A Case of Deoxycorticosterone-Producing Adrenal Adenoma
NORIO WADA, MITSUMASA KUBO, HIROMICHI KIJIMA, YASUAKI YAMANE*,
TETSUO NISHIKAWA ** , HIRONOBU SASANO *** , AND TAKAO KOIKE
Department of Medicine II, Hokkaido University School of Medicine, Sapporo 060, *Department of Internal Medicine, Kitami Red Cross Hospital, Kitami 090, ** Department of Internal Medicine, Yokohama Rosai Hospital, Kanagawa 222, and *** Second Department of Pathology, School of Medicine, Tohoku University, Sendai 980, Japan
Abstract. A 29-year-old woman with deoxycorticosterone (DOC)-producing adrenocortical adenoma had hypertension and hypokalemia but without Cushingoid features. Plasma renin activity and the aldosterone concentration were low, while the DOC concentration was high (6.10-10.3 ng/ml; normal range 0.03-0.33). Plasma cortisol, androgens, and estrogens as well as urinary 17-OHCS and 17-KS were within normal limits. Furosemide administration and two hours upright posture resulted in a 3-fold increase in plasma DOC, but the administration of ACTH, dexamethasone, or angiotensin III had no effect on plasma DOC. Following resection of a right adrenal tumor weighing 70 g, the hypertension and hypokalemia disappeared. DOC content in the tumor was high. On light microscopic examination, the tumor was encapsulated, composed of cells with clear cytoplasm and large nuclei and there were extensive areas of fibrosis and infiltration of lymphocytes. According to Weiss’s criteria, the tumor was considered to be an adrenocortical adenoma. Immunohistochemically, P450scc, 3ßHSD, P450c21 and P45011; were positive with heterogeneity of intra-tumoral expression. No immunoreactivity for P45017a in this adenoma was detected. This is different from a previous report in which a relatively small number of cells in DOC-secreting adrenocortical carcinoma were positive for P45017a-
Key words: Deoxycorticosterone, Adrenocortical adenoma, Immunohistochemistry, Steroid content (Endocrine Journal 42: 637-642, 1995)
ALTHOUGH the mineralocorticoid activity of deoxycorticosterone (DOC) is quite weak compared with that of aldosterone, excessive production of DOC was reported to result in hypertension, hy- pokalemia, and suppression of plasma renin activity (PRA) [1, 2]. Increased secretion or high plasma levels of DOC were occasionally noted in patients with adrenocortical carcinoma which overproduced cortisol or estrogens [2-4]. DOC- producing adrenal tumors without overproduction
of cortisol, estrogens, and androgens are extreme- ly rare. Eight cases of DOC-producing adrenal tumor were reported in the literature in English; four cases were malignant [5-8] and four were be- nign [7, 9-11]. On the other hand, DOC excess, hypertension and hypokalemia have been detect- ed in cases of congenital adrenogenital syndrome resulting from 11ß-hydroxylase or 17a-hydoxylase deficiency. The diminished or reduced activity of those steroidogenic enzymes may therefore be re- sponsible for the overproduction of DOC in DOC-producing adrenocortical tumor. Findings suggestive of these changes have been reported by Kelly et al. [5], Saha et al. [11], and Shibata et al. [12].
The site of specific steroid production in the ad-
renal cortex has been successfully localized by im- munohistochemical analysis of steroidogenic enzymes [13-18]. This has been reported previ- ously in DOC-secreting adrenocortical carcinoma [8], but not in adenoma. Here we report a case of a DOC-producing adrenocortical adenoma with en- docrinological and pathological findings, including immunohistochemical analysis of steroidogenic enzymes.
Methods
Plasma DOC, 18-OH-11-deoxycorticosterone (18- OH-DOC), and 11-deoxycortisol were measured by radioimmunoassay (RIA) preceded by extraction with dichloromethane and purification by Sepha- dex LH-20 column chromatography as described eleswhere [19-21]. Plasma corticosterone and cor- tisone were measured by RIA preceded by extraction with ether and purification by Sepha- dex LH-20 column chromatography, and plasma dehydroepiandrosterone and androstendione were measured by solid phase RIA after extraction with dichloromethane and ethylether, respectively (Med- ical System Service Kanagawa Co.). PRA, aldosterone, cortisol, progesterone, and testoster- one were measured with commercial RIA kits (Dinabott Radioisotope Institute Co., Daiichi Ra- dioisotope Laboratory Co., Baxter Co., Japan DPC Co., Eiken Chemistry Co.).
