Studies on the Efficiency of Adrenocortical 118-Hydroxylation in the Human Subject
G. L. NICOLIS AND J. L. GABRILOVE
Endocrine Research Laboratory of the Department of Medicine, The Mount Sinai Hospital and School of Medicine, New York, New York 10029
ABSTRACT. The efficiency of 118-hydroxyla- tion by the adrenal cortex was studied in normal subjects, women with hirsutism and/or virilism and patients with various types of adrenocortical hyperfunction by simultaneous measurement of the secretory rates of cortisol (F) and 11-deoxy- cortisol (S).2 The ratio F/F +S×100 was found to be 95.1 ±2.2 in subjects without abnormalities in 118-hydroxylation. This ratio was not altered by the administration of ACTH nor in 8 subjects
with Cushing’s syndrome due to nontumorous adrenocortical hyperfunction. Decreases in the ratio were found in 2 subjects with adrenocortical carcinoma, in 1 patient with Cushing’s syndrome secondary to a bronchogenic carcinoma, in 1 of 2 subjects with Cushing’s syndrome due to an adrenocortical adenoma, and in 2 patients given SK&F-12185, an 118-hydroxylation inhibitor. (J Clin Endocr 29: 831, 1969)
R ECENT investigations in this lab- oratory in regard to feminizing adre- nocortical tumors in men (1) and more par- ticularly the delayed manifestation of adrenocortical 116-hydroxylation defi- ciency in women (2) prompted us to study further the efficiency of this enzymic step. We were primarily interested in the fre- quency of such a defect in 118-hydroxyla- tion in women with post-puberal hirsutism and/or virilization and whether the ad- ministration of corticotropin might accen- tuate and make easier to demonstrate minor degrees of the defect. At first the studies concerned the urinary excretion of tetra- hydro-11-deoxycortisol (THS), tetrahydro- cortisol (THF) and tetrahydrocortisone (THE), but it soon became clear that si- multaneous secretory rates of 11-deoxycor- tisol (S) and cortisol (F) would afford a more precise evaluation of the efficiency of 116-hydroxylation. During the course of these studies, other investigations, paral- leling ours in some aspects, have appeared (3-5).
Received January 7, 1969.
Aided by grants from the NIH (HD 02764 and FR-71).
1 USPHS Special Fellowship 2-F3-AM33,139-02.
Materials and Methods
The subjects studied included normal con- trols, patients sent for endocrine evaluation and found to be free of adrenocortical disease, women with hirsutism and/or virilism or with the Stein-Leventhal syndrome, patients with an adrenogenital syndrome due to adrenocorti- cal carcinoma or nontumorous hyperfunction as well as those with Cushing’s syndrome due to tumor or nontumorous hyperfunction.
Some of these subjects were studied during a control period and on the third day of the in- tramuscular administration of corticotropin- Zinc, 40 U/day.
Urinary THS, THE and THF were mea- sured as previously described (6). The secre- tory rates of F and S were calculated from the specific activities of urinary THE and THS after the simultaneous intravenous administra- tion of 3H-1,2,F (25 Ci/mmole) and 3H-7,S (10 Ci/mmole). One half of the urine collected during the 24 hr following the administration of the radioactive tracers was hydrolyzed with ß-glucuronidase, extracted with ethyl acetate and chromatographed on a thin layer plate (7). The fraction containing THE was chromato-
2 Cortisol (F) =118,17a,21-trihydroxypregn-4- ene-3,20-dione; 11-deoxycortisol (S) =17a,21- dihydroxypregn-4-ene-3,20-dione; tetrahydrocorti- sone (THE) =3a,17a,21-trihydroxy-58-pregnane- 11,20-dione; tetrahydrocortisol (THF) =3a,118, 17a,21-tetrahydroxy-58-pregnan-20-one; allo-te- trahydrocortisol (Allo-THF) =3a,118,17a,21-te- trahydroxy-5@-pregnan-20-one; tetrahydro-11-de- oxycortisol (THS) =3a,17a,21-trihydroxy-58-preg- nan-20-one.
