Steroid 21-Hydroxylase and 17a-Hydroxylase in Microsomal Fraction of Functioning Adrenocortical Tumors
HIROSHI NAGANUMA and NOBUAKI SASANO Department of Pathology, Tohoku University School of Medicine, Sendai 980
NAGANUMA, H. and SASANO, N. Steroid 21-Hydroxylase and 17a-Hydroxylase in Microsomal Fraction of Functioning Adrenocortical Tumors. Tohoku J. Exp. Med., 1989, 158 (3), 253-262 - In order to survey the enzymic activities of steroidogenesis in functioning adrenocortical tumors, we investigated the activities of steroid 21-hydroxylase and 17a-hydroxylase in microsomal fractions of 12 surgically resected adrenocortical tumors associated with Cushing’s syndrome (5 adenomas and one carcinoma), primary aldosteronism (5 adenomas) and adrenogenital syndrome (AGS) (one carcinoma), and one adrenocortical hyper- plasia resulting from Cushing’s disease. Seven adrenal cortices from the patients with mammary carcinoma, renal cell carcinoma or pheochromocytoma were used for normal control. In normal controls 21-hydroxylase activities with progester- one as a substrate were 1.61+0.25 nmole/min/mg protein and those with 17a- hxdroxyprogesterone were 5.22+1.06 nmole/min/mg protein. The activity of 21-hydroxylase was higher in four cases of 5 aldosteronomas than in normal controls. Those activities in Cushing’s adenomas were in the range of normal controls in this study. 17a-hydroxylase activities were much variable from case to case even though in normal controls (4.50±2.40 nmole/min/mg protein), and in most cases of adenomas 17a-hydroxylase activities were in the range of normal controls. Activities of both hydroxylase in carcinomas were lower than in normal controls. The present paper showed the abnormal steroidogenic enzyme activities in aldosteronomas and adrenocortical carcinomas. --- adrenals ; steroid hydroxylase ; enzyme activity ; adrenocortical tumors ; microsomal fraction
The excessive production of steroids in functioning adrenocortical tumors may be related to any abnormal activities of the enzyme for steroidogenesis which locates on the membrane of mitochondria, smooth or rough endoplasmic reticulum (ER). The histologic features in adrenocortical tumors have been mentioned in detail according to clinical symptoms. Ultrastructural studies have revealed the morphological abnormalities of mitochondria, smooth and rough ER in tumor cells (Kano and Sato 1977; O’Hare et al. 1979 ; Sasano et al. 1980 ; Silva et al. 1982), and it is known that adrenocortical carcinomas have the obvious deficiency of the enzyme of steroidogenesis including 30-hydroxysteroid dehydrogenase, 21-
hydroxylase, C17-20 lyase and/or 116-hydroxylase (Lipsett and Wilson 1962 ; Martin 1962 ; West et al. 1964 ; O’Hare et al. 1979). However, only a few reports (Degenhart et al. 1982 ; D’Agata et al. 1987) about steroidogenic enzyme activity in adenomas have been published. Therefore it is still difficult to determine its enzymic function from the histological investigation in each case. We have been studying the relationship between morphology and function of adrenocortical tumors to know whether the morphological abnormality cause following excessive production of steroid. We documented the abnormality of 116-hydroxylase and 18-hydroxylase in mitochondrial fraction of adrenocortical tumors previously (Naganuma et al. 1988). In the present paper we report the abnormal activities of steroid 21-hydroxylase and 17a-hydroxylase in functioning adrenocortical tumors manifesting Cushing’s syndrome, primary aldosteronism or AGS.
MATERIALS AND METHODS
Materials
The surgical materials from 12 adrenocortical tumors, one adrenocortical hyperplasia and 7 normal adrenal glands were used. Sex and age of the patients along with the relevant clinical data and the histology of the adrenocortical tumors are listed in Table 1. Five of the adrenocortical adenomas are from the patients with primary aldosteronism and Cushing’s syndrome, respectively. There were one carcinoma resulting in Cushing’s syn- drome and one resulting in AGS. The normal adrenal glands were taken from 2 patients with mammary carcinoma, 2 with pheochromocytoma, 2 with renal cell carcinoma and one with adrenal cyst without any hormonal symptoms as controls.
