STEROID DETERMINATIONS IN SIX CASES OF HYPERPLASIA AND THREE CASES OF TUMOUR OF THE ADRENAL CORTEX
N. Norman, K. R. Reksten and J. H. Vogt
From the Hormone Laboratory and Medical Department B, Aker Sykehus, Oslo, Norway
Abstract. A report is given of nine cases of adreno- cortical hyperfunction; six of these were due to hyper- plasia, two to carcinoma, and one to adenoma of the adrenal cortex. The cases were subjected to tests of ACTH stimulation, 118-hydroxylase inhibition with meto- pirone, and suppression with dexamethasone. In urine col- lections obtained before the tests, the following corti- costeroid metabolites were determined: tetrahydro S, preg- nanediol, pregnanetriol, dehydroepiandrosterone, aetio- cholanolone, androsterone, aldosterone, oestriol and “free” cortisol. Plasma cortisol values were obtained at 8 a.m. and 8 p.m.
The experience gained in this series can be summarized as follows:
Plasma cortisol determined at night is a useful screen- ing test in suspected adrenocortical hyperfunction. The “resting” 17 ketosteroid (17-KS) and 17-ketogenic steroid (17-KGS) excretion is too inclusive for this purpose, as slight elevations are seen in obesity and anxiety. The occasional adrenal adenoma may furthermore be missed. Suppression tests performed with dexamethasone and evaluated on the basis of corticosteroid determinations in plasma or urine conclude the screening procedures. The patients who remain suspect of having adrenocortical disease subsequent to the screening procedures should be investigated with as complete a set of procedures as are available. An extensive spectrum is necessary if a firm and differential diagnosis is to be made on the bio- chemical evidence.
The ACTH stimulation has been of value in the dif- ferentiation between normal function and the excessive function of adrenal hyperplasia, but not in the distinc- tion between hyperplasia and tumours, as the latter cases also responded with increases in output.
Metopirone is very useful in differential diagnosis, particularly if supplemented with tetrahydro S determina- tion. The tumour cases had elevated values of tetra- hydro S in the “control” urine samples, and showed no increase in 17-KS and 17-KGS during the test.
Dexamethasone, 2 mg q.i.d. reduced the urinary 17-KS and 17-KGS in the hyperplasia cases, but not in the tumours.
The metabolites from the different “levels” of adreno- cortical steroid synthesis were elevated at various degrees,
and rather irregularly in the carcinoma cases. In the cortisol-producing adenoma the urinary metabolites of adrenal androgens were excreted in subnormal amounts.
During recent years an extensive spectrum of chemical steroid analyses has become available to clinicians at larger medical centres. The applica- tion of these analytical methods to the various forms of adrenocortical hyperfunction has been of help to the clinician in explaining some of the pathophysiology of the conditions, and in provid- ing diagnostic tests. The proper application of these tests, and their interpretation and evaluation, are current problems.
Lipsett and Wilson (20) gave a clear account of the abnormal quantitative distribution of urinary steroid metabolites in ten cases of adrenal cortical carcinoma. They emphasized the value of the metabolite tetrahydro S as a specific indicator of carcinoma, an observation made earlier by Jailer et al. (16). Pal and James (28) demonstrated an enormous increase in DHA and pregnenetriol in the urine of one case of adrenocortical car- cinoma, a less marked increase in another case and moderate increases in several cases of hyper- plasia. In the case described by Harrison et al. (15) the high DHA excretion was combined with an increased output of oestrogen. Martin and Hamman (21) determined the metabolites preg- nanedial and pregnanetriol before and after ACTH stimulation. They found the results of great help in assessing the aetiology of Cushings’ syndrome, thereby supporting the findings of Damkjær Nielsen et al. (6). In view of the wide spectrum of steroids normally secreted from the adrenal cortex (8) and the number of enzymatic systems involved, it is possible that similar ob-
servations can be made regarding other metabo- lites.
