrapone because urinary steroid levels were not determined immediately prior to administration of the drug. There was a normal plasma 11-OHCS response to a 5 hour infusion of ACTH, to a 2 hour infusion of lysine-vasopressin, to insulin-induced hypoglycaemia and to intravenously administered pyrogen (see Table 2).
Pathological findings. The histological report on the lymph node removed sur- gically was >>Almost the entire gland is replaced by an oat-cell carcinoma. The tumour is spreading into the periglandular fatty tissue and a small pulmonary artery contains tumour« (Dr. E. Neumark).
At autopsy the bronchial neoplasm was found to have invaded the surrounding tissues extensively and to have involved the hilar and mediastinal lymph nodes. The
| When performed | Patient | Control subjects | Procedure employed | ||
|---|---|---|---|---|---|
| No. | Range of responses | ||||
| Maximum plasma 11-OHCS | a) Prior to | 58 | 14.5-36.7 | Landon et al. | |
| increment (ug/100 ml) | Metyrapone | 23.2 | (1965 b) | ||
| above the basal level | |||||
| during a 5 hour infusion | b) Post | ||||
| of ACTH (10 IU/hour) | Metyrapone | 15.0 | |||
| Maximum plasma 11-OHCS | a) Prior to | ||||
| increment (ug/100 ml) above the basal level | Metyrapone | 8.5 | 19 | 5.6-33.5 | Landon et al. (1965 a) |
| during a 2 hour infusion | |||||
| of lysine-vasopressin | |||||
| (5 pU/hour) | |||||
| Maximum plasma 11-OHCS | a) Prior to Metyrapone | 22.8 | |||
| increment (ug/100 ml) above the basal level | 9 | 10.1-39.7 | Melby (1959) | ||
| following an intravenous | |||||
| injection of pyrogen (0.005 µg/kg body weight) | |||||
| Maximum plasma 11-OHCS | a) Prior to | 9.0 | |||
| increment (ug/100 ml) above the basal level | Metyrapone | 28 | 7.7-24.2 | Greenwood et al. (1966) | |
| following an intravenous injection of insulin (0.20 units/kg body weight) | |||||
| Minimum plasma 11-OHCS | a) Prior to | 104% | |||
| level during a 3 hour infusion of dexamethasone | Metyrapone | 10 | 23-37% | James et al. (1965) | |
| (1 mg/hour) expressed | |||||
| as a percentage of the | b) Post | ||||
| basal level | Metyrapone | 108% | |||
liver and peritoneum were the site of multiple metastases but neither the pituitary gland nor the adrenals contained macroscopic tumour deposits. Both adrenals were enlarged (combined weight 32 g) and showed marked hypertrophy of the zona reticu- laris and fasciculata.
Hormone assays. The primary tumour, a lymph node largely replaced by tumour tissue and the adrenals (to act as control material) were deep frozen prior to assay. After lyophilisation each was subjected to the procedure described by Lee et al. (1961)
Elution with ammonium acetate
0’005 M
0’020 M
0’075 M
0’20 M
PH 5’8
PH 5’8
PH 58
PH 70
Pituitary
ACTH
0’04
d MSH
₿ MSH
0
280 mp
Synthetic ACTH
1.0
0
0
Synthetic B
1- 24
Density
ACTH
005
Optical
0
Primary
Tumour
004
0
Metastasis
004
0
60
120
180
240
Tube
No.
for the isolation and purification of ACTH and a- and -MSH from pituitary glands. The final stage in this procedure involves discontinuous elution of these materials from a carboxymethylcellulose column with ammonium acetate of increasing molarity and pH. Pituitary extracts treated in this way give several peaks including one for B-MSH another for a-MSH and a third for ACTH (see Fig. 3). A discrete peak running in the position of ACTH was obtained from the primary tumour extract (containing 0.98 mg of material) and from the extract of the metastasis (containing
0.39 mg of material). This was immunologically similar to human pituitary ACTH by radioimmunoassay (Yalow et al. 1964). The extract from the metastasis gave a small peak corresponding in position to a-MSH (containing 0.031 mg of material). This was bioassayed by a modification of the method of Shizume et al. (1954) based on direct microscopic observation of melanocyte expansion in frog skin. The material extracted from the metastasis had an activity that was equivalent to that produced by synthetic a-MSH and 300 times greater than the melanocyte-stimulating activity of the tumour »ACTH«. The physicochemical, biochemical and immunological properties of materials obtained from this and other patients’ tumours will be the subject of a future com- munication.
