Effects of Synthetic Corticotropin-releasing Factor in Normal Individuals and in Patients with Hypothalamic-pituitary-adrenocortical Disorders
H. JØRGENSEN, S. SKARE, H. FREY, K. F. HANSSEN and N. NORMAN From Medical Department B and Hormone and Isotope Laboratory, Aker Hospital, University of Oslo, Oslo, Norway
ABSTRACT. Jørgensen H, Skare S, Frey H, Hanssen KF, Norman N. (Medical Depart- ment B and Hormone and Isotope Laboratory, Aker Hospital, University of Oslo, Oslo, Norway.) Effects of synthetic corticotropin-releasing factor in normal individuals and in patients with hypothalamic-pituitary-adrenocortical disorders. Acta Med Scand 1985; 218: 79-84.
Plasma adrenocortical hormone (ACTH) and cortisol response to four dose levels (25, 50, 100 and 300 µg) corticotropin-releasing factor (CRF) were studied in 5 healthy men, and the response to 100 µg CRF in 12 patients with various disorders of the hypothalamic-pituitary- adrenocortical function. In normals, mean plasma ACTH and cortisol concentration rose at all dose levels of CRF and peaked at 30 and 60 min respectively. The increment in plasma cortisol at 60 and 90 min was significantly higher on 100 and 300 µg CRF than on 25 ug, but the total cortisol concentration was not. Seven patients had Cushing’s syndrome. In 2 patients with adrenocortical carcinoma the basal plasma ACTH was suppressed. After CRF a small increase was seen in plasma ACTH and cortisol in one patient successfully treated with mitotane, while the other patient did not respond. In 1 patient with ectopic ACTH syndrome an increase in plasma ACTH 15 min after CRF was not accompanied by any increase in plasma cortisol. One patient with bilateral multinodular adrenocortical hyperplasia did not respond to CRF. The plasma ACTH and cortisol response to CRF was supernormal in 2 patients with Cushing’s disease, while a third patient responded in the normal range. In 2 patients with Nelson’s syndrome the plasma ACTH response was excessive. Two out of three hypophysectomized patients did not respond to CRF, while one patient with a slightly positive response to hypoglycemia also responded (subnormally) to CRF. Our data indicate that CRF in doses of 50-100 µg will be a valuable substance in the differential diagnosis of Cushing’s syndrome. Some overlap in the response is, howev- er, seen between patients with Cushing’s disease and other patients with Cushing’ syn- drome. CRF will possibly be of value also for the diagnosis of secondary adrenocortical failure. Key words: corticotropin-releasing factor, dose response, normals, Cushing’s syndrome, Nelson’s syndrome, secondary adrenocortical failure.
Recent reports (1-5) indicate that corticotropin-releasing factor (CRF) may be a valuable tool in the differential diagnosis of Cushing’s syndrome. Studies on the dose-related response of plasma adrenocortical hormone (ACTH) and cortisol to CRF have recently been reported (6, 7). More studies are, however, needed before the optimal test dose of CRF is established.
In the present study we examined the plasma ACTH and cortisol response to four different dose levels of synthetic CRF in normal men. We also report on the response to 100 µg CRF in 7 patients with Cushing’s syndrome of various etiology, 2 with Nelson’s syndrome and in 3 hypophysectomized patients.
Abbreviations: CRF = corticotropin-releasing factor, ACTH = adrenocortical hormone, GH = growth hormone, LH = luteotropic hormone, FSH = follicle-stimulating hormone, TSH = thyroid- stimulating hormone.
Plasma ACTH
140
(ng/()
CRF i.v.
CRF dose(µg) :
120
100
100
50
300
80
0
25
60
50
0
0
5
15
30
60
90
Time after CRF (min.)
STUDY POPULATION AND METHODS
Five healthy men (aged 37-50 years) were given four different doses (25, 50, 100 and 300 µg) of synthetic ovine CRF (Bachem Inc., Fine Chemicals, Torrance, California, USA) in 5 ml isotonic saline, pH 2. Each subject was tested at intervals of at least one week.