The other approach to the differential diagnosis of the various causes of Cushings’ syndrome is provided by tests of the functional capacity of the pituitary-adrenal axis, with use of ACTH, dexamethasone and metopirone. Birke et al. (2) demonstrated an enhanced response to ACTH in adrenal hyperplasia, but reduced responses in adrenal adenoma and carcinoma, the parameter of the measurement being the urinary excretion of 17-hydroxycorticosteroids. Ernest (14), on the other hand, did not find this test particularly helpful in the differentiation of diagnoses. The suppression test using dexamethasone at the dose levels of 0.5 mg or 2 mg q.i.d. was found very useful by Liddle (18) in a large series of patients. Others (13, 17) maintain that the response to the test must be evaluated with caution, as misleading results can be obtained, particularly in nodular hyperplasia. Irregular responses may also occur due to marked variation in basal excretion of steroids. Inadequate specificity of the methods generally used for the determination of urinary corticosteroids may also give rise to misleading results. The evidence presented by Jailer et al. (16), that the usefulness of the metopirone test for the diagnosis of adrenal carcinoma is de- pendent upon the simultaneous determination of tetrahydro S and 17-ketogenic steroids, is sup- ported by our observations (26). An excellent survey of the various tests of function has re- cently been presented by Cope (5); Ross et al. (31) have successfully related corticosteroid analy- ses and tests (or at least some of them) to clinical and biochemical aspects of Cushings’ syndrome.
In the cases to be presented in this paper a rather wide spectrum of urinary steroids has been determined, as well as the response to ACTH, metopirone and dexamethasone. This then allows a certain comparison and evaluation of the merits of the different analytical approaches. The diag- noses have been verified by operation.
MATERIAL
A survey of the case material is presented in Table I. Cases 1 and 2 had large carcinomas; case 3 was found to have a small adenoma, the remainder has been classi- fied as bilateral hyperplasia.
METHODS
ACTH stimulation
ACTH in the form of a long-acting preparation (Jaton Prolongatum A.L.) was given intramuscularly in a dose of 120 IU at 10 p.m. for a sequence of three days. The urine collections started at the time of injection and con- tinued for the following 24 hours. 17-KS and 17-KGS determinations were performed in the three 24 hour urine collections during stimulation, as well as in samples from the two 24 hour periods preceding the ACTH administra- tion. The results presented in this article were obtained on the third day of stimulation.
Metopirone test
Metopirone was given at 8 a.m., 10 a.m., 12 a.m., 2 p.m., 4 p.m., 6 p.m., 8 p.m. and 10 p.m. at a dose level of 250 mg; this sequence of administration was continued with 750 mg at midnight and at 4 a.m. The total dose therefore amounted to 3.5 g within 24 hours. Urine was collected for 24 hour periods before, during and after the metopirone administration. In these urine collections 17-KS and 17-KGS, as well as the metabolite tetrahydro S, were determined. The figures shown in the tables are from the first and the last day of the test.
Dexamethasone suppression
Dexamethasone was given at two dose levels, 0.5 mg q.i.d. for two days being followed by 2 mg q.i.d. for two days. Urine was collected for 17-KS and 17-KGS deter- mination during administration, as well as for the two days preceding the test. The figures presented in the tables were obtained on the last day of each dose.
Chemical methods for steroid determination (in outline) 17-KS and 17-KGS were determined by the method of Norymberski et al. (27) as modified by Diczfalusy et al. (7). The Zimmermann reaction was performed using the organic base N-benzyl-trimethyl ammonium methoxide ac- cording to Bongiovanni et al. (3).
Tetrahydro S was measured by the Porter-Silber reac- tion after initial hydrolysis with 8-glucuronidase, chloro- form extraction, purification of the extract, and chro- matography in the systems toluene/propylene glycol fol- lowed by a modified Bush B5. The procedure is described in more detail elsewhere (24).
Oestriol was measured by the modified method of Eberlein et al. (10) based on the original procedure of Brown and Bauld.
The procedure for aldosterone closely resembled the method of Neher and Wettstein (23), but the chromatog- raphy was continued in a third system, Bush Bs, and quantitation obtained by the isoniazide colour reaction (33). Further details of the procedure have been presented elsewhere (25).
