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Functional and Nonfunctional Adrenocortical Tumors Demonstrate a High Responsiveness to Low-Dose Adrenocorticotropin

Division of Endocrinology (T.M., B.K., G.A.), Department of Internal Medicine, University of Ancona, 60100 Ancona, Italy; and Division of Endocrinology (F.M.), Department of Internal Medicine, University of Padua, 35100 Padua, Italy

Address all correspondence and requests for reprints to: Dr. Giorgio Arnaldi, Clinica di Endocrinologia, Azienda Ospedaliera Umberto I, Sede Torrette, 60100 Ancona, Italy. E-mail: arnaldi.giorgio{at}libero.it, g.arnaldi{at}ao-umbertoprimo.marche.it.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Aldosterone-producing adenomas (APAs) demonstrate exquisite sensitivity to endogenous ACTH. We previously showed an ACTH receptor overexpression in APAs compared with the other adrenal tumors.

To evaluate the meaning of such findings, we investigated the response of aldosterone, cortisol, and 17OH progesterone (17OHP) to 1 µg ACTH in 42 patients with adrenocortical tumors (23 NHAs, 9 APAs, and 10 CPAs) and 10 normal subjects (C). All 52 subjects were responsive to ACTH, and hormone peak levels were reached at 30 min. The aldosterone peak level was significantly higher in APAs [mean ± SEM: 84.3 ± 13.1 ng/dl (2335.1 ± 362.9 pmol/liter)] than in other tumors and control (C). Cortisol peak levels was higher in CPAs [37.1 ± 3.9 µg/dl (1023.9 ± 107.6 nmol/liter)] than in NHAs (P < 0.01), in C (P < 0.01) and in APAs (P = n.s.). 17OHP peak levels were significantly higher in patients with adrenocortical tumors toward C.

In summary: 1) low-dose ACTH induces an important stimulation in all tumors, suggesting preservation of high responsiveness to ACTH; 2) this is especially true for aldosterone in APA and could be of primary importance when performing diagnostic tests for hyperaldosteronism; and 3) 17OHP-hyperresponsiveness to low-dose ACTH is the most common alteration both in functional and nonfunctional tumors.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
THE ROLE OF ACTH in the physiological regulation of aldosterone secretion is not as important as the renin-angiotensin system or serum potassium concentrations.

Physiologically, administration of an iv bolus of ACTH is associated with a brisk rise in serum aldosterone levels. The prolonged administration of pharmacological amounts of ACTH has been shown to produce an initial increase with a subsequent decline in aldosterone secretion. When physiological concentrations of ACTH are maintained in plasma, the zona glomerulosa responds in a parallel manner to that of the zona fasciculate (1, 2, 3).

Al least one variety of hyperaldosteronism, glucocorticoid-suppressible aldosteronism, appears to be related to ACTH, and it can be promptly reversed by administration of small doses of dexamethasone. In aldosterone-producing adenomas (APAs), the tumor tends to demonstrate exquisite sensitivity to endogenous ACTH. This is reflected in several phenomena including parallel changes in plasma aldosterone levels with the circadian fluxes of plasma ACTH and the increase in aldosterone levels following an ACTH infusion (4, 5, 6, 7).

In summary, whereas ACTH plays a minimal role in the day-to-day regulation of aldosterone secretion, in some pathological states it may assume a significant role.

The presence of ACTH receptor mRNA in both functioning and nonfunctioning adrenocortical tumors has been demonstrated (8, 9). In our previous study, an RT-PCR quantitative evaluation of its distribution showed an overexpression in APAs, whereas cortisol-producing adenomas (CPAs) and nonhypersecreting adenomas (NHAs) showed an ACTH receptor expression similar to that of normal adrenal tissue (9).

To evaluate the functional meaning of such findings, with the present study we tried to systematically investigate the responsiveness to acute ACTH stimulation in different adrenocortical tumors with an in vivo study.

The ACTH test is widely used in clinical practice for the diagnosis of adrenal insufficiency, but the optimal dose that has to be administered is still a matter of debate. In the classical ACTH test, a dose of 250 µg ACTH 1–24 (tetracosactin) is administered, but it has been well known for many years that this is a supramaximal dose. In the present study, we have chosen a low dose of ACTH 1–24 (1 µg) because it is more physiological for the assessment of the hypothalamic-pituitary-adrenal (HPA) axis functionality (10, 11, 12).

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subjects (Table 1)

Forty-two patients (25 women and 17 men, aged 18–81 yr) with an adrenocortical tumor were studied over the last 5 yr and were evaluated before the start of any treatment. Twenty-three patients had an NHA, 9 an APA, and 10 a CPA. The clinical data of patients are reported in Table 1.