An i.v. cannula was inserted at 7.30 a.m. after an overnight fast, and the subjects were kept supine throughout the studies. After 30 min rest, basal blood samples were collected, immediately followed by i.v. injection of CRF/saline through the cannula within 1 min. Blood samples were then collected after 5, 15, 30, 60 and 90 min and analyzed for ACTH, cortisol, luteotropic hormone (LH), follicle- stimulating hormone (FSH), thyroid-stimulating hormone (TSH), growth hormone (GH) and prolac- tin. The subjects were examined and observed with regard to possible side-effects during the tests.
CRF, 100 µg i.v., was given to 12 patients, employing the same procedure. Blood samples were, however, collected only at 0, 15, 30 and 60 min and analyzed only for ACTH and cortisol. Among 7 patients with active Cushing’s syndrome, 3 had Cushing’s disease. Since pituitary adenomas were not demonstrated radiologically (by CT) in any of these, they were treated with bilateral adrenalectomy, as was one patient with bilateral, multinodular adrenocortical hyperplasia. Two patients had adreno- cortical carcinoma; one of them, no. 6 (Table I), was successfully treated with mitotane. One patient had ectopic ACTH secretion from an oat-cell carcinoma of the lung. The adrenocortical function in this patient was normalized after removal of the lung tumour. We also studied 2 patients with Nelson’s syndrome, 1 respectively 21 years after bilateral adrenalectomy for Cushing’s disease. Both patients were doing well on cortisone, 25 mg in the morning and 12.5 mg at 6 p.m. together with Florinef, 0.10 mg i.d. The CRF test was done at 8 a.m., 14 hours after the last dose of cortisone. Three patients, nos. 10, 11 and 12 (Table I), were studied 1-3 years after hypophysectomy, and the CRF test was performed 38 hours after the last substitution dose of cortisone. Two of these patients showed, according to extensive endocrine testing, complete functional hypopituitarism after total hypophysec- tomy for ACTH-producing adenoma in one and a large craniopharyngeoma in the other. The third patient, in whom hypophysectomy was performed for Cushing’s disease due to an invasive pituitary adenoma, responded to insulin hypoglycemia with a small increase in plasma ACTH and cortisol.
Specific radioimmunoassay methods were used for measurement of cortisol, ACTH, LH, GH, prolactin and TSH (8, 9, 10).
Wilcoxon test for paired differences was used for statistical analysis. Results were expressed as mean ± SEM, p-values ≤0.05 were considered significant.
Plasma cortisol (nmol /l)
500
CRF dose(ug)
300
I
50
100
400.
CRF i.v.
25
300.
200.
0
0
5
15
30
60
Time in min after CRF
90
RESULTS
Controls
Basal mean plasma ACTH concentration in three normal subjects at 0 min was 63±6.0 ng/1 and did not change after injection of acid saline. After CRF, plasma ACTH rose within 5 min at all dose levels of CRF (Fig. 1). In 8 of 12 individual observations the peak plasma ACTH was seen at 30 min irrespective of the CRF dose. Unfortunately, the plasma samples for ACTH determinations from two of the five controls were lost.
Basal mean plasma cortisol concentration was 235±41 nmol/l at 0 min (n=5) and declined after acid saline injection. Following all dose levels of CRF, the mean plasma cortisol concentration more than doubled. Plasma cortisol increased after 15 min and was significantly (p<0.05) elevated at 30, 60 and 90 min with a peak at 60 min (Fig. 2).
The differences in mean plasma cortisol concentrations after the various doses of CRF were not significant at any time point. Irrespective of the CRF dose, peak plasma cortisol was in 15 of 20 tests found at 60 min, in 4 tests at 90 min and in one test at 30 min. When
the CRF response was expressed as increment from the basal value at 0 min, the peak increment in plasma cortisol at all dose levels of CRF was also seen at 60 min, and was in the range of 216-297 nmol/l. It was significantly (p<0.05) higher after 100 and 300 µg CRF than after 25 µg, while no significant difference was found between the incremental cortisol response to 50 µg CRF and the other dose levels of CRF. The increment in plasma cortisol at 90 min was significantly (p<0.05) higher at 300 µg than at the other dose levels of CRF. There were no significant changes after any dose of CRF in the plasma concentra- tions of LH, FSH, GH, TSH and prolactin; neither were there any changes in supine blood pressure or pulse rate.