In the cases of pregnanediol and pregnanetriol, the urine samples were hydrolyzed with 6-glucuronidase and extracted with chloroform. After purification and evap- oration of the extracts, chromatography was performed on paper with petroleum ether (100°-120°)/toluene (2 : 1) as the mobile phase, and 85% methanol in water as the stationary phase. The final quantitation was achieved with the bisulfite-sulfuric acid reaction (9). For pregnan-
| Case | Age | Sex | Symptoms and signs | Adrenal pathology | Adrenal weight |
|---|---|---|---|---|---|
| 1 | 24 | 4 | Moderate hirsutism for 2 years. Metrorrhagia. No features of Cushing's syndrome. Four years postop. without recurrence | Encapsulated, irregular tumour, large areas of necrosis, nodules 4-5 cm diameter with cells slightly resembling zona fasc., marked criteria of malignancy in cell nuclei | 1700 g Right side |
| 2 | 62 | 9 | For 6 months moderate hirsutism. Periods of failing mental orientation. Incipient features of Cushing's syndrome | Encapsulated tumour with necrosis, studded with pea sized nodules of carcinomatous cells of rather low differentiation | 480 g Right side |
| 3 | 31 | 우 | Tendency to oedema formation during 3 years. Oligo menorrhoea. Development of features of Cushing's syndrome during period of observation | Encapsulated tumour, cells resembling those of zona reticularis, well differentiated. Marked atrophy of cortical tissue adherent to tumour | 9.5 g Left side |
| 4 | 28 | ? | For 1.5 to 2 years typical features of Cushing's syndrome, complete. Amenorrhoea for 6 months | Slight hyperplasia right side, left side areas of necrosis. Fragment of right adrenal left in situ | Right 5.2 g Left 5.0 g |
| 5 | 33 | ? | For 6 months typical features of Cushing's syndrome in complete form | Histological signs of hyperplastic overactivity both sides. Fragment left in situ on right side | Right 7.4 g Left 6.3 g |
| 6 | 49 | ? | Diagnosed Cushing's syndrome for 15 years. Right adrenal removed 14 years ago, admission for recurrence | Histologically irregular hyperplasia both sides | Left 12.9 g |
| 7 | 45 | 우 | Typical features of Cushing's syndrome for 6 years | Hyperplasia with signs of ACTH stimulation both sides. Fragment of left gland left in situ | Right 7.9 g Left 6.3 g |
| 8 | 61 | 우 | Hirsutism and reddish-cyanotic colour of face, increasing during last 2 years | Bilateral hyperplasia, hyperplastic nodule | Right 6.0 g Left 6.3 g |
| 9 | 45 | 3 | Features of Cushing's syndrome for 5 years | Bilateral hyperplasia, signs of active ACTH stimulation | Right 12.0 g Left 14.2 g |
ediol the colour was read at 390, 425 and 460 mu; for pregnanetriol at 405, 440 and 475 mu, the specific ex- tinctions being obtained by application of the correction of Allen (1).
To determine dehydroepiandrosterone, aetiocholanolone, androsterone, the urinary sulphates were precipitated with barium chloride, the urine centrifuged and the super- natant incubated with a mixture of 8-glucuronidase and sulphatase (Glusulase, Endo Laboratories) for 48 hours, extra enzyme being added after 24 hours of incubation. After an initial extraction with ether, the urine was boiled with hydrochloric acid and re-extracted. The combined ether extract was washed with 2 N NaOH and distilled water, evaporated to dryness, and subjected to paper chromatography in the system isooctane/methanol/water (10 : 9 : 1). The ketosteroids were stained on the paper, and the selected coloured spots eluted according to the method of Epstein and Zak (12). Quantitation was ob- tained by photometry at 430, 515 and 600 mu with the application of the correction of Allen (1).
The urinary “free” cortisol was determined after a procedure of hydrolysis, extraction and chromatography corresponding to that of aldosterone in these cases. Plasma “cortisol” was measured by a method similar to that of Peterson et al. (30). For both of these cortisol determina-
tions the final measurement was made with the Porter- Silber reaction, reading at 370, 410, and 450 mu, and using the correction of Allen (1).
The urine samples for the determination of the spec- trum of steroid metabolites were collected shortly after admission to the hospital, and before the patients were subjected to tests or treatment.