All patients with CPA had typical signs and symptoms of Cushing’s syndrome such as elevated urinary free cortisol excretion, absent cortisol rhythm, lack of suppression of cortisol after dexamethasone (1 mg) and suppressed basal levels of ACTH. The adrenal tumor size, evaluated with a CT or MRI scan, was 2.9 ± 0.5 cm (mean ± SEM) ranging from 2–3.8 cm.

We also evaluated 23 adrenal NHAs incidentally discovered by noninvasive abdominal imaging techniques performed for reasons other than suspected adrenal disease. The adrenal tumor size at CT or MRI was 3.0 ± 0.4 cm (mean ± SEM) ranging to 1.5–10 cm. Inclusion criteria were: 1) absence of specific signs and/or symptoms of hormone excess; 2) normal tests of HPA axis or no more than one abnormal test; 3) morphological aspect of the tumor suggesting the presence of a cortical adenoma (round shape with smooth edges, homogeneous with relatively low density; Ref.13). All subjects received an extensive endocrine evaluation (clinical and hormonal) before being included in the NHA, to exclude a functioning adrenal tumor. Baseline data included determination of urinary free cortisol, plasma ACTH, serum dehydroepiandrosterone sulfate, serum 17OH progesterone (17OHP), serum testosterone, upright plasma aldosterone/PRA, urinary catecholamines excretion, plasma cortisol rhythm. Dynamic tests included overnight 1 mg and 8 mg dexamethasone test and CRH test in some cases.

Ten healthy control (C) subjects (10 women, mean age 34 ± 4; range 18–70 yr) with no chronic diseases were also recruited over the same period of time.

Full informed consent was obtained from each subjects and the study was approved by our local Ethics Committee.

Study design

Vials containing 250 µg of ACTH 1–24 (tetracosactin, Synacthen) were purchased from Novartis-Pharma (Huningue, France). To prepare a dose of 1 µg, 250 µg of ACTH were diluted in 100 ml saline and 9 ml of saline were added to 1 ml of this solution; 4 ml of this final solution were injected iv. A butterfly needle was positioned in an antecubital vein and blood samples for cortisol, aldosterone, and 17OHP assay were collected at baseline (0 min) and then 30 and 60 min after the ACTH injection. Dilutions of ACTH were done by the same nurse, supervised by one of the doctors involved in the study, avoiding any loss of ACTH material. For ACTH collection, plastic tubes were used so that no protein was necessary for prevention of ACTH glass absorption.

Basal levels were also evaluated 30 min after positioning the needle (-30 min). The patient was in a recumbent position during the test. The test was performed in the morning, starting at 0830–0900 h after an overnight fast. No side effects were recorded after ACTH administration.

Cycling women were studied in the follicular phase. All patients were on normal sodium intake as monitored by urinary sodium excretion and were not receiving any drug therapy interfering with hormonal evaluation.

Hormone peak levels and percentage rise ( = [(peak levels/basal levels) x 100] - 100) were evaluated.

Hormone assays

Serum cortisol (µg/dl) was measured by chemiluminescence (Immulite, Diagnostic Products Corp., Los Angeles, CA). The intra-assay and interassay coefficients of variation were 9.0% and 10.3% respectively. The sensitivity of the assay was 0.2 µg/dl.

Serum aldosterone (picograms/milliliter) was measured by RIA (Aldosterone Maia, Biodata Diagnostics, Guidonia, Italy). The intra-assay and interassay coefficients of variation were 5.4% and 6.38%, respectively. The sensitivity of the assay was 6.0 pg/ml.

Serum 17OHP (ng/ml) was measured by RIA (Coat-A-Count, Diagnostic Products Corp.). The intra-assay and interassay coefficients of variation were 6.7% and 11%, respectively. The sensitivity of the assay was 0.07 ng/ml.

Statistical analysis

Data are expressed as mean ± SEM. Statistical comparison between groups were made by ANOVA. Post hoc analysis (Fisher’s protected least significance) and Bonferroni correction was applied for multiple means comparison.

Correlations were evaluated by Spearman analysis. Levels of statistical significance were set at P < 0.05. Statistical analysis were performed with the Statview 4.1 statistical package (SAS Institute Inc., Cary, NC).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
All 52 subjects were responsive to ACTH; and aldosterone, cortisol, and 17OHP peak levels were reached at 30 min.

Peak levels and percentage rises for each hormone were higher in 42 patients with adrenocortical tumors (ATs; all adrenal masses included) than in C subjects even if significance was reached only for 17OHP peak levels [mean ± SEM: 8.0 ± 0.8 ng/ml (26.4 ± 2.7 nmol/liter) in AT vs. 2.9 ± 0.3 ng/ml (9.6 ± 0.9 nmol/liter) in C, P = 0.007].

