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Original Studies |
Division of Endocrinology, Department of Internal Medicine, University of Turin, 10126 Torino, Italy
Address correspondence and requests for reprints to: Ezio Ghigo, M.D., Divisione di Endocrinologia, Ospedale Molinette, C.so Dogliotti 14, 10126 Torino, Italy. E-mail: camanni{at}pianeta.net
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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areas under
response curve (AUCs) after all ACTH doses, apart from 0.01 µg,
were significantly higher (P < 0.02) than that after
placebo, showing a clear dose-response relationship (P
< 0.001). The doses of 0.03 and 1.0 µg ACTH were the minimal and
maximal effective doses, respectively. The cortisol response to 250.0
µg ACTH was not modified by pretreatment with 0.01, 0.03, and 0.06
µg ACTH doses, whereas it was progressively reduced by increasing the
dose of ACTH pretreatment (P < 0.001). The aldosterone
AUCs to all but 0.01 µg ACTH doses were significantly higher
(P < 0.02) than that after placebo, showing a clear
dose-response relationship (P < 0.001). The dose of
0.03 µg was the minimal effective stimulating dose, whereas 25.0 µg
showed the same aldosterone-releasing effect of 250.0 µg. The
aldosterone response to 250.0 µg ACTH, preceeded by placebo, was not
modified by pretreatment with 0.01 and 0.03 µg ACTH doses, whereas it
was reduced by increasing the dose of ACTH pretreatment
(P < 0.05–0.02). The DHEA AUCs to all ACTH doses
were significantly higher (P < 0.01) than that after
placebo, showing a clear dose-response relationship (P
< 0.001). The doses of 0.01 and 1.0 µg ACTH were the minimal and
maximal effective dose, respectively. The DHEA response to 250.0 µg
ACTH was not modified by pretreatment with 0.01, 0.03, 0.06, and 0.125
µg ACTH doses, whereas it was progressively reduced by pretreatment
with 0.5, 1.0, and 25.0 µg ACTH doses (P < 0.01). In
conclusion, these results show that an extremely low ACTH dose is
needed to stimulate adrenal steroids and, among them, DHEA seems the
most sensitive to corticotropin stimulation. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The classical 250.0 µg ACTH dose is largely supramaximal also for stimulation of aldosterone and dehydroepiandrosterone (DHEA) (14, 15, 16). Interestingly, the sensitivity of aldosterone to ACTH has been reported to be even greater than that of cortisol, which, in turn, seems to be similar to that of DHEA (14).
ACTH dose of 1.0 µg has been proposed to verify the function of the hypothalamo-pituitary-adrenal (HPA) axis in patients with suspected secondary adrenal insufficiency, although controversial results have been obtained (1, 2, 3, 4, 5, 12, 17, 18, 19). Thus, it may be hypothesized that lower ACTH doses could better demonstrate the existence of pituitary ACTH insufficiency.
Based on the foregoing, we aimed to study the doseresponse effect of eight ACTH doses on cortisol, aldosterone, and DHEA levels in humans of both sexes. Specifically, we aimed to define the minimal and maximal stimulatory ACTH dose on adrenal steroid secretion.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Vials containing 250.0 µg ACTH (ACTH 1–24, tetracosactin, Synacthen) were purchased from Novartis-Pharma (Huningue, France).
ACTH dose preparations were performed as: the standard ACTH dose was prepared by adding 250 µg to 2 mL 0.9% NaCl. The 25.0 µg ACTH dose was prepared by taking 0.2 mL of the standard ACTH solution, by adding 0.8 mL saline. For ACTH doses ranging from 1.0–0.125, 250 µg in 250 mL saline were diluted; a 1.0 µg ACTH dose was prepared by taking 1 mL; 0.5 and 0.125 µg were prepared by taking 0.5 and 0.125 mL (in a insulin syringe) and by adding 0.5 and 0.875 mL saline, to obtain 1 mL to be injected. For lower ACTH doses (ranging from 0.06–0.01 µg), 9 mL saline to 1 mL saline containing 1 µg ACTH were added; then, we took volumes of this mixture ranging from 0.6–0.1 and added saline to obtain 1 mL of volume. The resulting solutions were used immediately after preparation.