RESULTS
The amounts of the steroid metabolites excreted in the urine of the patients are presented in Table II. Most of the figures are means of two values obtained in separate 24 hour urine samples, a few are from one urine collection only, and some are based on three or more collections. The values for the same metabolite in each patient were usually in close agreement and always in the same range. It is to be noted that the figures for pregnanediol and pregnanetriol in case 1 were
| Carcinoma | Adenoma | Hyperplasia | Normal | |||
|---|---|---|---|---|---|---|
| Cases 4-9 | ||||||
| Case 1 | Case 2 | Case 3 | Mean | Range | Range | |
| 17-ketogenic steroids, mg/24 h | 37.9 | 50.7 | 16.7 | 36.9 (6 cases) | 65.6 | |
| 23.1 | 5-15 | |||||
| 17-ketosteroids, mg/24 h | 42.5 | 24.6 | 2.8 | 14.0 (6 cases) | 28.1 | |
| 7.0 | 5-15 | |||||
| Pregnanediol, mg/24 h | 46.9ª | 3.7 | 0.5 | 1.9 (3 cases) | 2.2 | |
| 0.6 | < 1b | |||||
| Pregnanetriol, mg/24 h | 46.3ª | 1.4 | 0.2 | 0.3 (3 cases) | 0.6 | |
| 0.1 | <: 0.56 | |||||
| Tetrahydro S, mg/24 h | 5.9 | 7.5 | 0.58 | 0.26 (5 cases) | 1.2 | |
| 0 | < 0.2 | |||||
| Aldosterone, ug/24 h | 8.1 | 24 | 5-28 | 8.1 (3 cases) | 14.0 | |
| 4.6 | 5-12 | |||||
| Dehydroepiandrosterone, mg/24 h | 8.5 | 2.3 | 0.1 | 1.1 (5 cases) | 5.1 | |
| 0.3 | 0.2-1.5 | |||||
| Androsterone, mg/24 h | 22.0 | 1.9 | 0.1 | 2.0 (5 cases) | 4.0 | |
| 0.8 | 1.5-4.5 | |||||
| Aetiocholanolone, mg/24 h | 10.4 | 10.7 | 0.5 | 4.3 (5 cases) | 8.9 | |
| 1.4 | 1.5-4.5 | |||||
| Oestriol, µg/24 h | 81 | 256 | 11.0 | 17.1 (2 cases) | 27.3 | |
| 9.8 | < 106 | |||||
| Urinary "free" cortisol, µg/24 h | 80 | 62.5 (2 cases) | 80.0 | |||
| 44.5 | ≤30 | |||||
| Plasma Morning | 27 | 36 | 37.6 | 28.5 (4 cases) | 44.0 | |
| cortisol | 21.6 | 15-28 | ||||
| Night | 40 | 41.6 | 36.5 (4 cases) | 64.0 | ||
| 22.2 | 5-10 | |||||
” Determined in a different urine sample, separated from the rest of the analyses in this case by a time interval of 6 weeks. b Range for males, and women without cyclic ovarian function.
obtained in a different urine collection, separated by six weeks, from the urine collections on which the other results from this patient are based.
The range of normal values to the right in Table II are modified slightly from the standard normal range in order to be more closely appli- cable to the presented group of cases. The values for oestriol and pregnanediol are given without any consideration to the cyclic ovarian increases in output.
As can be seen in Table II, some of the param- eters seem to be useful guides in separating the tumour cases, as well as the hyperplasias, from normal individuals. The increased level of plasma cortisol at night with the resulting obliterated diurnal variation is common to both types of hyperfunction. The urinary “free” cortisol also showed an increase in both types in the few cases in which it was carried out. The 17-KGS excre- tion was above normal in all instances, and in most of the cases very markedly so. Extensive overlap
into the normal range was found for the plasma cortisol values obtained in the morning, and also for the 24 hour 17-KS excretion. For the latter parameter this applies mainly to the hyperplasias.