| Age | Sex | Patient | SR S mg/24 hr | SR F mg/24 hr | F/(F +S) X100 | TG ug /24 hr | Clinical diagnosis or symptoms |
|---|---|---|---|---|---|---|---|
| 19 | f | MF | 0.8 | 10.9 | 93.2 | Primary amenorrhea | |
| 46 | f | AN | 0.6 | 23.6 | 97.5 | Absence of endometrium Oligomenorrhea, hypertension, obesity | |
| 34 | f | HH | 0.7 | 5.6 | 88.9 | "Weakness." Normal 17- hydroxycorticoid response to ACTH | |
| 28 | f | IN | Hirsutism. Anovulatory menses | ||||
| 0.3 | 4.3 | 93.5 | 10.1 | on prednisone | |||
| 0.4 | 9.9 | 96.1 | 6.1 | off prednisone for 3 weeks | |||
| 25 | f | AM | 1.0 | 16.5 | 94.3 | Stein-Leventhal syndrome | |
| 20 | f | MB | 0.6 | 12.7 | 95.5 | Stein-Leventhal syndrome | |
| 21 | f | IH | 0.8 | 7.0 | 89.7 | Oligomenorrhea, hirsutism, obesity, hypertension | |
| 21 | f | PP | 0.8 | 19.5 | 96.1 | 45.0 | Hirsutism, oligomenorrhea |
| 22 | f | BB | Hirsutism | ||||
| 6.7 | 10.5 | 61.0 | 10.3 | on prednisone (5 mg bid) | |||
| 0.6 | 10.1 | 94.4 | off prednisone 1 month | ||||
| 20 | f | EB | 0.8 | 2.3 | 74.2 | 118-hydroxylase deficiency (2) on prednisone (5 mg bid) | |
| 21 | f | SK | 0.6 | 9.5 | 94.1 | 7.0 | Oligomenorrhea, hirsutism |
| 23 | f | JB | 0.8 | 14.5 | 94.8 | 2.7 | Hirsutism |
| 16 | f | TM | 0.7 | 14.0 | 95.2 | 7.3 | Hirsutism, obesity |
| 56 | f | SD | 0.9 | 22.6 | 96.2 | Acromegaly | |
| 19 | m | PR | 1.3 | 24.2 | 94.9 | 110 | Klinefelter's syndrome (chromatin negative) |
| 17 | m | KT | 1.5 | 16.4 | 91.6 | 31 | Klinefelter's syndrome (chromatin positive) |
| 28 | m | MJ | 0.8 | 12.2 | 93.0 | 89 | Gynecomastia; oligospermia |
| 17 | f | RK | Congenital adrenal hyperplasia | ||||
| (pregnanetriol 1.4 mg/24 hr) | |||||||
| (sib of | 4.4 | 5.6 | 56.8 | 8.2 | on prednisone 7.5 mg qd | ||
| LK) | 0.6 | 13.1 | 95.6 | off prednisone 2 months | |||
| 0.5 | 19.8 | 97.5 | 11.1 | off prednisone 5 months | |||
| 0.4 | 24.9 | 98.4 | 14.7 | off prednisone 6 months | |||
| 20 | f | LK | 3.5 | 1.8 | Congenital adrenal hyperplasia | ||
| (sib of | (pregnanetriol 4.6 mg/24 hr) | ||||||
| RK) | 0.4 | 15.8 | 97.5 | 3.1 | off prednisone 2} months | ||
| 0.3 | 16.4 | 98.2 | 7.9 | off prednisone 6 months | |||
| 11.6 | off prednisone 9 months | ||||||
| Mean | 0.65±0.28 | 15.2±5.5 |
graphed on paper in the toluene/propylene gly- col and in the ethylene dichloride/ethylene gly- col systems. THS was chromatographed in the same systems and in addition in the ethyl acetate:toluene (6:4)/formamide system. Ste- roids were located by scanning for 3H and by staining a portion of the chromatograms with blue tetrazolium. Specific activities were mea- sured by liquid scintillation counting and by the blue tetrazolium reaction (7). For THS this measurement was usually carried out on both of the last 2 chromatograms and the as- say was repeated if the difference exceeded 15%. In 4 cases the urine was additionally collected from 24 to 48 hr after the radioactive tracers. The THE and THS fractions obtained
from this urine contained less than 1% of the radioactivity found in the urine collected during the first 24 hr. No significant radioactivity was found in the THS fraction after administration of 3H-F alone, nor in the THE fraction after administration of 3H-S (3 cases). Urinary tes- tosterone glucuronide was measured as pre- viously described (6).