Case Report
A 29-year-old woman was first found to have hypertension one week prior to her second obstet- ric delivery in 1992. Three months after delivery she was still hypertensive, and hypokalemic, and subsequent CT scan revealed a right adrenal tu- mor. She was referred to Hokkaido University Hospital for further examination.
She was 150 cm tall, weighed 43.5 kg and Cush- ingoid features were absent. Blood pressure was 154/98 mmHg and the pulse rate 72/min. Labo- ratory examination revealed hypokalemia (2.5 mEq/L) even with the administration of KCI 750 mg/day. Blood urea nitrogen was 8 mg/dl and serum creatinine was 0.6 mg/dl. The arterial pH was 7.41, Po2 89 mmHg, Pco2 46 mmHg and HCO3- 29.2 mM/L.
Hormonal examination showed PRA and the plasma aldosterone concentration (PAC) on nor- mal sodium intake (190 mEq/day) to be 0.01 ng/ ml/h (normal range; 0.2-2.0) and 45.2 pg/ml (nor- mal range; 50-150), respectively. The plasma cortisol level was 14.7 ug/dl with a normal diur- nal rhythm. Urinary 17-OHCS and 17-KS were within normal limits. Adrenaline, noradrenaline, and dopamine concentrations in plasma, and lev- els of their metabolites in the urine were normal. The profile of plasma steroid hormones is summa- rized in Fig. 1. Plasma DOC was remarkably
Pregnenolone
17-OH-Pregnenolone
DHEA 2.5 ng/ml (0.8-7.0)
Progesterone 0.9 ng/ml (0.2-31.6)
17-OH-Progesterone
Androstenedione 1.4 ng/ml (0.6-5.0)
18-OH-DOC 0.11 ng/ml (0.01-0.07)
DOC 11.6 ng/ml (0.03-0.33)
11-Deoxycortisol 1.00 ng/ml (0.11-0.60)
Testosterone 30.8 ng/dl (6.0-86.0)
Corticosterone 10.70 ng/ml (0.21-8.48)
Cortisol 14.7 mg/dl (4.0-18.3)
Aldosterone 45.2 pg/ml (50.0-150.0)
Cortisone 28.10 ng/ml (2.61-19.2)
increased: 6.10-10.3 ng/ml in 7 occasions in the morning (normal range; 0.03-0.33) and 11.6 at 2300 h. Corticosterone, 18-OH-DOC, 11-deoxycortisol, and cortisone levels were slightly high while lev- els of other steroids were normal. The administration of 8 mg of dexamethasone had no effect on plasma DOC levels (10.3 to 6.98 ng/ml). ACTH administration (250 µg i.v.) resulted in no increase in plasma DOC (11.2 to 4.61 ng/ml) and PAC (47.0 to 52.2 pg/ml) with a normal increase in plasma cortisol (20.1 to 38.3 ug/dl). Furosemi- de administration (1 mg/kg B.W. i.v.) and 2 h upright posture resulted in approximately a 3-fold increase in plasma DOC (8.19 to 22.6 ng/ml) and a 2-fold increase in PAC (32.7 to 65.6 pg/ml), but no increase in PRA. Angiotensin III infusion (20 ng/ kg B.W./h) did not alter the plasma DOC concen- trations. On adrenal venous sampling examination, the levels of DOC and cortisol, and the DOC/cor- tisol ratio in the right adrenal vein were 246 ng/ ml, 6.2 µg/dl and 40, but 6.29 ng/ml, 18.4 ug/dl and 0.34 in the left adrenal vein.
Abdominal CT scan revealed a right adrenal tu- mor, measuring 28 x 44 mm, and MRI showed that this tumor was heterogeneous (Fig. 2). 131]- adosterol uptake was evident in bilateral adrenals but to a lesser extent in left adrenal.
A right adrenal tumor weighing 70 g was ex- cised. The cut surface of the tumor was yellowish (Fig. 3).
Plasma DOC levels and the serum potassium concentration were normalized and hypertension disappeared during the early post-operative peri-
od. She was discharged well from the hospital and her post-operative course was uneventful.
Histopathology
Light microscopic examination revealed that the tumor was well circumscribed and encapsulated. The tumor was composed of the cells with clear cytoplasm and relatively large nuclei (Fig. 4). Ex- tensive areas of fibrosis and infiltration of lymphocytes were observed in the specimen (Fig. 4). According to Weiss’s criteria [22], the tumor was considered to be a benign adrenocortical ade- noma. The adjacent normal adrenal cortex was not atrophied.