Distinguishing features in the two cases of car- cinoma are the pronounced elevation of the tetra- hydro S excretion, and the high DHA, aetiocho- lanolone, androsterone and oestriol. In case 1 the amount of pregnanediol excreted is higher than during the last three months of a normal preg- nancy, and pregnanetriol has a corresponding ex- cessive rate of excretion. These two parameters also show a clear increase above the normal in case 2, when age and lack of cyclic ovarian func- tion are considered. The adenoma case presents a different pattern; tetrahydro S is only moderately increased, and the excretion of 17-KS, androster- one, aetiocholanolone and DHA is below normal. Plasma cortisol is clearly elevated with absent diurnal variation; the 17KGS excretion is, on the other hand, slightly above the upper normal range.
Acta med. scand. 183
| ACTH | Metopirone | Dexamethasone | ||||
|---|---|---|---|---|---|---|
| Resting | Response | Resting | Response | 0.5 mg × 4 | 2 mg × 4 | |
| Carcinoma (case 1) | ||||||
| 17-ketogenic steroids, mg/24 h | 37.9 | 36.8 | 33.6 | 43.2 | 78.9 | |
| 17-ketosteroids, mg/24 h | 42.5 | 122.8 | 82.6 | 86.4 | 29.6 | |
| THS, mg/24 h | 5.9 | 14.9 | ||||
| Carcinoma (case 2) | ||||||
| 17-ketogenic steroids, mg/24 h | 50.7 | 107.3 | 38.9 | 37.8 | 61.7 | 63.8 |
| 17-ketosteroids, mg/24 h | 24.6 | 37.4 | 13.9 | 18.3 | 30.5 | 25.0 |
| THS, mg/24 h | 7.5 | 15.3 | ||||
| Pregnanetriol, mg/24 h | 1.4 | 6.3 | ||||
| Pregnanediol, mg/24 h | 3.7 | 7.6 | ||||
| Aldosterone, ug/24 h | 24 | 38 | ||||
| "Free" cortisol, ug/24 h (urine) | 80 | 80 | ||||
| Oestriol, ug/24 h | 258 | 39.2 | ||||
| Adenoma (case 3) | ||||||
| 17-ketogenic steroids, mg/24 h | 16.7 | 31.6 | 20.8 | 14.0 | 19.4 | 19.3 |
| 17-ketosteroids, mg/24 h | 2.8 | 6.4 | 4.2 | 1.8 | 2.2 | 6.3 |
| THS, mg/24 h | 0.53 | 2.0 | ||||
| Plasma cortisol, ug/100 ml | 33.6 | 50.6 | ||||
| Starting value (mg/24 h) | Response value (mg/24 h) | Percent variation response value × 100 starting value | ||||
|---|---|---|---|---|---|---|
| Mean | Range | Mean | Range | Mean | Range | |
| ACTH, 5 cases | ||||||
| 17-ketogenic steroids | 34.6 | 55.2 | 104.8 | 136.0 | 304 | 560 |
| 22.6 | 45.7 | 151 | ||||
| 17-ketosteroids | 10.4 | 16.9 | 25.7 | 37.7 | 247 | 390 |
| 6.9 | 15.3 | 192 | ||||
| Metopirone, 3 cases | ||||||
| 17-ketogenic steroids | 32.3 | 38.6 | 69.5 | 105.1 | 215 | 272 |
| 28.9 | 46.2 | 160 | ||||
| 17-ketosteroids | 15.1 | 19.1 | 20.3 | 22.2 | 134 | 215 |
| 10.3 | 18.2 | 107 | ||||
| Tetrahydro S | 0.39 | 1.2 | 20.4 | 40.5 | ||
| 0 | 9.7 | |||||
| Dexamethasone, 0.5 mg q.i.d., 4 cases | ||||||
| 17-ketogenic steroids | 45.9 | 65.6 | 31.4 | 39.6 | 77 | 91 |
| 33.2 | 29.7 | 60 | ||||
| 17-keto steroids | 18.4 | 28.1 | 16.7 | 27.3 | 93 | 121 |
| 10.2 | 11.9 | 71 | ||||
| Dexamethasone, 2 mg q.i.d., 4 cases | ||||||
| 17-ketogenic steroids | 45.9 | 65.6 | 25.5 | 41.4 | 54 | 63 |
| 33.2 | 14.0 | 42 | ||||
| 17-ketosteroids | 18.4 | 28.1 | 12.7 | 18.6 | 74 | 88 |
| 10.2 | 8.5 | 58 | ||||
The results of the functional tests as applied to the tumour cases are presented in Table III. In both cases of carcinoma there is a response to ACTH. In case 1 this response is qualitatively abnormal, being limited to the 17-KS excretion; in case 2 the response is closely similar to that seen in hyperplasias, involving both 17-KS and 17-KGS to the same degree. Metopirone produces very little change in the 17-KS and 17-KGS values. THS excretion is characterized by a markedly abnormal starting value and an increase above the normal range. There is no suppression with dexamethasone in these cases. The adenoma responds rather weakly to ACTH; the resulting values from this stimulation are in the lower normal range. Metopirone gives a fall in 17-KS and 17-KGS excretion, and the THS output after metopirone is below the normal range (5-12 mg/ 24 h). There is no suppression by dexamethasone.