Plasma renin activity (PRA), plasma aldosterone concentration (PAC) and plasma cortisol (F) were measured by radioimmunoassay, 17-hydroxycorticoids (17-OHCS) by Porter-Silber’s method and 17-ketosteroids (17-KS) by Zimmermann-Kanbegawa’s method in the commercial laboratory institute.
Isolation of microsomal fraction
Adrenocortical tumors and adrenal cortex removed surgically were rapidly placed in an ice bath. Tissues of the tumor, adjacent adrenal cortex, medulla and capsule were each isolated. The microsomal fraction of the tumor and adrenocortical tissues was prepared according to the method of Cammer et al. (1968) with some modifications. A part of microsomal fraction was fixed in 3% glutaraldehyde with cacodylate buffer (pH 7.4) and examined by electron microscopy (JEM 100B, JEOL, Ltd., Tokyo). Protein concentration was determined by the method of Lowry et al. (1951) using bovine serum albumin as the standard.
Enzyme assay
General conditions of the incubation were as follows; The triplicated microsomal preparations, 0.1 mg/ml of 20 mM Tris-HCl buffer (pH 7.4) containing 11.5 mM NaCl, 15.7 mM KCI, 0.5 mM NADPH were incubated aerobically at 37℃ for 10 min in the presence of 5 nM of progesterone or 17a-hydroxyprogesterone as a substrate. Then the reaction was terminated by an addition of 1.0 ml methanol and samples were stored at -20℃ until analysis by high-performance liquid chromatography (HPLC).
The incubation medium was extracted with five times volumes of dichloromethane. The extracts were evaporated to dryness and the residue was dissolved in 20 ul of tetrahy- drofuran (THF). An aliquot was subjected to HPLC for determination of products. The apparatus used was a Waters Model 510 pump (Water Assoc., Milford, MA, USA) with a
| Case | Age | Sex | Dssease | PRA (ng/ml/hr) | PAC (pg/ml) ) | F (µg/100 ml) | (mg/day) 17-OHCS | 17KS (mg/day) | Histology | Tumor size (cm) | |
|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | R.T. | 57 | F | MC | |||||||
| 2 | T.S. | 60 | F | MC | |||||||
| 3 | F.H. | 51 | M | RCC | Normal | ||||||
| 4 | R.M. | 36 | F | Pheo | adrenocortex | ||||||
| 5 | K.N. | 42 | F | Pheo | |||||||
| 6 | M.O. | 73 | F | RCC | |||||||
| 7 | Y.A. | 50 | F | Ad. cyst | |||||||
| 8 | Y.H. | 27 | F | PA | 0.2 | 310 | 3.3 | 4.6 | 1.5×1.5×1.0 | ||
| 9 | C.I. | 28 | F | PA | 0.3 | 354 | 8.7 | 2.3×2.2×1.0 | |||
| 10 | M.I. | 39 | F | PA | 2.2 | 370 | 26.2 | 5.8 | 3.5 | Adenoma | 2.1×1.6×1.4 |
| 11 | T.K. | 40 | M | PA | Trace | 186 | 13.0 | 6.9 | 6.8 | 1.0×1.2×1.4 | |
| 12 | K.W. | 45 | F | PA | Trace | 285 | 4.7 | 8.1 | 6.1 | 1.5×1.8×2.2 | |
| 13 | S.I. | 30 | F | Cushing | 25.6 | 16.2 | 8.8 | 3.2×3.0×2.5 | |||
| 14 | T.S. | 27 | M | Cushing | 21.0 | 23 | 9.3 | 9.2 | 9.3 | 3.0×2.5×2.2 | |
| 15 | T.Y. | 36 | M | Cushing | 38.5 | <1 | 36.0 | 24.0 | 12.9 | Adenoma | 2.7×3.2×2.9 |
| 16 | E.N. | 34 | F | Cushing | 0.2 | 54 | 26.0 | 12.5 | 4.3 | 3.0×2.0×1.8 | |
| 17 | R.O. | 64 | F | Cushing | 22.2 | 10.5 | 4.0 | 1.5×2.0×2.4 | |||
| 18 | K.W. | 35 | F | CD | 25.4 | 62 | 37.2 | 35.3 | 7.0 | Hyperplasia | |
| 19 | I.S. | 41 | F | AGS | 3.9 | 262 | 24.3 | 4.0 | 125.3 | Carcinoma | 17.0×12.0×5.5 |
| 20 | T.T. | 56 | F | Cushing | 5.7 | 85 | 19.9 | 22.4 | 23.7 | Carcinoma | 10.5×8.5×7.0 |
PRA, plasma renin activity ; PAC, plasma aldosterone concentration ; F, plasma cortisol ; 17-OHCS, 17-hydroxocorticoids ; 17-KS, 17-ketosteroids ; MC, mammary carcinoma; RCC, renal cell carcinoma ; Pheo, pheochromocytoma ; Ad. cyst, adrenal cyst ; PA, primary aldosteronism ; Cushisg, Cushing’s syndrome ; CD, Cushing’s disease ; AGS, adrenogenital syndrome ; - , not examined.