Immunohistochemistry of steroidogenic enzymes
Immunolocalization of steroidogenic enzymes including P450scc (cholesterol side-chain cleavage), 3BHSD (3-hydroxysteroid dehydrogenase), P450c21 (21-hydroxylase), P450118 (11-hydroxylase), and P45017a (17a-hydroxylase) was performed as previously described [13-18]. P450scc, P450C21, P450118, and 3HSD immunoreactivity was ob- served in the tumor cells (Fig. 5). Intra-tumoral heterogeneity of immunolocalization of ste- roidogenic enzymes was observed, i.e., the number of tumor cells positive for the above mentioned steroidogenic enzymes differed from area to area. Immunoreactivity for P45017% was not detected in any of the tumor cells examined.
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Steroid contents
The amounts of various steroids in the tumor and five normal adrenal tissues (histologically con- firmed as normal) which had been excised from patients with renal carcinoma because of close ad- herence to the carcinoma tissues were estimated as previously described [23, 24]. The results were as follows: progesterone 284.6 ng/g wet tissue (nor- mal range; 203.3 + 126.2: n=5, mean ± SEM), DOC 417.9 ng/g wet tissue (normal range; 37.6 ± 18.1), corticosterone 105.8 ng/g wet tissue (normal range; 307.5 ± 209.3), and aldosterone 719.6 ng/g wet tis- sue (normal range; 5323.8 ± 1136.5).
Activities of 21-hydroxylase and 11 ß-hydroxylase
Mitochondrial and microsomal fractions were prepared from normal and tumor tissues, and the activities of P450c21 in the microsomal fraction and P45011; in the mitochondrial fraction were assayed as described elsewhere [25]. The protein concen- tration was determined by the method of Lowry et al. [26] with bovine serum albumin as the stan- dard. Activities of P450c21 and P450118 in this tumor were 0.4 nmol/mg protein/2 min (normal adrenals; 3.6 and 4.8) and 1.14 µg 11-OHCS formed/mg pro- tein/10 min (normal adrenals; 4.9 and 5.1).
Discussion
Immunohistochemical examination of ste-
roidogenic enzymes has become increasingly im- portant for the evaluation of steroid hormone-producing tumors. In our patient, im- munoreactivities of P450scc, 3฿ HSD, P450c21, and P450118 but not P45017g were detected in tumor cells. This finding indicates that the tumor did not ac- tively produce cortisol, a notion consistent with the non-atrophied adjacent normal adrenal cortex, 131I-adosterol uptake in the left adrenal, no require- ment of hydrocortisone replacement after right adrenalectomy, and mineralocorticoid excess. This is in contrast to the report of a DOC-producing adrenocortical carcinoma in which 5 to 10% of car- cinoma cells expressed P45017x [8]. This difference may be due to the difference between an adreno- cortical adenoma and a carcinoma, but it awaits further investigation for clarification.
In humans, two genes encoding 11ß-hydroxy- lase, CYP11B1 (P45011B gene) and CYP11B2 (P450aldo gene; aldosterone synthase cytochrome P450 gene) were identified [27-29]. P450aldo shows all three catalytic activities (11ß-hydroxylase, 18-hydroxy- lase, and 18-oxidase) on DOC, a substrate of aldosterone, when P450aldo cDNA was expressed in COS-7 cells [28]. Since the antibody to P450118 we used here crossreacts with P450aldo, it is possi- ble that this antibody may detect P450aldo, but specific mRNA, enzyme activities, and the immu- noreactivity of P450aldo are generally below the level of detection in the normal adrenal [27, 29]. They are demonstrable only in aldosterone-producing adrenal tumors [29]. Moreover, the aldosterone concentration in the plasma and the tumor from
this patient was noticeably low, so that immunore- activity of P450118 may not represent that of P450aldo in this case.
Endocrinological examination of our patient re- vealed high DOC, slightly increased 18-OH-DOC, corticosterone, 11-deoxycortisol, and cortisone lev- els in plasma, and low PRA and PAC. Exogenous ACTH administration did not alter plasma DOC levels. In three patients with DOC-producing ade- noma there was responsiveness of DOC to ACTH [7-9], but in another unresponsiveness [10]. In DOC-producing carcinoma, one patient showed no response of DOC to ACTH [6] while one showed a 1.4-fold increase [7]. The suppression of DOC by dexamethasone was demonstrated in one patient with adenoma and one with carcinoma [6, 9], but was not in the others with adenoma and carcino- ma [7, 8]. There was no apparent correlation between ACTH responsiveness and dexamethasone suppression in DOC-producing adrenocortical neo- plasm.