DISCUSSION
It is now clearly recognised that a variety of tumours possess the ability to synthesise and release peptides whose biological activity is, in many cases, indistinguishable from hormones which are normally produced in significant quantities only by endocrine tissues. More than two hundred cases have been reported in which there has been presumptive evidence for the production of corticotrophin or a corticotrophin-like material in that Cushing’s syndrome was present coincidentally with an extrapituitary tumour.
In a patient presenting with this condition the demonstration of the presence of »ACTH« in the tumour, or its metastases, is obligatory before it may be concluded that the association is not fortuitous. The observation of elevated plasma »ACTH« levels is, by itself, not sufficient since »ACTH« of pituitary and of ectopic origins cannot be distinguished. »ACTH« activity has been demonstrated in a variety of such neoplasms by bioassay. These include car- cinomata of the trachea and bronchus, thymus, ovary, pancreas, parotid, prostate and adrenal medulla (Holub & Katz 1961; Meador et al. 1962; Liddle et al. 1963; Marks et al. 1963; Pfohl & Doe 1963; Tretbar & Cawley 1963; Hallwright et al. 1964; Jarett et al. 1964; Liddle et al. 1964; Lemon et al. 1966; Nelson et al. 1966). In the present case, using radioimmunoassay, elevated plasma >ACTH« levels were found and the presence of >>ACTH« was demonstrated in an oat-cell bronchial neoplasm and its metastases. This material could not be distinguished immunologically from human corticotrophin. A material with melanocyte-stimulating activity equivalent to that of pure synthetic @-MSH was detected, by bioassay, in the metastases. Although only present in small amounts it was 300 times more active in MSH-activity than ACTH and may explain the marked degree of pigmentation in this patient. Hallwright et al. (1964) and Shimizu et al. (1965) have also presented evidence for the presence of two distinct peptides, one with melanocyte-stimulating activity and the other with corticotrophic activity, in tumours removed from patients with this syndrome.
The ability of certain neoplasms to produce peptides which, in some cases
at least, are indistinguishable from hormones originating in normal endocrine tissues is a puzzling phenomenon. Although it is possible that such tumours may arise from embryological cell rests, there is no support for this idea from morphological studies. A more attractive hypothesis would seem to derive from the theories of Jacob (1961) and Monod (1963), based on the supposition that whilst all body cells contain the genetic information necessary for the synthesis of peptide hormones this information is only expressed in cells of the appropriate endocrine tissues, and is masked in others. Ectopic hormone pro- duction would imply a loss of this genetic repressor mechanism in certain neoplasms.
Detailed information on the structure of hormones produced by tumour cells would be of considerable value in that dissimilarities from normally produced hormones would argue against the hypothesis discussed above. With regard to corticotrophin, available evidence (Liddle et al. 1964; Jarett et al. 1964) sug- gests that ‘ACTH’ of tumour and pituitary origin are identical. The present study supports this view. Thus while it is possible that the tumour might secrete peptides containing only that part of the ACTH molecule necessary for biological activity, namely the N-terminal sequence, studies using a carboxy-methyl cellulose column (Fig. 3) show that tumour »ACTH« is eluted in a similar position to pituitary ACTH, and its behaviour is different chroma- tographically from synthetic §1-24 ACTH. The finding that the »@-MSH« in the present study had equivalent melanocyte-stimulating activity to synthetic «-MSH is also relevant since it has been shown that slight alterations in the structure of this hormone, for example the absence of the N-terminal acetyl group, are associated with a marked loss of activity (Hoffman & Yajima 1961; Guttman & Boissonnas 1961).
Usually the signs and symptoms of Cushing’s syndrome occur as a com- plication of a pre-existing tumour or of a malignancy easily recognised at the time of diagnosis. There are several recorded cases, however, in which bilateral adrenal hyperplasia and Cushing’s syndrome has predated, sometimes by years, the clinical recognition of an extra-pituitary neoplasm. It is important from a prognostic and therapeutic standpoint to differentiate these cases from the more common group in which bilateral adrenal hyperplasia and Cushing’s syndrome is the result of excessive ACTH production by the pituitary gland (Cushing’s disease). Certain clinical features help differentiate these two con- ditions and the finding of hypokalaemic alkalosis should alert the physician to the possibility of an underlying neoplasm (Bagshawe 1960). Nevertheless, the presence of a normal electrolyte pattern does not exclude this syndrome (Thompson et al. 1962; Friedman et al. 1965). A survey of the literature was, therefore, undertaken to determine which tests of pituitary-adrenal function, if any, are of value in differentiating patients with this syndrome from those with Cushing’s disease. Although 116 separate communications were found
concerning more than 200 patients, data relating to the results of pituitary- adrenal function tests were available in only a minority.