Subjects and methods
Twelve healthy young volunteers (6 males and 6 females; age, 22–34 yr; BMI 20–25 kg/m2; body surface 1.6–1.9 m2) were studied. All subjects gave their informed consent to participate in the study, which had been approved by the independent Ethical Committee of the University of Turin.
All subjects underwent the following nine testing sessions: 1) placebo (1 mL saline 0.9% as iv bolus at 0 min) + ACTH 250.0 µg (as iv bolus at 60 min); 2) 0.01 µg ACTH (at 0 min) + 250.0 µg ACTH (at 60 min); 3) 0.03 µg ACTH (at 0 min) + 250.0 µg ACTH (at 60 min); 4) 0.06 µg ACTH (at 0 min) + 250.0 µg ACTH (at 60 min); 5) 0.125 µg ACTH (at 0 min) + 250.0 µg ACTH (at 60 min); 6) 0.5 µg ACTH (at 0 min) + 250.0 µg ACTH (at 60 min); 7) 1.0 µg ACTH (at 0 min) + 250.0 µg ACTH (at 60 min); 8) 25.0 µg ACTH (at 0 min) + 250.0 µg ACTH (at 60 min); and 9) 250.0 µg ACTH (at 0 min).
The tests were performed in the morning starting at 0830–0900 h after an overnight fasting. Tests were done in random order and at least 3 days apart. Women were studied in their early follicular phase. None of the subjects was on sodium restriction or potassium loading. None was taking any medication from at least 1 month. In each session, blood samples for cortisol, aldosterone, and DHEA assay were collected at baseline (0 min) and then at +15, +30, +60, +90, and +120 min.
Serum cortisol (nmol/L) was measured in duplicate by RIA (CORT-CTK125; DIA Sorin, Diasorin Diagnostics, Saluggia, Italy). The sensitivity of the assay was 11.0 nmol/L. The inter- and intra-assay coefficients of variation ranged from 4.3–14.6% and from 4.2–8.96%, respectively. Serum aldosterone (pmol/L) was measured in duplicate by RIA (ALDO-MAIA; Biochem Diagnostics, Guidonia, Italy). The sensitivity of the assay was 16.2 pmol/L. The inter- and intra-assay coefficients of variation ranged from 11.96–14.06% and from 4.21–9.57%, respectively. Serum DHEA (nmol/L) was measured in duplicate by RIA (DSL-9000 ACTIVE DHEA; Diagnostic Systems Laboratories Inc., Webster, TX). The sensitivity of the assay was 0.068 nmol/L. The inter- and intra-assay coefficients of variation ranged from 5.6–10.6% and from 7.0–10.2%, respectively.
All samples from the same subject were analyzed together. The results
are expressed as mean ± SEM of either absolute 
change above baseline or areas under response curve (AUC)
calculated by trapezoidal integration. The statistical evaluation was
performed by nonparametric ANOVA (Friedman) and then with the Wilcoxon
test.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Cortisol response to various ACTH doses
Placebo did not elicit any significant increase in cortisol
levels. The dose of 0.01 µg ACTH significantly increased cortisol
levels at 15 min (P < 0.05), whereas those of 0.03 and
0.06 µg did it at 15 min and 30 min (P < 0.01) with
peak at 15 min. The higher doses of ACTH from 0.125–250.0 µg
increased cortisol levels at all time points (P <
0.01) with peak at 30 min for 0.125 and 0.5 µg doses and at 60 min
for higher ones (Fig. 1
).
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increase above baseline) in all subjects after
placebo and in 2 of 12 (16%) subjects after 0.01 µg ACTH. All the
other ACTH doses induced a positive increase in all subjects.
When the data were evaluated as AUC, the cortisol responses to all
but 0.01 µg ACTH dose were significantly higher (P <
0.02) than that after placebo, showing a clear dose-response
relationship (P < 0.001). The doses of 0.03 and 1.0
µg ACTH were the minimal and maximal effective doses, respectively
(Fig. 1
).