In case 2 pregnanediol and pregnanetriol have been determined during the ACTH stimulation; the distinct increase in the levels of these lends support to the assumption that they are of adrenal origin in this instance.
The term “exaggerated normal responses” would tend to describe the majority of the results presented in Table IV, showing the functional tests in the adrenocortical hyperplasias. In four out of five cases the stimulation values for 17-KS and 17-KGS are beyond the normal range after ACTH, as they are for all three cases following metopirone. In this latter test response the THS excretion is at the upper normal limit for one case, and well above for the other two. Dexa- methasone at the high dose level (2 mg q.i.d.) provides a fair suppression of 17-KGS output (37-58 %), but it is to be noted that some sup- pression (9-40%) is also obtained by the lower dose of 0.5 mg q.i.d. As for the response in 17- KS excretion there is a mean suppression of 9 % on the low dosage and 26% on the high dosage.
DISCUSSION
The complete set of tests as presented in this paper, is obviously too large to be useful as a tool in handling the primary problem of the diagnostic unit, namely to establish the probability of adrenocortical disease. A small set of screening tests that single out patients for further study and possibly treatment is clearly needed. For this
screening purpose the determination of 17-KS and 17-KGS in the 24 hour urine collection of an unstimulated patient, has been widely used. As can be seen from the presented results in Table II, the method would have clearly placed the carcinoma cases, as well as the hyperplasias, into the group for further study. This cannot be said quite so safely for the adenoma case, her excretion values for 17-KGS being just above the normal limit. This sort of value is too frequently found by this method. It occurs in young, obese individuals, and may easily be brought on by very slight emotional stimuli, as, for example, admission to a hospital. It is not possible to act with an all-out diagnostic effort on this evidence alone.
The danger of depending on the plasma cortisol determination for screening when performed on a morning sample is evident from the figures in Table II. Some of these cases have values in the normal range. As pointed out as early as in 1956 by Lindsay et al. (19) and Mellinger and Smith (22), the obliterated diurnal variation in steroid excretion (and plasma concentration) is a good sign of adrenocortical overactivity; this, rather than the elevated values, is the important feature, as shown in the cases of Ekman et al. (11). As can be seen, plasma cortisol in the evening sample was always elevated in our cases. In our hands, however, this test gives a number of positive responses which are not later supported by the accumulated evidence, even if this is less pro- nounced than for the 17-KS and 17-KGS determi- nation. Of these two tests, the determination of the diurnal variation more efficiently excludes the young, obese individuals from further study. Brooks et al. (4) also found the plasma cortisol morning values unreliable for screening. They furthermore emphasized the need for at least two abnormal indices in a given case in order to distinguish from the situation in young people with generalized obesity and pink striae.
We have recently been trying the short-term dexamethasone suppression as described by Pav- latos et al. (29) as a part of out spectrum of diagnostic tests. This method is sufficiently simple to be suitable for screening purposes, requiring at its minimum only one administration of dexamethasone (1 mg at 10 or 11 p.m.) and one plasma cortisol determination the following morn- ing at 8 a.m., at which time it should normally be
less than 5 ug/100 ml. Our experience with this test has been encouraging, although failure of suppression occurred in cases in which hyper- andrenocorticism was not confirmed by the total evidence.
