Concurrent adrenocortical carcinoma and Conn’s adenoma in a man with primary hyperaldosteronism. In vivo and in vitro studies
Y Touitou, A Boissonnas1, A Bogdan and A Auzéby
Biochemical Department, Faculté de Médecine Pitié 91 boulevard de l’Hôpital, 75013, Paris, France and Hôpital Cochin1, Paris
Touitou Y. Boissonnas A, Bogdan A, Auzéby A. Concurrent adrenocortical carcinoma and Conn’s adenoma in a man with primary hyperaldosteronism. In vivo and in vitro studies. Acta Endocrinol 1992;127:189-92. ISSN 0001-5598
This is a report of a rare and unusual case of adrenal pathology. A patient presented with clinical and biological signs of primary aldosteronism and computed body tomography scan led to our suspecting the presence of a left adrenocortical carcinoma. The in vitro studies performed on the resected tumour showed very low synthesis of mineralocorticoids and glucocorticoids. The patient could not be re- examined until 15 months later, when he still suffered hypertension; another tomography scan revealed a mass on the right adrenal gland. The studies performed on this second tumour confirmed the diagnosis of Conn’s adenoma: active in vitro biosynthesis of 18-hydroxy-corticosterone and aldosterone from exogenous tritiated precursors.
Y Touitou, Biochemical Department, Faculté de Médecine Pitié 91 boulevard de l’Hôpital, 75013, Paris, France
Primary aldosteronism is a clinical condition attributed in 90% of cases to an overproduction of aldosterone by a single adrenocortical adenoma (1). Among the minera- locorticoid secreting tumours, corticosterone-secreting adrenal adenoma has been reported (2) as well as 18- hydroxycorticosterone secreting variants of aldostero- nomas (3). Although approximately 50% of patients with adrenocortical adenoma have no clinical signs of increased steroid production (4), these tumours have also been found responsible for hypertension due to mineralocorticosteroid effect (5). We report here the in vitro demonstration of the origin of an hyperaldostero- nism in an unusual case of adrenal pathology in a patient with both adrenocortical carcinoma and Conn’s adenoma.
Case report
A 44-year-old Iranian male was admitted to hospital suffering from high blood pressure, headaches and right hemiparesia. Two months before admission he com- plained of physical weakness but had no history of cramps, polyuria, nycturia or diabetes mellitus and was not taking any drugs. Physical examination revealed right pyramidal signs and obesity but no Cushingoid aspect. The electrocardiogram showed negative T waves in DI-VL, ST segment depression in V4, V5, V6 and U waves in V2 through V4.
The patient’s main biological data are given in Table 1. Blood pressure was 250/150 mmHg, plasma sodium was 147 mmol/l and plasma potassium 2.6 mmol/l. The urinary excretions of sodium and potassium were
respectively 105 and 40 mmol/24 h. Baseline levels of urinary free cortisol, plasma cortisol and ACTH were in the normal range. Dynamic studies of the pituitary- adrenal function showed normal responses to metyra- pone and dexamethasone tests. On normal sodium diet, plasma aldosterone was high, with no change in the upright position; plasma renin activity was low. After the administration of [13]]] 6 B-methyl-19-norcholes- terol as a single shot and of dexamethasone (3 mg/day) for eight days, adrenal scintiscan showed no image on the left side and, unexpectedly, a low uptake in the right gland. A computed body tomography scan showed a 6 cm mass in the left adrenal gland and a slightly hyperplastic right adrenal gland. With the diagnosis of left adrenocortical carcinoma the patient underwent surgery. To avoid adrenal insufficiency and because no tumour was found in the right adrenal gland it was not removed.
A 150 g tumour was removed from the left side and sections were used for in vitro studies. On microscopic examination the tumour was described as an adrenal carcinoma with necrotic foci and cellular pleomorphism. In some areas, large polygonal cells with eosinophilic cytoplasm and irregular nuclei were arranged in cords. There was no capsular or vascular invasion and no local extension of the tumour was observed. The attached adrenal cortex was normal and there was no hyperplasia of the zona glomerulosa.