Patients
The response to 100 µg CRF in 7 patients with untreated Cushing’s syndrome and 2 patients with Nelson’s syndrome, 1 respectively 21 years after bilateral adrenalectomy for Cushing’s disease, are shown in Table I.
The basal plasma ACTH concentration was increased in 2 of the 3 patients with Cushing’s disease and rose more than three-fold after CRF. In the third patient, both plasma ACTH concentration and ACTH response to CRF were within the range of normal controls. Plasma cortisol concentration rose after CRF in all patients with Cushing’s disease, and the peak concentrations seen at 30 or 60 min were nearly two-three times higher than in the controls. The increment in plasma cortisol (from the basal value) was also supernormal in two of the patients and within the normal range in one patient with Cushing’s disease.
| Pat. no. | Sex | Diagnosis | Plasma ACTH (ng/1) | Plasma cortisol (nmol/l) | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 0' | 15' | 30' | 60' | 1 maxb | 0' | 15' | 30' | 60' | 1 maxb b | |||||
| Ở | Normal controlsª | |||||||||||||
| Mean | 65 | 92 | 108 | 94 | 43 | 235 | 317 | 413 | 484 | 294 | ||||
| SEM | 5 | 7 | 11 | 7 | 44 | 29 | 13 | 8 | ||||||
| 1 | Ở | Cushing's disease | 142 | 454 | 380 | 491 | 349 | 256 | 617 | 670 | 801 | 545 | ||
| 2 | Ở | Cushing's disease | 118 | 231 | 366 | 182 | 248 | 1 024 | 153 | 224 | 409 | 385 | ||
| 3 | 9 | Cushing's disease | 59 | - | 92 | 80 | 21 | 609 | 730 | 875 | 643 | 266 | ||
| 4 | 9 | Bilateral nodular adrenocortical hyperplasia | 72 | 80 | 63 | 59 | 8 | 269 | 235 | 246 | 258 | - | ||
| 5 | ㅎ | Adrenocortical carcinoma | <50 | <50 | <50 | <50 | 0 | 1 223 | 298 | 1 277 | 1 409 | 178 | ||
| 6 | 우 | Adrenocortical carcinoma | <50 | 64 | 62 | 69 | 19 | 323 | 349 | 356 | 371 | 48 | ||
| 7 | ? | Ectopic | ||||||||||||
| ACTH syndrome | 154 | 189 | 166 | 159 | 35 | 791 | 793 | 781 | 734 | 2 | ||||
| 8 | 8 | Nelson's syndrome | 530 | >16 | 000 | >16 000 | >16 000 | 9 470 | 89 | 84 | 68 | 72 | - | |
| 9 | Ở ☒ | Nelson's syndrome | 760 | 1 | 530 | 1 950 | 256 | 770 | 63 | 55 | 67 | 67 | 4 | |
| 10 | 9 | Hypophysectomy | <50 | <50 | <50 | <50 | 0 | <30 | <30 | <30 | <30 | 0 | ||
| 11 | 9 | Hypophysectomy | <50 | <50 | <50 | 52 | 2 | <30 | <30 | <30 | <30 | 0 | ||
| 12 | Hypophysectomy | <50 | - | 60 | 94 | 44 | 55 | - | 152 | 208 | 153 | |||
“Plasma ACTH n=3, plasma cortisol n=5. ‘Peak increment from the basal value. The lower limit of detection of plasma ATCH=50 ng/1.
In the patient with multinodular adrenocortical hyperplasia, basal plasma ACTH was within the normal range, but neither plasma ACTH nor cortisol rose after CRF. In 2 patients with adrenocortical tumour, plasma ACTH was suppressed. In one of them (no. 6), successfully treated with mitotane, a slight increase in plasma ACTH and cortisol was seen after CRF, while the other did not respond. In the patient with ectopic ACTH syndrome, a small increase in the elevated basal plasma ACTH was not accompanied by any increase in plasma cortisol after CRF.