Results
As discussed by Cope (3), the expression F/(F+S) ×100 may be assumed to repre- sent the per cent of 11-deoxycortisol that is further hydroxylated at C-11 to form cor- tisol in the adrenal cortex. In this sense it
| Age | Sex | Patient | SR S mg/24 hr | SR F mg/24 hr | F/(F+S) X100 | TG ug/24 hr | Clinical diagnosis or symptoms |
|---|---|---|---|---|---|---|---|
| 18 | f | ST | 0.8 5.0 | 16.5 | 95.4 | 6.4 | Stein-Leventhal syndrome |
| p ACTH | 69.3 | 93.2 | 12.0 | ||||
| 23 | f | HV | 0.9 | 14.6 44.9 | 94.2 | 30.0 | Hirsutism |
| p ACTH | 0.6 | 98.7 | 52.0 | ||||
| 32 | f | ZS | 0.4 | 19.2 86.4 | 97.9 | 58.0 | Oligomenorrhea, hirsutism, |
| p ACTH | 9.2 | 90.4 | infertility | ||||
| 18 | f | RW | 0.6 | 15.4 124.5 | 96.2 | 4.0 | Oligomenorrhea |
| p ACTH | 4.1 | 96 9 | 67.0 | ||||
| 28 | f | EK | 0.8 | 31.9 | 97 5 | 1.4 | Oligomenorrhea, hirsutism, |
| p ACTH | 3.9 | 75.2 | 95.1 | 3.0 | obesity | ||
| 21 | f | IS | 0.4 | 20.8 | 98.1 | 10.3 | Oligomenorrhea, hirsutism |
| p ACTH | 3.2 | 79.0 | 96.1 | 2.8 | |||
| 29 | f | EMcG | 0.6 | 13.7 273.0 | 95.8 | 7.0 | Hirsutism |
| p ACTH | 12.6 | 95.6 | 56.0 | ||||
| 25 | f | PP | 0.4 | 11.3 87.0 | 96.6 | 6.3 | Obesity, hirsutism |
| p ACTH | 5.3 | 94.3 | 18.0 | ||||
| 42 | f | HE | 0.4 | 6.9 67.0 | 94.5 | 10.0 | "Weakness" |
| p ACTH | 1.2 | 98.2 | 3.1 | ||||
| 29 | m | HK | 2.1 | 30.7 | 93.6 | 7.0 | Chromatin positive |
| p ACTH | 2.2 | 92.0 | 97.7 | 51.0 | Klinefelter's syndrome | ||
| 29 | m | BG | 0.3 | 15.4 49.5 | 98.1 | 106.0 | Oligospermia |
| p ACTH | 0.7 | 98.6 | 1060.0 | ||||
| 38 | m | DG | 0.4 | 19.2 | 97.9 | 142.0 | High urinary 17-KS |
| p ACTH | 2.8 | 115.0 | 97.6 | 128.0 | (29 mg/24 hr) | ||
| Mean | Control | 0.67 ±0.5 | 17.9±3.9 | ||||
| p ACTH | 4.2 ±3.7 | 96.9±59.2 |
may be used as an index of the efficiency of the adrenocortical 116-hydroxylase sys- tem. From Tables 1 and 2, consisting of pa- tients with and without various endocrine disorders, it can be seen that the per cent F/(F+S) is usually greater than 90. If the patients treated with prednisone are omit- ted as well as EB, a patient with an 113- hydroxylase deficiency (2), the mean per- centage for 26 subjects is 95.1±2.2,3 a value statistically identical with that found for normal subjects by Cope et al. (3) and by Vermeulen et al. (5). The administra- tion of ACTH had no effect on the effi- ciency of 118-hydroxylation since in 12 sub- jects tested in this fashion the control ratio was 96.3 ±2.5% and after ACTH 96.0 ± 2.5%. Similarly, eight patients with Cush- ing’s syndrome due to nontumorous adre- nocortical hyperfunction converted S to F efficiently as evidenced by normal ratios (mean 97.0 ±1.6%) (Table 3). However,
patient MC, with Cushing’s syndrome associated with a bronchogenic carcinoma, showed reduced 118-hydroxylase activity (71%). Of the two patients with an adre- nocortical adenoma in association with Cushing’s syndrome, the ratio was normal in one and low (83.5%) in the other. A marked reduction in the conversion of S to F is clearly seen in both our patients with feminizing (HM) and virilizing (JK) adre- nocortical carcinoma. In a third patient with carcinoma of the adrenal and Cush- ing’s syndrome in whom secretory rates were not measured, the ratio of urinary THS/(THE+THF)×100 was 13.6 (nor- mal less than 2%; unpublished data).