Waters Model 441 UV detector (254 nm). A radial-pak phenyl (8-10 um) column (10 cm × 8 mm I.D.) (Waters Assoc.) was employed as the mobile phase of THF : H2 O=30 : 70 at a flow-rate of 3 ml/min. The products were qualified with the authentic steroids. The value was identified using external standard curve.
Comparison of results were made with Student’s t-test. Results are expressed as the mean ± S.D.
Reagents
Progesterone, 17a-hydroxyprogesterone, 11-deoxycorticosterone, 11-deoxycortisol and 16-hydroxyprogesterone were obtained from Sigma Chemical Company (St. Louis, MO, USA). Other reagents were commercial products of analytical grade.
RESULTS
Electron microscopical findings
The microsomal fraction obtained was the homogeneous fraction without contamination of mitochondria by electron microscopic examination (data not shown).
Steroid hydroxylation
The results of the partition chromatography of the extracts from the incuba- tion of the microsomal fraction of the control adrenal cortex with progesterone are shown Fig. 1. Accordisng to authentic steroids five peaks were identified as progesterone, 17a-hydroxyprogesterone, deoxycorticosterone, 11-deoxycortisol and 16-hydroxyprogesterone. As shown in Fig. 2, 17a-hydroxyprogesterone and 11-deoxycortisol were detected in the incubation of every microsomal fractions
Absorbance at 254 nm
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Absorbance at 254 nm
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with 17a-hydroxyprogesterone.
21-hydroxylase
The activities of 21-hydroxylase with progesterone and with 17a- hydroxyprogesterone were compared. As shown in Table 2, the activities of 21-hydroxylase with progesterone and 17a-hydroxyprogesterone in control adrenal cortex were 1.31-1.93 nmole/min/mg protein (mean+s.D .= 1.61+0.25 nmole/min/mg protein) and 3.12-6.61 nmole/min/mg protein (mean+s.D .= 5.22±1.06 nmole/min/mg protein), respectively. The 21-hydroxylase activity with progesterone was higher in four cases of five aldosteronomas than in normal control (3.28±1.52 vs. 1.61±0.25, p<0.05). Those activities with progesterone in Cushing’s adenomas except two cases (No. 13 and No. 17) were in the range of normal controls. Case 13 showed lower 21-hydroxylase activities with progester- one and 17a-hydroxyprogesterone (0.50 nmole/mg protein/min and 2.02 nmole/ min/mg protein, respectively). Case 17 showed higher activities of 21- hydroxylase with both substrates (5.80 and 14.59 nmole/min/mg protein). In contrast, these 21-hydroxylase activities were lower in carcinomas than in normal controls. The activities of 21-hydroxylation of 17a-hydroxyprogesterone in most of adenomas were in the range of normal controls.