The cortisol responses to ACTH were not statistically different in both
sexes, although the response to the lowest tetracosactin dose in men
seemed higher than in women (Table 1).
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).
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Placebo did not elicit any significant increase in aldosterone
levels. The doses of 0.01, 0.03, and 0.06 µg ACTH significantly
(P < 0.05) increased aldosterone levels at 15 min. The
higher ACTH doses, from 0.125–1.0 µg, increased aldosterone levels
(P < 0.01) at 15 and 30 min with peak at 15 min,
whereas those of 25.0 and 250.0 µg increased hormonal levels at all
time points with peak at 30 min (Fig. 2).
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increase in all
subjects.
When the data were evaluated as AUC, the aldosterone responses to
all but 0.01 µg ACTH doses were significantly higher
(P < 0.02) than that after placebo, showing a clear
dose-response relationship (P < 0.001). The dose of
0.03 µg was the minimal effective stimulating dose, whereas 25.0 µg
showed the same aldosterone-releasing effect of 250.0 µg (Fig. 2).
The aldosterone responses to ACTH were not statistically different in
both sexes, although the response to the lowest tetracosactin doses in
men seemed higher than in women (Table 1).
The aldosterone response to 250.0 µg ACTH preceded by placebo was not
modified by pretreatment with 0.01 and 0.03 µg ACTH doses, whereas it
was reduced by increasing the dose of ACTH pretreatment
(P < 0.05 from 0.06–1.0 µg and P <
0.02 by 25.0 µg) (see Fig. 4).
DHEA response to various ACTH doses
Placebo did not elicit any significant increase in DHEA levels.
The doses of 0.01 as well as that of 0.03 µg ACTH significantly
(P < 0.01) increased DHEA levels at 15 min. The ACTH
doses ranging from 0.06–0.5 µg increased DHEA levels
(P < 0.01) at 15 and 30 min (peak at 15 min for 0.06-
and 0.125 µg doses and at 30 min for the 0.5 µg dose), whereas
those ranging from 1.0–250.0 µg increased hormonal levels at all
time points with peak at 60 min (Fig. 3).
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increase in
all the subjects studied.
When the data were evaluated as AUC, the DHEA responses to all ACTH
doses were significantly higher (P < 0.01) than that
after placebo, showing a clear dose-response relationship
(P < 0.001). The doses of 0.01 and 1.0 µg ACTH were
the minimal and maximal effective dose, respectively (Fig. 3). The DHEA increase after each ACTH
dose was similar in both sexes (Table 1).
The DHEA response to 250.0 µg ACTH preceded by placebo was not
modified by pretreatment with 0.01, 0.03, 0.06, and 0.125 µg ACTH
doses, whereas it was progressively reduced by pretreatment with 0.5,
1.0, and 25.0 µg ACTH doses (P < 0.01) (Fig. 4).
Side effects
No side effect was recorded after all ACTH administrations.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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That 250 µg ACTH dose is fairly supramaximal is widely accepted (1, 2, 3, 4, 5). There is evidence that the maximal cortisol response is coupled with plasma ACTH levels as low as 13–14 pmol/L; these circulating ACTH levels are fairly exceeded by injection of an ACTH dose as low as 0.5 µg (1). On the other hand, other authors found that the cortisol response to 0.6 µg is lower than that after 1.0 µg (10), which, in turn, is similar to that recorded after 250 µg ACTH (6, 7, 8, 9, 10, 11, 12). Our present results confirm these latter findings showing that 1.0 µg is the maximal effective ACTH dose on cortisol secretion.
There is increasing clinical evidence suggesting that submaximal more than maximal ACTH dose could more usefully evaluate the adrenal function in patients suspected for subclinical primary or secondary adrenal insufficiency (1, 2, 3, 4, 5). Our results show that circulating cortisol levels are significantly increased by extremely low ACTH doses, 0.03 µg being the lowest stimulatory dose when the glucocorticoid response is evaluated as AUC. In agreement with our present findings, a cortisol increase after 0.03 µg or even lower ACTH doses has been observed in some normal subjects by other authors (13, 20).