For personal reasons the patient had to return to Iran two weeks after surgery. Although with a sodium intake of 120 mmol/day, supine plasma aldosterone and plasma renin activity were normal and blood pressure
| Normal range | Blood pressure mmHg | Plasma Na mmol/l 135-147 | Plasma K mmol/l 3.5-5 | Diet dependent | Aldosterone supine/upright pmol/l 560-1100 | PRA supine/upright ng . 1-1.s -1 0.14-0.42 | Plasma cortisol 8:00 am nmol/l 250-650 | Plasma 11-deoxycortisol 8:00 am Urinary Na 14-40 | Urinary free cortisol Urinary K 65-250 | 17 KS mg/24 h 12-18 | 17 OHCS mg/24 h 4-8 | ACTH pmol/l <20 | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Urinary Na mmol/24 h | Urinary K mmol/24 h | ||||||||||||
| Before surgery Normal sodium diet | 250/150 | 147 | 2.6 | 105 | 100 | 2275/2358 | 0.06/0.06 | 193 | 28 | 55 | 8.0 | 2.5 | <2 |
| 5 days after left adrenalectomy Normal sodium diet | 170/120 | 142 | 3 | 120 | 80 | 943/ND | 0.25/ND | 166 | 28 | 7 | 13.1 | 2.2 | 2.2 |
| 17 months after left adrenalectomy Normal sodium diet | 160/110 | 148 | 2.8 | 175 | 75 | 3468/ND | 0.03/ND | 138 | ND | 121 | 4.5 | 3.9 | 4.0 |
| 21 days after right adrenalectomy Normal sodium diet (40 mg/day hydrocortisone) | 140/70 | 138 | 4.8 | 225 | 60 | Not detectable | 0.92/ND | 83 | 1] | ND | ND | ND | ND |
ND: not determined. 17 KS: 17 ketosteroids. 17 OHCS: 17 hydroxycorticosteroids.
was still 170/120 mmHg. With a low sodium intake (42 mmol/day) plasma aldosterone rose more than three- fold. For the next 15 months the patient was treated in Iran with 300 mg/day spironolactone because of persist- ent high blood pressure. He was then readmitted to hospital two months after stopping treatment. No remarkable signs were observed on clinical examin- ation. Blood pressure was 160/110 mmHg, plasma sodium 148 mmol/l, plasma potassium 2.8 mmol/l; urinary sodium and potassium were respectively 175 and 75 mmol/24 h. Plasma cortisol, urinary free cortisol and ACTH were in the normal range. Further hormonal studies showed basal plasma deoxycorticosterone and plasma corticosterone within the normal range (respect- ively 708 pmol/l and 9.8 umol/l) and high plasma aldosterone in the supine position. A threefold increase in plasma aldosterone and deoxycorticosterone was observed after the injection of 250 µg of synthetic ACTH. A computed body tomography scan showed a mass on the right adrenal gland. Adrenal scintiscan revealed a high uptake in the gland. Adrenalectomy was performed and a 6.4 g well encapsulated golden yellow tumour was removed. Histological examination of the tumour con- cluded a Conn’s adenoma. The tumour had a fibrotic capsule and was composed of irregular and polyedric clear cells with vacuolated nuclei. The cells were arranged in small cords or lobules separated by fine connective tissue containing few capillaries. Three weeks after right tumour removal, plasma renin activity rose to 0.92 ng ·1-1 . s-1, plasma aldosterone was unde- tectable and blood pressure was 140/70 mmHg. The patient left hospital with adequate treatment for acute adrenal insufficiency. A few months later an abdominal mass with ascite was discovered and the patient died shortly thereafter.
Materials and methods
Steroids and reagents
The following [1,2-3H] steroids were used for the experiments: 17«-hydroxyprogesterone (40.4 Ci/ mmol), 11-deoxycortisol (58.5 Ci/mmol), 11-deoxycor- ticosterone (46.8 Ci/mmol) and corticosterone (50 Ci/ mmol) were purchased from the New England Nuclear Corporation, 18-hydroxy-11-deoxycorticosterone (51.8 Ci/mmol) and 18-hydroxycorticosterone (52 Ci/mmol) were purchased from Amersham, UK. The following [4-14C] steroids were used: aldosterone (55.0 Ci/mol), cortisol (55 Ci/mol), cortisone (59.8 Ci/mol) and cortico- sterone (59.3 Ci/mol) were purchased from the New England Nuclear Corporation. [4-14C] 18-hydroxycorti- costerone and [4-14C] 18-hydroxy-11-deoxycorticoster- one were obtained from [4-14C] deoxycorticosterone by incubation of rat adrenal homogenates and purity was achieved as described previously (6). The radiochemical purity of all the labelled steroids was checked by paper chromatography shortly before use. Unlabelled steroids,
nicotinamide adenine nucleotide phosphate (NADP+), potassium hydroxide and solvents were analytical grade as supplied by Merck, malic acid was from Sigma, sterile Earle’s medium was from Institut Pasteur (Paris) and liquid scintillation counting solution was from Amer- sham.
Experimental procedure
Adrenal tissues were immediately transferred on ice to the laboratory and kept at - 20℃ until processed (left) or immediately incubated (right). Adrenals tissues were cleared of fat and connective tissue, then weighed and homogenized with a teflon glass homogenizer in Earle’s medium buffer. Duplicate incubations (150 mg from right and 1 g from left, wet weight) were performed as previously described (7, 8). Incubation flasks contained exact amounts of one of the following tritiated pre- cursors: 17a-hydroxyprogesterone, 11-deoxycortisol, 11-deoxycorticosterone, corticosterone, 18-hydroxy- 11-deoxycorticosterone (18-OHDOC). 18-hydroxy- corticosterone (18-OHB) as ethanolic solutions. The precursors were evaporated to dryness then dissolved in 2.0 ml Earle’s solution 1 h before starting the incubation.