Aldosterone response to ACTH test

Both basal and peak aldosterone levels were significantly higher in APA than in other groups (Fig. 1). The aldosterone basal level (mean ± SEM) was 33.1 ± 2.4 ng/dl (916.9 ± 66.5 pmol/liter) in APA vs. 7.8 ± 1.6 ng/dl (216.1 ± 44.3 pmol/liter) in CPA, 9.5 ± 1.1 ng/dl (263.1 ± 30.5 pmol/liter) in NHA and 11.6 ± 1.7 ng/dl (321.3 ± 47.0 pmol/liter) in C (P < 0.0001). The aldosterone peak level (mean ± SEM) was 84.3 ± 13.1 ng/dl (2335.1 ± 362.9 pmol/liter) in APA vs. 23.6 ± 6.8 ng/dl (728.5 ± 188.4 pmol/liter) in CPA, 25.1 ± 3.1 ng/dl (695.3 ± 85.9 pmol/liter) in NHA, and 26.2 ± 5.3 ng/dl (725.8 ± 146.8 pmol/liter) in C (P < 0.001).

View larger version (16K): [in this window] [in a new window]   Figure 1. Mean (±SEM) plasma aldosterone response after 1 µg 1–24 ACTH in APAs, in C, in CPAs, and in NHAs. Conversion factor to SI units: 27.7.

Cortisol response to ACTH test

CPA had higher cortisol basal levels than other groups even if significance was not reached (Fig. 2) [mean ± SEM: 18.0 ± 3.8 µg/dl (496.8 ± 104.9 nmol/liter) in CPA, 12.3 ± 1.0 µg/dl (339.5 ± 27.6 nmol/liter) in APA, 10.6 ± 1.0 µg/dl (292.6 ± 27.6 nmol/liter) in NHA, 13.7 ± 2.4 µg/dl (378.1 ± 66.3 nmol/liter) in C].

View larger version (17K): [in this window] [in a new window]   Figure 2. Mean (±SEM) plasma cortisol response after 1 µg 1–24 ACTH in APAs, in C, in CPAs, and in NHAs. Conversion factor to SI units: 27.6.

A high degree of cortisol percentage response after ACTH administration was observed in all groups, ranging from 148–293%. Patients with NHA had a higher but not significant cortisol percentage rise (mean ± SEM: 293 ± 99%) than other groups (mean ± SEM: APA = 162 ± 22%, CPA = 148 ± 36%, C = 153 ± 42%).

17OHP response to ACTH test

17OHP basal levels were lower in healthy C subjects [0.9 ± 0.1 ng/ml (2.9 ± 0.3 nmol/liter)] vs. both patients with AT considered as a single group [1.7 ± 0.2 ng/ml (5.6 ± 0.6 nmol/liter)] and vs. each type of tumor considered separately, although significance was not reached [mean ± SEM: 2.2 ± 0.4 ng/ml (7.3 ± 1.3 nmol/liter) in APA, 2.1 ± 1.0 ng/ml (7.0 ± 3.3 nmol/liter) in CPA, 1.4 ± 0.2 ng/ml (4.6 ± 0.7 nmol/liter) in NHA, 0.9 ± 0.1 ng/ml (3.0 ± 0.3 nmol/liter) in C].

17OHP peak levels were significantly higher in patients with adrenocortical tumors respect to normal subjects [Fig. 3; mean ± SEM, C = 2.9 ± 0.4 ng/ml (9.6 ± 1.3 nmol/liter); APA, 7.2 ± 1.4 ng/ml (23.8 ± 4.6 nmol/liter), P = 0.05 vs. C; CPA, 8.3 ± 1.6 ng/ml (27.4 ± 5.3 nmol/liter), P = 0.01 vs. C; NHA, 8.5 ± 1.1 ng/ml (28.0 ± 3.6 nmol/liter), P < 0.002 vs. C].

View larger version (18K): [in this window] [in a new window]   Figure 3. Mean (±SEM) plasma 17OHP response after 1 µg 1–24 ACTH in APAs, in C, in CPAs, and in NHAs. Conversion factor to SI units: 3.3.

Interestingly, the percentage of adrenal tumors with 17OHP hyperresponsiveness to low-dose ACTH was very high: 24% (10/42) with a cut-off more than 10 ng/ml and 57% (24/42) with a cut-off more than 5 ng/ml.