17a-hydroxylase
The activity of 17a-hydroxylase were much variable from tissue to tissue in control adrenocortices (2.03-8.49 nmole/min/mg protein) and from case to case in adrenocortical tumors. There was no significant differences among the groups of the cases. However, aldosteronomas of cases 11 and 12, a Cushing’s adenoma of case 13 and two cases of carcinomas showed the lower activity than normal
| Case | 21-hydroxylase substrate | 17 a-hydroxolase | ||
|---|---|---|---|---|
| Progesterone | 17 a-OH-prog. | |||
| Normal control | ||||
| 1 | R.T. | 1.32* | 5.05 | 5.61 |
| 2 | T.S. | 1.31 | 3.12 | 8.49 |
| 3 | F.H. | 1.74 | 5.55 | 5.02 |
| 4 | R.M. | 1.46 | 5.12 | 3.21 |
| 5 | K.N. | 1.93 | 5.60 | 2.03 |
| 6 | M.O. | 1.72 | 5.48 | 2.64 |
| 7 | Y.A. | 1.82 | 6.61 | 3.72 |
| Aldosteronoma | ||||
| 8 | Y.H. | 5.16 | 0.63 | 3.64 |
| 9 | C.I. | 2.04 | 6.00 | 4.53 |
| 10 | M.I. | 3.78 | 8.93 | 4.03 |
| 11 | T.K. | 3.99 | 6.86 | 0.94 |
| 12 | K.W. | 1.45 | 5.69 | 1.50 |
| Cushing's adenoma | ||||
| 13 | S.I. | 0.50 | 2.02 | 1.79 |
| 14 | T.S. | 1.29 | 4.06 | 6.14 |
| 15 | T.Y. | 1.95 | 6.36 | 6.95 |
| 16 | E.N. | 1.95 | 5.82 | 4.06 |
| 17 | R.O. | 5.80 | 14.59 | 3.38 |
| Hyperplasia | ||||
| 18 | K.W. | 1.25 | 3.46 | 6.79 |
| Carcinoma | ||||
| 19 | I.S. | 0.03 | 0.73 | 1.97 |
| 20 | T.T. | 0.69 | 2.13 | 0.50 |
*Values are mean for triplicated samples and are expressed in nmole/min/mg protein.
17a-OH-prog., 17a-hydroxyprogesterone.
controls.
DISCUSSION
It is generally accepted that the pathway from pregnenolone to 17a- hydroxyprogesterone through progesterone is dominant in human adrenal gland. Therefore glucocorticoid is produced more than mineralocorticoid in human. In this study we confirmed the theory using progesterone and 17a- hydroxyprogesterone as the substrates from the enzyme studies in the normal subjects. The activity of 21-hydroxylase with 17a-hydroxyprogesterone was three times as much as that with progesterone in normal controls (mean±s.D .=
| Case | C21-17P/C21-P | C17/C21 | |
|---|---|---|---|
| Normal control | |||
| 1 | R.T. | 3.8 | 4.3 |
| 2 | T.S. | 2.4 | 6.5 |
| 3 | F.H. | 3.2 | 2.9 |
| 4 | R.M. | 3.5 | 2.2 |
| 5 | K.N. | 2.9 | 1.1 |
| 6 | M.O. | 3.2 | 1.5 |
| 7 | Y.A. | 3.6 | 2.0 |
| Mean±S.D. | 3.2±0.4 | 2.9+1.8 | |
| Aldosteronoma | |||
| 8 | Y.H. | 0.1 | 0.7 |
| 9 | C.I. | 2.9 | 2.2 |
| 10 | M.I. | 2.4 | 1.1 |
| 11 | T.K. | 1.7 | 0.2 |
| 12 | K.W. | 3.9 | 1.0 |
| Mean±S.D. | 2.2±1.3 | 1.0±0.66 | |
| Cushing's adenoma | |||
| 13 | S.I. | 4.0 | 3.6 |
| 14 | T.S. | 3.1 | 4.8 |
| 15 | T.Y. | 3.3 | 3.6 |
| 16 | E.N. | 3.0 | 2.1 |
| 17 | R.O. | 2.5 | 0.6 |
| Mean+S.D. | 3.1±0.5 | 3.4±1.6 | |
| Hyperplasia | |||
| 18 | K.W. | 2.8 | 5.4 |
| Carcinoma | |||
| 19 | I.S. | 24.3 | 2.7 |
| 20 | T.T. | 3.1 | 0.7 |
C21-17P/C21-P, ratio between 21-hydroxylase with 17a- hydroxyprogesterone and 21-hydroxylase with progesterone ; C17/ C21, ratio between 17a-hydroxylase with progesterone and 21- hydroxylase with progesterone.
3.2±0.4) as shown in Table 3. Nelson and Bryan (1975) have also reported the similar findings in microsome from normal human adrenocortex. These results suggest that the pathway to glucocorticoid from progesterone in dominant over the pathway to mineralocorticoids in human adrenocortical microsomes.