An NADPH generating system made up of NADP + (1 mmol/l) and malic acid (5 mmol/l) neutralized with a 0.1 mol/l potassium hydroxide solution was added to each incubation flask (6); the final volume of each incubation flask was 10 ml. Aerobic incubations were performed in a Dübnoff metabolic shaker incubator at 37℃ for 2 h. The proteins were precipitated with 15.0 ml acetone, and trace amounts of [4-14C] steroids, depending on the tritiated steroid precursor used, were added to the incubation flasks to be used as internal standards. Extraction of steroids and measurement of radioactivity were performed as previously described (7). The results were calculated as the percentage conversion of the total radioactivity; data were corrected for pro- cedural losses.
Isolation and characterization of steroids
Details on the isolation and characterization procedures have been published previously (7-9). The steroids were separated by descending paper chromatography. Indi- vidual isolated metabolites were further run in suitable chromatographic systems to assess their isopolarity with the carbon-14 internal standards. The isolated steroids were then characterized through derivative formation with the following methods. Chromic acid oxidation: chromium dioxide (20 g/l) in water-pure acetic acid (50:50), 3 h in darkness at laboratory temperature, extraction with chloroform; periodic acid oxidation : periodic acid (2 g/l in water-pyridine 98:2), overnight at laboratory temperature, extraction with chloroform; sodium bororhydride reduction: sodium bororhydride (2 g/l in methanol) 35 min at laboratory temperature, extraction with ethyl acetate; acetic anhydride acetylation: dried extracts dissolved in three drops pure pyridine, then addition of three drops acetic anhydride, overnight at laboratory temperature, extraction with chloroform; acetate hydrolysis: Na2CO3 (5 g/l in water) 1 h at laboratory temperature, extraction with chloroform. The radiochemical purity of synthe- sized steroids and conversion products was assumed from the constancy of 3H/14C ratios at different stages of purification.
Results and discussion
The in vitro study of the adrenocortical carcinoma showed unexpectedly low synthesis of steroids whatever the precursor (Table 2). The synthesis of cortisol was 3.0% from 17«-hydroxyprogesterone and 0.6% from 11-deoxycortisol. The synthesis of aldosterone from 11- deoxycorticosterone and corticosterone was 0.3 and 1.3% respectively. These results proved the non- secreting nature of the tumour and that the origin of the hyperaldosteronism had to be looked for in the contrala-
| Precursor | 11-deoxycortisol | Cortisol | E | |||
|---|---|---|---|---|---|---|
| Cancer | Conn | Cancer | Conn | Cancer | Conn | |
| 17-hydroxyprogesterone | 1.5 | 0.3 | 3.0 | 46.6 | ND | ND |
| 11-deoxycortisol | - | - | 0.6 | 45.5 | 0.7 | 10.2 |
| Precursor | Corticosterone | 18-OHDOC | 18-OHB | Aldosterone | ||||
|---|---|---|---|---|---|---|---|---|
| Cancer | Conn | Cancer | Conn | Cancer | Conn | Cancer | Conn | |
| 11-deoxycorticosterone | 0.4 | 17.3 | 0.9 | 1.6 | 1.1 | 49.6 | 0.3 | 13.6 |
| Corticosterone | - | - | - | - | ND | 47.4 | 1.3 | 12.4 |
| 18-OHDOC | - | - | - | - | ND | 41.7 | ND | 2.3 |
| 18-OHB | - | - | - | - | - | - | ND | 8.0 |
ND: not determined.
teral adrenal. But since the patient had returned to Iran before the results of the in vitro study, further clinical and biological exploration could not be performed until 17 months later.
The in vitro study of the adenomatous tissue obtained with the second adrenalectomy showed a synthesis of glucocorticosteroids in the usual range and very similar to that observed previously in another Conn’s adenoma (7). These data are also coherent with those obtained by Brode et al. (10) with progesterone as a precursor. The conversion rates to aldosterone and other mineralocorti- coids observed with various precursors confirmed the origin of the primary hyperaldosteronism, since aldo- sterone yields were approximately 13% thus 10 times larger than with the adrenocortical carcinoma (Table 2). Such active conversion of corticosterone into 18-hy- droxycorticosterone and aldosterone have been reported by others (11, 12). The same differences were observed in the synthesis of corticosterone and 18-hydroxycorti- costerone from 11-deoxy-corticosterone when compar- ing the two adrenal tissues.
This is a rare and unusual case of adrenal pathology with a Conn’s syndrome related to an aldosterone producing adenoma in a man with an adrenocortical carcinoma whose non-secreting characteristics were demonstrated in vitro.
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Received December 27th, 1991 Accepted March 16th, 1992