In patient DF with Cushing’s syndrome the ratio of F/(F+S) was low (83.1%), but unfortunately the nature of the under- lying adrenal lesion could not be established since the patient was lost to follow up. Two patients with the adrenogenital syndrome (BB and RK) had an increased secretion of S (6.7 and 4.4 mg) while receiving predni-
3 +Standard deviation throughout the text.
| Age | Sex | Patient | SR S mg/24 hr | SR F mg/24 hr | F/(F+S) ×100 | TG ug/24 hr | Diagnosis |
|---|---|---|---|---|---|---|---|
| 40 | m | SW | Cushing's syndrome; nontumorous | ||||
| 3.3 | 60.0 | 94.8 | hyperfunction off all Rx | ||||
| 63.0 | 28.5 | 31.1 | on SK&F-12185 -- 19 days | ||||
| 71.1 | 30.0 | 29.9 | 69.0 | on SK&F-12185-2 months | |||
| 0.9 | 36.0 | 97.6 | 30.0 | off all Rx | |||
| 1.8 | 56.0 | 96.9 | Zinc-ACTH 40 U qd X3 days | ||||
| 47 | f | SL | Cushing's syndrome; nontumorous | ||||
| hyperfunction | |||||||
| 11.3 | 39.0 | 77.5 | on SK&F-12185 | ||||
| 7.3 | 36.5 | 83.3 | on SK&F-12185 | ||||
| 8.5 | 33.0 | 79.5 | 60.0 | on SK&F-12185 | |||
| 47.0 | off SK&F-12185-6 days | ||||||
| 0.3 | 33.5 | 99.1 | off Rx | ||||
| 20 | f | AG | 1.4 | 45.0 | 97.0 | 17.0 | Cushing's syndrome; nontumorous hyperfunction |
| 37 | m | BS | 5.3 | 127.0 | 96.0 | 74.0 | Cushing's syndrome; nontumorous hyperfunction |
| 41 | f | BF | 0.32 | 39.8 | 99.0 | Cushing's syndrome; nontumorous | |
| 60 | m | DG | 1.3 | 47.5 | 97.5 | hyperfunction Cushing's syndrome; nontumorous hyperfunction | |
| 39 | f | BF | 0.8 | 40.2 | 98.0 | 2.0 | Cushing's syndrome; nontumorous hyperfunction |
| 22 | f | MF | 1.4 | 79.5 | 98.3 | Cushing's syndrome; nontumorous hyperfunction | |
| 48 | f | MC | 18.5 | 45.0 | 70.1 | Cushing's syndrome; nontumorous hyperfunction 2º to bronchogenic carcinoma | |
| 33 | f | DF | 8.9 | 43.9 | 83.1 | Cushing's syndrome; underlying lesion not known | |
| 48 | f | BM | 9.25 | 47.0 | 83.5 | Cushing's syndrome; adrenal adenoma | |
| 9.60 | 50.0 | 84.0 | After 8 mg dexamathasone | ||||
| 29 | f | MC | 4.7 | 72.0 | 93.8 | 29.3 | Cushing's syndrome; adrenal adenoma |
| 55 | m | HM | 13.5 | 8.0 | 37.2 | 65.0 | Feminizing adrenal Ca |
| 19 | f | JK | Virilizing adrenal Ca | ||||
| 4.7 | 2.0 | 29.8 | control | ||||
| 13.4 | 1.5 | 10.1 | control | ||||
| 28.0 | 1.0 | 3.48 | on o,p'DDD | ||||
| 27.0 | 33.0 | 55.0 | 23.0 | on o,p'DDD +cortisol | |||
| (37.5 mg/day) |
sone. This became normal after the drug was discontinued. In two similar patients (IN and EB) receiving prednisone, S secre- tion was normal. Patient SW with Cush- ing’s syndrome had an S secretion of 0.08 mg after bilateral adrenalectomy and while receiving 50 mg of prednisone a day.