That extremely low ACTH doses are more likely to explore the sensitivity of the adrenal gland is also suggested by evidence that the timing of the cortisol peak response after ACTH administration was dependent on the tetracosactin dose. In fact, the lowest ACTH doses showed cortisol peaks between 15 and 30 min whereas the highest doses induced progressive cortisol increase peaking later on. This evidence agrees with physiological studies of ACTH and cortisol pulsatility (21), showing that cortisol increase generally follows ACTH pulses after 15-min latency.
Another interesting aspect of our findings is that the cortisol response to 250 µg ACTH is inversely associated with the dose of the previous tetracosactin challenge. These findings suggest two potential explanations. Theoretically, the adrenal cortisol reserve could have been exhausted by pretreatment with medium-high tetracosactin doses. Alternatively, a self-protective adrenal mechanism against overstimulation by ACTH could be hypothesized. In agreement with this latter hypothesis, there is already evidence that the ACTH response to the most potent stimuli of HPA axis (such as hypoglycemia or CRH and Arginine-vasopressin coadministration) is far greater than that of cortisol (21).
Present and previous findings (14, 22) clearly show also that the aldosterone response to ACTH is dose dependent. In the present study, like cortisol, aldosterone AUC significantly increased after an ACTH dose as low as 0.03 µg. On the other hand, the evidence that a 25.0 µg but not a 1.0 µg ACTH dose has the same releasing effect of 250.0 µg on aldosterone secretion does not support the hypothesis that aldosterone is more sensitive than cortisol to tetracosactin (14), although it confirms that the glomerulosa zone responds to extremely low ACTH variations. Interestingly, our findings also show that the aldosterone response to ACTH is almost invariably recorded 15 min after ACTH administration. This evidence indicates that the timing of the mineralocorticoid response is independent of the ACTH dose, at variance with the glucocorticoid response. In fact, aldosterone synthesis and secretion mainly depend on the renin-angiotensin system (23), and, in this context, it is noteworthy that pretreatment with Angiotensin-converting enzyme inhibitors abolishes the aldosterone response but not the cortisol response to ACTH in humans (24).
Like cortisol and aldosterone, circulating DHEA levels showed clear dose-dependent response to ACTH, according to previous studies (14, 16, 20). DHEA had been reported as sensitive as cortisol to the stimulatory effect of ACTH, the lowest dose tested being 0.5 µg (14). In contrast with these data, in our study the maximal DHEA response was recorded after administration of 1.0 µg tetracosactin dose but even a 0.01 µg ACTH dose was able to elicit significant increase of circulating DHEA levels when evaluated as AUC as well as mean peak. Thus, DHEA seems more sensitive than either cortisol or aldosterone to ACTH stimulation; the peculiar sensitivity of the reticular adrenal zone to extremely low ACTH doses agrees with clinical evidence indicating that DHEA is a very sensitive marker of corticotropin secretion (25).
It has been suggested that the activity of the HPA axis shows gender-related differences, probably reflecting estrogenic influence (26, 27). Moreover, sex-specific differences in cortisol production rates have been reported, with women having a cortisol production rate lower than men (27). We did not find any sex-related difference in the adrenal responsiveness to stimulation with ACTH. This evidence indicates that the influence of gender on the HPA axis activity reflects differences in the neural control of corticotroph function.
In conclusion, our study allowed us to define the maximal and the minimal effective ACTH dose on adrenal secretion. An extremely low ACTH dose is needed to stimulate adrenal steroids, and, among them, DHEA seems the most sensitive to corticotropin. The hypothesis that extremely low ACTH doses could be useful to verify the existence of primary or secondary subclinical adrenal insufficiency needs to be verified. Testing with sequential administration of the minimal and maximal effective ACTH dose could allow concomitant investigation of adrenal sensitivity to ACTH and steroid-releasable pool.
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Acknowledgments |
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Footnotes |
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Received November 17, 1999.
Revised March 2, 2000.
Revised May 15, 2000.
Accepted May 24, 2000.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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