There has been no report on DOC in response to furosemide and upright posture in DOC-produc- ing tumors. In this case a significant increase in DOC without any change in PRA in response to furosemide and upright posture was demonstrat- ed. Plasma ACTH and cortisol levels on the loading examination were not measured. ACTH, however, does not seem to be a stimulating factor in this case, in contrast to primary aldosteronism, because exogenous ACTH administration elicited no increase in DOC. The mechanism of this phe- nomenon is inexplicable at present.
Deficiency of P450118 or P45017a in patients with
congenital adrenogenital syndrome results in ex- cessive production of DOC, in association with hypertension and hypokalemia. Therefore, over- production of DOC in adrenal tumors has heretofore been attributed to the diminished or re- duced activity of these enzymes. Kelly et al. [5] investigated in vitro steroid biosynthesis by using cultured tumor cells incubated with [3H]pregnenolone, and they noted the absence of P450118 and P45017g activity in the DOC-producing carcinoma. Saha et al. [11] reported low P450118 activity in a DOC-producing adrenal adenoma. Shibata et al. [12] demonstrated that the activities of P450118, P45017%., and P450scc were lower in DOC- producing adenoma than in normal adrenals, in association with decreased amounts of those en- zymes, determined by immunoblotting.
The activity of P450118 in this adenoma was low, in accord with other reports [5, 11, 12]. It is curi- ous that the activity of P450c21 in the tumor was also attenuated. This evidence might be related to the large size of this tumor as well as the marked fibrosis and heterogeneity of intra-tumoral P450118 and P450c21 expression. On the other hand, a study of the steroid contents of this tumor revealed that the DOC content was high and the corticosterone and aldosterone contents were scanty. In immu- nohistochemical analysis of steroidogenic enzymes, immunoreactivities to P450118 and P450c21 were de- tected, but not to P45017g. Thus the factor most likely to be responsible for excessive production of DOC in this DOC-producing adrenocortical ade- noma is the absence of P45017g and possibly the relatively low degree of P450118 expression.
References
1. Kahn M, Melby JC, Jacobs DR (1966) Isolated DOC excess: a unique syndrome simulating hyperaldosteronism with marked fluid retention. Clinical Research 14: 282-284.
2. Biglieri EG, Slaton PE, Schambelan M, Kronfield SJ (1968) Hypermineralocorticoidism. Am J Med 45: 170-175.
3. Solomon SS, Swersie SP, Paulsen CA, Biglieri EG (1968) Feminizing adrenocortical carcinoma with hypertension. J Clin Endocr 28: 608-612.
4. Saadi H, Bravo EI, Aron D (1990) Feminizing adrenocortical tumor: Steroid hormone response to ketoconazole. J Clin Endocrinol Metab 70: 540-543.
5. Kelly WF, O’Hare MJ, Loizou S, Davies D, Laing I
(1982) Hypermineralcorticism without excessive al- dosterone secretion: An adrenal carcinoma producing deoxycorticosterone. Clin Endocrinol 17: 353-361.
6. Powell-Jackson JD, Calin A, Fraser R, Graham R, Mason P, Missen GAK, Powell-Jackson PR, Wilson A (1974) Excess deoxycorticosterone secretion from adrenocortical carcinoma. Brt Med J 2: 32-33.
7. Irony I, Biglieri EG, Perloff D, Rubinoff H (1987) Pathophysiology of deoxycorticosterone-secreting adrenal tumor. J Clin Endocrinol Metab 65: 836-840.
8. Yamamoto A, Naroda T, Kagawa S, Umaki Y, Shintani Y, Sano T, Sasano H (1993) Deoxycorticosterone-secreting adrenocortical carci-
noma. Endocr Pathol 4: 165-168.
9. Kondo K, Saruta T, Saito I, Yoshida R, Maruyama H, Matsuki S (1976) Benign deoxycorticosterone producing adrenal tumor. JAMA 236: 1042-1044.
10. Ishikawa S, Saito T, Kaneko K, Okada K, Fukuda S, Kuzuya T (1988) Hypermineralocorticism without elevation of plasma aldosterone: Deoxycorticosterone-producing adrenal adenoma and hyperplasia. Clin Endocrinol 29: 367-375.