Discussion
11-Deoxycortisol is considered the main direct precursor of cortisol in the human adrenal. Since there is no evidence that it is
converted into other major biosynthetic products, the adrenal secretion of 11-deoxy- cortisol relative to that of cortisol has been taken as a measure of the completeness of hydroxylation at C-11.
In 1954 THS was isolated from human urine and was identified as the major a- ketolic excretory product of S (8, 9). In- creased urinary excretion of this compound has since been found in a large proportion of patients with adrenal carcinoma (8, 9), in patients with the hypertensive form of
congenital adrenal hyperplasia (10), and in subjects receiving metyrapone (11) or similar 116-hydroxylase inhibitors (6). Compared to normal daily excretions of 0.02-0.1 mg (12, 13) urinary THS in these conditions usually exceeded 2.0 mg and often reached much higher titers (12, 14- 16). These increases were not accompanied by similar rises in the urinary metabolites of cortisol and indicated a relative or abso- lute defect in the enzymatic system that converts 11-deoxycortisol to cortisol. Smal- ler increases in urinary THS (usually to less than 1.0 mg/day) were also occasionally found in patients with Cushing’s syndrome due to nontumorous hyperfunction and after stimulation with ACTH (12, 17). In these cases, however, there was no clear change in the proportion of the urinary metabolites of S and F and it seemed prob- able that 113-hydroxylation was proceed- ing normally.
In 1966, Cope et al. (3) studied the con- version of 11-deoxycortisol to cortisol by simultaneously administering these ste- roids in radioactive form and calculating their secretion rates from the specific activ- ity of their urinary metabolites. They sug- gested that the per cent efficiency of 118- hydroxylation could be conveniently ex- pressed by the ratio F/(F+S) ×100, where F and S represent the simultaneously de- termined secretion rates. In 13 subjects without endocrine dysfunction the daily secretion of S varied from 0.09 to 0.87 mg (mean 0.34 ±0.24) and that of F from 7.8 to 18.3 mg (mean 13.9 ±5.64). The mean F/(F+S) was 97±2.85%, indicating that by this criterion the conversion of 11-de- oxycortisol to cortisol was nearly quantita- tive. Of the nine patients with Cushing’s syndrome and nontumorous hyperfunction later studied by Cope and his associates (4), eight had normal or slightly elevated S secretion (0.2-2.4 mg) and normal 118- hydroxylation (mean 97.5%), and only one had markedly increased S secretion (24.6 mg) and reduced 118-hydroxylation (68%). A patient with Cushing’s syndrome and an
adrenal adenoma secreted 4.1 mg of S and had normal 116-hydroxylation efficiency (91%). Markedly elevated S secretion (49.0 and 165.8 mg) and reduced 113-hy- droxylation (53%) were found in two pa- tients with adrenal carcinoma and Cush- ing’s syndrome, but a third patient with virilization also due to an adrenal carci- noma had normal S secretion (1.4 mg) and normal 118-hydroxylation (91 %).
Vermeulen et al. (5), using the same ap- proach, obtained essentially the same re- sults. Their normal S secretion varied from 0.22 to 1.36 mg/day (mean 0.54 ±0.34) and the calculated conversion of 11-deoxycorti- sol to cortisol was 95.8 ±2.7%. After stim- ulation with zinc-ACTH (120 U/day for 3 days) the secretion of S rose to 1.38-17.9 mg but there was a similar increase in F secretion and F/(F +S) ×100 remained un- changed (94.0 ±4.2). Two patients with Cushing’s syndrome, one with an adrenal carcinoma and the other with benign hyperplasia, secreted 3.9 and 2.4 mg of S and had unimpaired 113-hydroxylation (95.5 and 97.7%).