In our opinion the patients that are still suspected of adrenocortical hyperfunction (irre- spective of aetiology) following a preliminary screening by the 24-hour 17-KS and 17-KGS excretion, by the determination of the diurnal plasma cortisol variation, and by a short-term dexamethasone suppression, ought to be in- vestigated with all available tests of the types outlined previously. This will provide a fair in- sight regarding the total pathophysiology of the adrenal cortexes of the patient, and the associated pathology can be predicted reasonably well. The responses to various diagnostic “spot tests” are sufficiently irregular to be misleading as sole criteria in deciding upon the final diagnosis and treatment.
In the separation between the carcinomas on the one hand, and the hyperplasias on the other, the urinary metabolites of intermediates from corticosteroid synthesis are of diagnostic signifi- cance. The quantity of these metabolites in the urine reflects a deficient synthesis of steroid hormones by the tumour tissue; whether the decisive factor in this respect is enzymatic, coen- zymatic or circulatory in nature has not been finally established (20, 32). The defective trans- formation at various stages of the steroid synthesis in the large masses of tumour tissue does not seem to prevent adequate or more than adequate secretion of cortisol from the tumorous glands. The increased amount of urinary “free” cortisol in case 2 is strong evidence for this. One must, however, proceed with caution when estimating cortisol excretion on the basis of the values for the plasma cortisol and the 17-KGS in the urine. Plasma “cortisol” will include steroids such as compound S, 6-hydroxycortisol, and the tetra- hydro metabolites of these and cortisol. The 17- KGS in the urine will include such compounds as pregnanetriol, tetrahydro S and others which may occur in large amounts in the urine from tumour patients.
Further support for the assumption that cor- tisol was secreted from the tumours is provided by the function tests. The results of these are easily explained if one assumes that the pituitary
ACTH-producing cells have been suppressed by cortisol from the tumours. This is perhaps best demonstrated in the metopirone test, in which there is no increase in the steroid output. The increase in THS excretion in the carcinoma cases following metopirone, is not to be interpreted as reflecting a response to an increased output of endogenous ACTH. It must rather be seen as the modified end-product of the continuously se- creting autonomous tumour tissue, affected by the 118-hydroxylase inhibitor. Both carcinoma cases responded to exogenous ACTH with an increase in the 17-KS and 17-KGS output.
All the findings in the adenoma case are ex- plained by assuming that this tumour secreted cortisol at a rate slightly exceeding the physio- logical level, probably even without abatement during the night. In addition it is likely that the tumour was responsible for the aldosterone ex- cretion. This point is, however, debatable, as the aldosterone excretion showed marked variations in response to alterations in electrolyte intake and to the administration of diuretics. The non- tumorous cortical tissue adherent to the removed tumour was thin and atrophic, but contained small cortical cells filled with lipid. In addition small clusters of cells were seen at the outer surface. These were believed to be remnants of the zona glomerulosa.
None of the tumour cases responded to exo- genous ACTH in the period of time shortly following the operation. The significance of this observation is obscured by the heavy cortico- steroid therapy during and after the operation. Up to the present time case 2 has been in need of supportive corticosteroid therapy (i.e. for a period of three years).
The biochemical evidence for cortisol produc- tion by the tumours contrasts with the clinical picture of the tumour cases. As a group the signs and symptoms of Cushing’s syndrome were in- distinct. The adenoma case with the lowest steroid values developed some features of the syndrome during the six months of observation before operation. The explanation may be that the cortisol from the tumour was balanced by a reduced secretion from the non-tumorous adreno- cortical tissue during the early stages of the disease. The period when cortisol was in excess in the organs of the tumour cases, may therefore have been of short duration. An explanation on
the basis of the assumption that the steroid secre- tion in the carcinoma cases consisted almost ex- clusively of products of low biological activity seems less satisfactory.
ABBREVIATIONS AND TRIVIAL NAMES
Tetrahydro S, THS=3a-,17a-,21-trihydroxy-58- pregnane-20-one.
Pregnanediol = 58-pregnane 3g-,20g-diol.
Pregnanetriol = 58-pregnane-3a-,17a-,20g-triol.
Pregnenetriol = pregn-5-ene 38-,17a-,20a-triol.
Dehydroepiandrosterone, DHA = 38-hydroxy-androst-5- en-17-one.
Etiocholanolone = 3g-hydroxy-58-androstan-17-one.