The very high plasma ACTH concentrations in 2 patients with Nelson’s syndrome more than doubled after CRF. There were no responses in plasma ACTH and cortisol in 2 patients subjected to total hypophysectomy, while the third patient responded in the low normal range in agreement with the presence of a positive response to Metopirone and to insulin hypoglycemia (Table I).
Side-effects
Immediately after injection of 100 and 300 µg CRF, two of the healthy controls experi- enced facial burning for 3-5 min accompanied by facial flush for 30 min in one. The same individuals felt concomitantly, after 300 µg CRF, transient shortness of breath for about 30 sec and breathed then more deeply and rapidly for 1/2-1 min. None of the patients tested complained of unpleasant sensations after CRF.
DISCUSSION
The time-related responses in plasma ACTH and cortisol in our controls are in accordance with the results reported by others (2, 3, 6, 7, 11). Our data also show that in normal individuals, within the dose range of CRF studied, the peak response is seen already on doses of 50 µg, and that increasing the dose to 300 ug only involves a longer duration of the response in plasma ACTH and cortisol and the risk of side-effects. Administration of 50-100 µg of CRF therefore appears to be a reasonable test dose in future work.
Until now, only a few dose-response studies have been reported. The study by Orth et al. (6) indicates a threshold dose of CRF of 0.01-0.03 ug/kg b.wt., a half maximal dose of 0.3-1.0 µg/kg and a maximally effective dose of 3-10 µg/kg. A biphasic response in plasma ACTH and cortisol has also been reported after CRF doses of ≥3 g/kg b.wt .: the first peak at 30-60 min probably reflecting secretion of readily accessible ACTH, while the second peak after 2-3 hours reflects secretion of newly synthesized ACTH (7). In another study (2) the response to 100 µg CRF was the same as to 200 µg, while Nakahara et al. (5) found both a higher peak response in plasma ACTH and cortisol and a more sustained response to 500 µg CRF than to 100 µg.
All of our patients were treated with 100 µg CRF. The unresponsiveness in patients with Cushing’s syndrome due to ectopic ACTH secretion as well as to adrenocortical tumours and multinodular adrenocortical hyperplasia is a consistent finding also in other studies (3, 4, 5, 12). This indicates that CRF is clinically useful in the confirmation of pituitary suppression by autonomously secreted cortisol. The modest response to CRF in one of our patients with adrenocortical carcinoma probably reflects the reduced tumour secretion of cortisol during treatment with mitotane, thus making the pituitary gland less suppressed, being able to react to CRF.
The response to CRF in patients with Cushing’s disease is more variable. We found hyperresponsiveness in two patients and a response in plasma ACTH and cortisol within the normal range in a third patient, an observation in agreement with findings in other studies (1, 2, 3, 5). The reason for the inconsistencies in response is open to speculation.
Variations in location of the lesion to the different areas of the anterior/middle pituitary lobe or hypothalamus may be of importance.
By means of the CRF test it seems, however, possible to separate cases of Cushing’s disease from those due to autonomously secreted cortisol, thus, hopefully, obviating the need for more time-consuming and often disagreeable stimulation and suppression tests for pituitary ACTH.
The excessive response to CRF in our patients with Nelson’s syndrome was as reported by others (5, 12, 13). This observation is of theoretical interest, suggesting that adenomas in Nelson’s syndrome are not autonomous, but possibly under the influence of hypotha- lamic CRF.
Judging from our observations in three hypophysectomized patients, CRF may also prove to be useful in the study of the pituitary secretory capacity of ACTH in patients with suspected hypopituitarism. The only patient who responded to CRF was the one who also responded in the low normal range to Metopiron and hypoglycemia. If these observations are confirmed, CRF might replace the more time-consuming and often unpleasant stimula- tion tests for pituitary ACTH secretion at least in some patients.
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Received April 9, 1984.
Correspondence: H. Jørgensen, M. D., Medical Department, Telemark Central Hospital, N-3900 Porsgrunn, Norway.