Functioning adrenocortical tumors produce excessive steroids. To examine
the cause of overproduction of steroids, we have investigated the enzyme activities for steroidogenesis including 116-hydroxylase and 18-hydroxylase in mito- chondria (Naganuma et al. 1988) and 21-hydroxylase and 17a-hydroxylase in microsome of functioning adrenocortical tumors.
In some aldosteronomas increment of 21-hydroxylase activity and decrement of 17a-hydroxylase activity were observed. The mean ratios between 21- hydroxylase with 17-hydroxyprogesterone and that with progesterone were under 3 (mean+S.D .= 2.2+1.27) (Table 3). The ratios between 17a-hydroxylase and 21-hydroxylase with progesterone were lower in aldosteronomas than in normal controls (mean+s.D .= 1.0±0.7 vs. 2.9+1.8, p<0.05) (Table 3). These indicate the relative decrement of 17a-hydroxylase activity in these adenomas. Sasano et al. (1980) reported the elevation of plasma progesterone, deoxycorticos- terone and 18-hydroxycorticosterone of the patients with aldosteronoma. They mentioned the decreased activity of 17a-hydroxylase by the results of plasma steroids level, and Dufau et al. (1968) have already reported it by enzyme assay in the tissue of aldosteronoma. Furthermore in our previous study 118- hydroxylase and 18-hydroxylase activity in mitochondrial fractions of aldoster- onomas were elevated (Naganuma et al. 1988). These results support the exces- sive production of aldosterone (mineralocorticoids) in the patients with aldoster- onoma. However, in some aldosteronomas no decrement activity was shown in this paper herein reported. In case 10 (clinically primary aldosteronism) 21- hydroxylase with 17a-hydroxyprogesterone was higher than those of other aldos- teronomas. This result of enzyme activity leads elevation of plasma cortisol in this case. In the clinical data of this case plasma cortisol level was also elevated (26.2 µg/100 ml) as if it was Cushing’s adenoma.
As the previous reports of ultrastructural findings of aldosteronomas have revealed in detail (Sommers and Terzakis 1970; Kano et al. 1979 ; Sasano et al. 1980 ; Mazzocchi et al. 1982), we also observed the morphology and quality of mitochondria and ER vary from case to case in aldosteronomas and the ER are not prominent in some cases. The fact of decreased amount of ER may cause low 17a-hydroxylase activity in these cases. These histological variation may be related to the various enzymic activity in the mitochondrial and microsomal fractions in aldosteronomas.
In most of Cushing’s adenomas and one case of adrenocortical hyperplasia the activities of 21-hydroxylase and 17a-hydroxylase were within the range of normal control. 116-hydroxylase activity was also in the range of normal controls in our previous report (Naganuma et al. 1988). Other investigators documented the increased activity of various enzymes in Cushing’s adenomas (Degenhart et al. 1982 ; D’Agata et al. 1987). Such increased enzyme activity was not observed in our study. From our data the overproduction of cortisol in Cushing’s adenomas is not supported, but histological examination may solve this problem. Compar- ing the tumor size in this study, Cushing’s adenomas were larger than aldoster-
onomas when they were surgically resected (Table 1). Ultrastructurally cortical cells in Cushing’s adenoma have much more abundant ER than those of normal adrenocortices. We speculate that a large amount of ER which have the enzyme lead glucocorticoid excess in Cushing’s adenomas.
In carcinomas complete defect or significant decrement of enzyme activities have been reported. Carcinomas show microscopically cellular atypism and ultrastructurally abnormal mitochondria and ER. Irregular shaped mitochon- dria and ER, or decreased numbers of ER were observed in our cases. In the case of carcinoma with AGS the activities of 21-hydroxylase and 17a-hydroxylase were low as well as the activity of 116-hydroxylase previously reported. How- ever, case 20 (carcinoma with Cushing’s syndrome) did not show prominent decrease of enzyme activities. The elevated serum cortisol level (19.9 µg/100 ml) in the clinical data of this patient was supported by these results. The abnormal features of mitochondria and ER observed morphologically in carcinomas may be strongly related to the enzyme defect or decreased activity of the enzyme.
Finally we found it is difficult to make clear the relationship between the morphological findings and enzymic activity, because the morphological changes vary from case to case and from cell to cell in the same tumor. However, it is obvious that both morphological and enzymic abnormalities lead overproduction or defect of certain steroids in the adrenocortical tumors.
References
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