The single most valuable differentiating test appears to be that based on the administration of dexamethasone. In adequate doses dexamethasone causes par- tial adrenal suppression in the majority of patients with Cushing’s disease (Liddle 1960), but caused no suppression in 32 of 40 patients with ectopic »ACTH« production. In those showing some suppression (Metzler & Flink 1956; Balls et al. 1959; Escovitz & Reingold 1961; Micić & Arsenijević 1963; Brooks et al. 1963; Pfohl & Doe 1963; Friedman et al. 1966; O’Riordan et al. 1966) it is possible that the lowered steroid values were the result of a spontaneous change in »ACTH« production. It should also be noted that some patients with bilateral adrenal hyperplasia due to pituitary dysfunction do not suppress with dexamethasone (James et al. 1965). A further complication arises from the fact that dexamethasone will not normally cause suppression of steroid ex- cretion in a patient in whom Cushing’s syndrome is caused by an adreno- cortical carcinoma, and this possibility must also be borne in mind. In this situation, plasma ACTH levels would not be expected to be elevated (Nelson et al. 1966). Further study of reported cases revealed that 20 of 30 cortico- trophin stimulation tests and 8 out of 12 metyrapone tests gave positive responses. A feature in the remainder, who had an impaired or absent response, was that the basal steroid values were usually very high with urinary cortico- steroid levels in excess of 100 mg/24 h. It would appear that the presence or absence of an adrenal response to these tests is related to basal adrenal activity. It is reasonable to suppose that if the ectopic secretion of »ACTH« is sufficient to cause maximum adrenal stimulation then no test will evoke a response. The patient reported here had a normal adrenal response to ACTH, lysine vasopressin, pyrogen, insulin hypoglycaemia and probably to metyra- pone. These findings may be interpreted as indicating a degree of adrenal and pituitary reserve, and a functional hypothalamic-pituitary-adrenal axis (Landon et al. 1965 a). In those patients in whom adrenal secretion is not maximal the determination of the plasma corticosteroid response to insulin- induced hypoglycaemia may be of differential diagnostic value, since patients with Cushing’s disease do not respond (Landon et al. 1965 b; Bethge et al. 1965).
The treatment of patients with this syndrome poses considerable problems. Ideally the tumour, and hence the source of ectopic ‘corticotrophin’ should be resected; however, this is seldom possible. Adrenalectomy has been employed in some cases and is a desirable procedure where the tumour is slow growing since the immediate hazard to life is the metabolic disorder arising from adrenocortical hyperfunction. This surgical procedure is not easily withstood by a severely ill patient, which emphasises the importance of early recognition of the condition. In advanced cases the 118-hydroxylation inhibitor Metyra-
pone has been used with varied success (Mattingly et al. 1964; O’Riordan et al. 1966). In the present study the administration of large amounts of this drug (500-750 mg 4 hourly) resulted in a correction of plasma corticosteroid and potassium levels and the cessation of glycosuria. Unfortunately, side-effects necessitated the cessation of therapy. Subsequently, urinary steroid values fell to within normal limits for more than three weeks. Although this may have been the result of a spontaneous partial remission it is of interest that Mattingly et al. (1964) report a similar experience.
ACKNOWLEDGEMENTS
We are grateful to Dr. G. F. Marrian, F. R. S. and Dr. F. C. Greenwood, for pro- viding one of us with the facilities and guidance to undertake the tumour extractions and ACTH and MSH assays at the Imperial Cancer Research Fund.
REFERENCES
Allott E. N. & Skelton M. O .: Lancet 2 (1960) 278.
Bagshawe K .: Lancet 2 (1960) 284.
Balls K. F., Nicholson J. T. L., Goodman H. L. & Touchstone J. C .: J. clin. Endocr. 19 (1959) 1134.
Bethge H., Winkelmann W. & Zimmermann H .: Acta endocr. (Kbh.) 51 (1966) 166.
Bornstein P., Nolan J. P. & Bernanke D .: New Engl. J. Med. 264 (1961) 363.