11. Saha PK, Ura T, Suzu H, Yamashita S, Yushita Y, Kanetake H, Saito Y, Tsuchiyama H, Shigematsu K, Hukuzi S, Kozima M (1990) A case of deoxycorticosterone-producing benign adrenocor- tical tumor. Urol Int 45: 367-369.
12. Shibata H, Suzuki H, Ogishima T, Ishimura Y, Saruta T (1993) Significance of steroidogenic en- zymes in the pathogenesis of adrenal tumor. Acta Endocrinol 128: 235-242.
13. Sasano H, Sasano N, Okamoto M (1989) Immuno- histochemical demonstration of cholesterol side-chain cleavage cytochrome P-450 in bovine and human adrenal. Pathol Res Pract 184: 337-342.
14. Sasano H, Mason JI, Sasano N, Nagura H (1990) Immunolocalization of 3ß-hydroxysteroid dehydro- genase in human adrenal cortex and in its disorder. Endocr Pathol 1: 94-101.
15. Sasano N, Sasano H (1990) In; Kovacs K, Asa SL (eds) The Adrenal Cortex in Functional Endocrine Pathology. Blackwell Scientific Publications, Bos- ton, vol. 2: 546-584.
16. Sasano H, Okamoto M, Sasano N (1988) Immuno- histochemical study of cytochrome P-450 11ß-hydroxylase in human adrenal cortex with mineralo- and glucocorticoid excess. Virchows Arch [A] 413: 313-318.
17. Sasano H, White PC, New MI, Sasano N (1988) Immunohistochemical localization of cytochrome P- 450c21 in human adrenal cortex and its relation to endocrine function. Hum Pathol 19: 181-185.
18. Sasano H, Mason JI, Sasano N (1989) Immunohis- tochemical study of cytochrome P-45017g in human adrenocortical disorders. Hum Pathol 20: 113-117.
19. Tochigi B, Tomita M, Yoshida T, Takagi S, Makino T, Kambegawa A (1976) A radioimmunoassay of plasma 11-deoxycorticosterone. Clin Endocrinol
(Tokyo) 24: 1151-1155 (In Japanese).
20. Den K, Honda M, Kambegawa A, Tomita M, Tochigi B, Yoshida T, Takagi S (1978) A radio- immunoassay of plasma 18-hydroxy-11- deoxycorticosterone. Endocrinol Japon 25: 171-175.
21. Ohashi T, Morioka M, Akaeda T, Mitsuhata N, Takeda K, Ohmori H, Saito T, Takami T, Kambegawa A (1981) Plasma 11-deoxycortisol re- sponse to single dose metyrapone in prostatic cancer patients. Folia Endocrinol Japon 57: 983-996 (In Japanese).
22. Weiss LM (1984) Comparative histologic study of 43 metastatizing and nonmetastatizing adrenocor- tical tumors. Am J Surg Pathol 8: 163-169.
23. Nishikawa T, Strott CA (1983) Unconjugated and sulfo-conjugated steroids in plasma and zones of the adrenal cortex of the guinea pig. Steroids 41: 105-119.
24. Nishikawa T, Strott CA (1984) Steroid concentra- tions in the outer and inner zones of the adrenal cortex of the guinea pig. J Steroid Biochem 20: 1123- 1127.
25. Sasano H, Suzuki T, Nagura H, Nishikawa T (1993) Steroidogenesis in human adrenocortical carcinoma. Hum Pathol 24: 397-404.
26. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193: 265-275.
27. . Mornet E, Dupont J, Vitek A, White PC (1989) Char- acterization of two genes encoding human steroid 11B-hydroxylase (P-450118). J Biol Chem 264: 20961- 20967.
28. Kawamoto T, Mitsuuchi Y, Ohnishi T, Ichikawa Y, Yokoyama Y, Sumimoto H, Toda K, Miyahara K, Kuribayashi I, Nakao K, Hosoda K, Yamamoto Y, Imura H, Shizuta Y (1990) Cloning and expression of a cDNA for human cytochrome P-450aldo as re- lated to primary aldosteronism. Biochem Biophys Res Commun 173: 309-316.
29. Ogishima T, Shibata H, Shimada H, Mitani F, Suzuki H, Saruta T, Ishimura Y (1991) Aldosterone synthase cytochrome P-450 expressed in the adrenals of patients with primary aldosteronism. J Biol Chem 266: 10731-10734.