In the present series excessive S secretion (4.7-13.5 mg) and reduced 116-hydroxyla- tion (10.1-37.2%) were present in two pa- tients, one with a feminizing and the other with a virilizing adrenal carcinoma. A sim- ilar degree of hypersecretion of S (18.5 mg) and reduction in 116-hydroxylation (71%) was observed in a patient with Cushing’s syndrome secondary to a bronchogenic carcinoma. A patient with a benign adrenal adenoma and Cushing’s syndrome se- creted 9.4 mg of S and also showed im- paired conversion of S to F (83%). To our knowledge an 116-hydroxylase defect had not been previously described in this latter condition. It is apparent that, although relative increases in urinary THS are more frequent and usually more marked in asso- ciation with adrenal carcinomas, they can also occur with nontumorous hyperfunc- tion and with benign adrenal adenomas. This parameter cannot therefore be used as an absolute criterion for the diagnosis of
adrenal carcinoma. In our remaining nine patients with Cushing’s syndrome (8 with nontumorous hyperfunction and 1 with an adrenal adenoma) the ratio of the secretion of F to S was normal. This ratio was also normal in the 20 females who were studied because of marked hirsutism and varying degrees of virilization, menstrual dysfunc- tion and obesity. Administration of ACTH to eight of these patients to demonstrate possible minor degrees of enzymic block was also unsuccessful. In this respect we were unable to detect new cases similar to those previously described (2) and it seems probable that 116-hydroxylase deficiency is uncommon as a cause of post-puberal vir- ilization. It was of interest that in two of our subjects (RK and BB), one with con- genital adrenal hyperplasia and the other with presumably an acquired form, pro- duction of S rose both in absolute and rela- tive terms compared to cortisol while they were being treated with prednisone. This finding may indicate that an early effect of suppression of ACTH is a decrease in 113-hydroxylation or that prednisone has a direct inhibitory effect on this enzymatic reaction.
Acknowledgment
We wish to thank Miss Frima Botnick for ex- cellent technical assistance.
References
1. Gabrilove, J. L., D. C. Sharma, H. H. Wotiz, and R. I. Dorfman, Medicine (Balt) 44: 37, 1965.
2. Gabrilove, J. L., D. C. Sharma, and R. I. Dorfman, New Eng J Med 272: 1189, 1965.
3. Cope, C. L., P. M. Dennis, and J. Pearson, Clin Sci 30: 249, 1966.
4. Dennis, P. M., C. L. Cope, and J. Pearson, J Endocr 39: 207, 1967.
5. Vermeulen, A., G. Verdouck, M. van der Straeten, and R. Daneels, J Clin Endocr 27: 365, 1967.
6. Gabrilove, J. L., G. L. Nicolis, and T. F. Gal- lagher, J Clin Endocr 27: 1550, 1967.
7. Nicolis, G. L., H. H. Wotiz, and J. L. Gabrilove, J Clin Endocr 28: 547, 1958.
8. Rosselet, J. P., L. Overland, J. W. Jailer, and S. Lieberman, Helv Chim Acta 27: 1933, 1954.
9. Touchstone, J. C., E. M. Richardson, H. Bula- schenko, I. Landolt, and F. C. Dohan, J Clin Endocr 14: 676, 1954.
10. Eberlein, W. R., and A. M. Bongiovanni, J Biol Chem 223: 85, 1956.
11. Liddle, G. W., D. Island, E. M. Lance, and A. P. Harris, J Clin Endocr 18: 906, 1958.
12. Touchstone, J. C., H. Bulaschenko, E. M. Richardson, and F. C. Dohan, J Clin Endocr 17: 250, 1957.
13. Cost, W. S., and J. J. M. Vegter, Acta Endocr (Kobenhavn) 41: 571, 1962.
14. Cost, W., Acta Endocr (Kobenhavn) 42: 39, 1963.
15. Lipsett, M. B., and B. Damast, Proc Soc Exp Biol Med 99: 285, 1958.
16. Lipsett, M. B., and H. Wilson, J Clin Endocr 22: 906, 1962.
17. Cost, W., Acta Endocr (Kobenhavn) 42: 61, 1963.