Brooks R. V., Dupre J., Gogate A. N., Mills I. H. & Prunty F. T. G .: J. clin. Endocr. 23 (1963) 725.
Brown W. H .: Lancet 2 (1928) 1022.
Christy N. P .: Lancet 1 (1961) 85.
Denny-Brown D .: J. Neurol. Neurosurg. Psychiat. 11 (1948) 73.
Escovitz W. E. & Reingold I. M .: Ann. intern. Med. 54 (1961) 1248.
Friedman M., Mikhail J. R. & Bhoola K. D .: Brit. med. J. 1 (1965) 27.
Friedman M., Marshall-Jones P. & Ross E. J .: Quart. J. Med. 35 (1966) 193.
Greenwood F. C., Landon J. & Stamp T. C. B .: J. clin. Invest. 45 (1966) 429. Guttman St. & Boissonnas R. A .: Experientia (Basel) 17 (1961) 265.
Hallwright G. P., North K. A. K. & Reid J. D .: J. clin. Endocr. 24 (1964) 496.
Hoffman K. & Yajima H .: J. Amer. chem. Soc. 83 (1961) 2289.
Holub D. A. & Katz F. H .: Clin. Res. 9 (1961) 194.
Jacob F .: J. molec. Biol. 3 (1961) 318.
James V. H. T. & Caie E .: J. clin. Endocr. 24 (1964) 180.
James V. H. T. & Fraser R .: J. Endocr. 34 (1966) 16.
James V. H. T., Landon J. & Wynn V .: J. Endocr. 33 (1965) 515.
Jarett L., Lacey P. E. & Kipnis D. M .: J. clin. Endocr. 24 (1964) 543.
Landon J., James V. H. T. & Stoker D. J .: Lancet 2 (1965 a) 1156.
Landon J., James V. H. T., Wood J. B. & Wynn V .: J. clin. Endocr. 25 (1965 b) 602.
Lee T. H., Lerner A. B. & Buettner-Janusch V .: J. biol. Chem. 236 (1961) 1390.
Lemon F. C., Fine M. B., Grasso S. G. & Kinsell L. W .: J. clin. Endocr. 26 (1966) 1. Liddle G. W .: J. clin. Endocr. 20 (1960) 1539.
Liddle G. W., Island D. P., Ney R. L., Nicholson W. E. & Shimizu N .: Arch. intern. Med. 111 (1963) 471.
Liddle G. W., Givens J. R., Nicholson W. E. & Island D. P .: Proc. 2nd Int. Congr. Endocr. 2 (1964) 1063.
Marks L. J., Rosenbaum D. L. & Russfield A. B .: Ann. intern. Med. 58 (1963) 143.
Mattingly D .: J. clin. Path. 15 (1962) 374.
Mattingly D., Keane P. M., Mccarthy C. F. & Reid A. E .: Bristol med. chir. J. 79 (1964) 6.
Meador C. K., Liddle G. W., Island D. P., Nicholson W. E., Lucas C. P., Nuckton J. G. & Luetscher J. A .: J. clin. Endocr. 22 (1962) 693.
Melby J. C .: J. clin. Invest. 38 (1959) 1025.
Metzler C. & Flink L. B .: J. Lancet 76 (1956) 242.
Mićić R. & Arsenijević M .: Lancet 2 (1963) 436.
Monod J .: J. molec. Biol. 6 (1963) 306.
Nelson D. H., Sprunt J. G. & Mims R. B .: J. clin. Endocr. 26 (1966) 722.
O’Riordan J. L. H., Blanshard G. P., Moxham A. & Nabarro J. D. N .: Quart. J. Med. 35 (1966) 137.
Pfohl R. A. & Doe R. P .: Ann. intern. Med. 58 (1963) 993.
Shimizu N., Ogata E., Nicholson W. E., Island D. P., Ney R. L. & Liddle G. W .: J. clin. Endocr. 25 (1965) 984.
Shizume K., Lerner A. B. & Fitzpatrick T. B .: Endocrinology 54 (1954) 553.
Thompson G. S., Horwich L. & Davis J. C .: Lancet 2 (1962) 534.
Tretbar H. A. & Cawley L. P .: J. Kansas med. Soc. 64 (1963) 487.
Yalow R. S., Glick S. M., Roth J. & Berson S. A .: J. clin. Endocr. 24 (1964) 1219.
Received on January 4th, 1967.