Androsterone = 3a-hydroxy-5a-androstan-17-one.
Dexamethasone = 9a-fluoro 16g-methylprednisolone.
Metopirone = Metapyrapone, Ciba Su 4885, 2-methyl-1,2- bis(3 pyridyl)-1-propanone.
ACTH = adrenocorticotrophic hormone.
21. Martin, M. M. & Hamman, B. L .: J. clin. Endocr. 26: 257, 1966.
22. Mellinger, R. C. & Smith, R. W .: J. clin. Endocr. 16: 350, 1956.
23. Neher, R. & Wettstein, A .: J. clin. Invest. 35: 800, 1956.
24. Norman, N .: Acta endocr. (Kbh) 40: 375, 1962.
25. Norman, N. & Vogt, J. H .: J. Oslo Cy Hosp. 13: 21, 1963.
26. Norman, N., Trygstad, O. & Vogt, J. H .: Acta endocr. (Kbh) Suppl. 100: 59, 1965.
27. Norymberski, J. K., Stubbs, R. D. & West, H. F .: Lancet 1: 1276, 1953.
28. Pal, S. B. & James, V. H. T .: Acta endocr. (Kbh) 46: 37, 1964.
29. Pavlatos, F. C., Smilo, R. P. & Forsham, P. H .: J. Amer. med. Ass. 193: 720, 1965.
30. Petersen, R. E., Karrer, A. & Guerra, S. L .: Analyt. Chem. 29: 144, 1957.
31. Ross, E. J., Marshall-Jones, P. & Friedman, M .: Quart. J. Med. 35: 149, 1966.
32. Symington, T .: Brit. med. Bull. 18: 117, 1962.
33. Umberger, E. J .: Analyt. Chem. 27: 768, 1955.
REFERENCES
1. Allen, W. M .: J. clin. Endocr., 10: 71, 1950.
2. Birke, G., Diczfalusy, E. & Plantin, L. O .: J. clin. Endocr. 20: 593, 1960.
3. Bongiovanni, A. M., Eberlein, W. R. & Thomas, P. Z .: J. clin. Endocr. 17: 331, 1957.
4. Brooks, R. V., Dupré, J., Gogate, A. N., Mills, I. H. & Prunty, F. T. G .: J. clin Endocr. 23: 725, 1963.
5. Cope, C. L .: Brit. med. J. 2: 841, 1966.
6. Damkjæer Nielsen, M., Binder, C., Pedersen, J. & Spechler, M .: Acta endocr. (Kbh.) Suppl. 100: 61, 1965.
7. Diczfalusy, E., Plantin, L. O., Birke, G. & Westman, A .: Acta endocr. (Kbh) 18: 356, 1955.
8. Dorfman, R. I .: Metabolism 10: 902, 1961.
9. Eberlein, W. R. & Bongiovanni, A. M .: J. clin. Endocr. 18: 300, 1958.
10. Eberlein, W. R., Bongiovanni, A. M. & Francis, C. M .: J. clin. Endocr. 18: 1274, 1958.
11. Ekman, H., Håkansson, B., McCarthy, J. D., Leh- mann, J. & Sjøgren, B .: J. clin. Endocr. 21: 684, 1961.
12. Epstein, E. & Zak, B .: J. clin. Endocr. 23: 355, 1963.
13. Ernest, I. & Håkansson, B .: Acta endocr. (Kbh) 48: 147, 1965.
14. Ernest, I .: Acta endocr. (Kbh) 51: 511, 1966.
15. Harrison, M. T., Brush, M. G. & MacFarlaine, M .: J. Endocr. 34: 61, 1966.
16. Jailer, J. W., Holub, D. A. & Frantz, A. G .: Proc. Soc. exp. Biol. 104: 243, 1960.
17. Levin, M. E .: Amer. J. Med. 40: 318, 1966.
18. Liddle, G. W .: J. clin. Endocr. 20: 1539, 1960.
19. Lindsay, F. A., Migeon, C. F., Nugent, C. A. & Brown, H .: Amer. J. Med. 20: 15, 1956.
20. Lipsett, M. B. & Wilson, H .: J. clin. Endocr. 22: 906, 1962.