Such hyperresponsiveness was present in both functional and nonfunctional adrenal tumors: APA 22% (2/9), CPA 33% (3/9); NHA 22% (5/23; cut-off more than 10 ng/ml); APA 55% (5/9), CPA 55% (5/9); NHA 61% (14/23; cut-off more than 5 ng/ml).

In normal subjects and in patients with adrenocortical tumors, we found a correlation between cortisol and 17OHP peak levels after ACTH (respectively r = 0.3, P < 0.0001 and r = 0.4 and P < 0.0001); this correlation persisted when the groups were considered separately (CPA: r = 0.8, P < 0.0001; APA: r = 0.6; P < 0.0001; NHA: r = 0.3, P < 0.0001).

Tumor size and hormone responses to ACTH

When we considered the size of the tumors and the hormonal changes after ACTH stimulation, a correlation was found only between cortisol or 17OHP peak levels and the size of CPA (r = 0.8, P < 0.0001 and r = 0.6, P = 0.02 respectively).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
It is widely accepted that the 250 µg ACTH dose, used for testing the HPA axis, exerts a supramaximal stimulation of adrenal function. In fact, there is evidence that a maximal cortisol response is coupled with plasma ACTH levels as low as 13–14 pmol/liter (11). Because the literature data have widely proved that 1 µg ACTH is the lowest dose that provokes a maximum cortisol secretion, similar to that recorded after 250 µg ACTH (10, 11, 12), we selected to use 1 µg ACTH dose to explore the responsiveness of normal and pathological adrenal glands.

It is well known that cortisol and aldosterone response to various doses of ACTH and in particular to 1 µg is independent of gender and/or age, whereas dehydroepiandrosterone sulfate and -androstenedione response is reduced with aging in both sexes (14, 15, 16, 17, 18, 19). A reduced response in elderly subjects was shown only for very low ACTH dose (0.06 µg; Ref.16).

Our results show that low-dose ACTH induces an important stimulation of cortisol, aldosterone and 17OHP in all patients with functional and nonfunctional adrenal tumors, suggesting that these tumors preserve a high sensitivity to ACTH, which is the most physiological stimulus.

Hormone response patterns (see Figs. 1–3) seem to be similar. In fact, cortisol, aldosterone, and 17OHP responses to ACTH were synchronous with a peak level reached at 30 min both in normal subjects and in patients with adrenocortical tumors.

The secretion of aldosterone in APA appears to be ACTH dependent. In fact, in patients with APA, aldosterone secretion is synchronous with cortisol circadian variations, regardless of posture, and these aldosterone changes are probably mediated by ACTH (4, 5, 6). In agreement with these observations, we have seen in APA the highest expression of ACTH receptor mRNA (9) and, in the present study, the highest aldosterone peak level and the greatest AUC. ACTH receptor hyperexpression and/or different aldosterone synthase sensitivity due to gene polymorphisms, could be possible mechanisms of aldosterone ACTH dependence (20, 21).

For these reasons, the simultaneous evaluation of cortisol and aldosterone could be of primary importance when performing diagnostic tests for hyperaldosteronism. This is especially important not only in long tests (for example, saline load) in which the circadiam rhythm could influence the results, but also in short tests (aldosterone/PRA ratio, orthostatism) in which acute stress could stimulate ACTH secretion.

In cultured human adrenocortical cells, ACTH receptors are positively regulated by ACTH (22) but on the contrary, CPA show normal (9) or even high (8, 23) expression of ACTH receptors in spite of chronic ACTH suppression. Furthermore, the sharp cortisol response to ACTH, observed in our patients with CPA, confirms the preservation of adrenal ACTH receptor activity in primary hypercortisolism where circulating ACTH levels are greatly reduced (mean value <10 pg/ml).

An exaggerated 17OHP response to ACTH test is probably the most common endocrine alteration observed in patients with adrenal incidentaloma (17–71% of patients) (24). Our results show that approximately 25% of patients with adrenal tumors present a 17OHP hyperresponsiveness (cut-off = 10 ng/ml) to low-dose ACTH. The interpretation of ACTH tests is sometimes controversial and an exaggerated response of 17OHP is not specific for 21-hydroxylase deficiency and could be merely a sign of altered intratumoral steroidogenesis. Several studies have failed to find the presence of CYP21 mutations in patients with adrenal tumors, suggesting that 21-hydroxylase deficiency is not implicated in the pathogenesis of most of such tumors (25, 26, 27). The disappearance of 17OHP hyperresponsiveness after removal of the tumor in a series of our patients (our unpublished data) may suggest that this phenomenon is not related to a genomic mutation of CYP21 but to an intrinsic feature of the tumor itself. Recently, these data have been refuted by Patócs et al. (28) and Baumgartner-Parzer et al. (29), whose studies do not rule out that CYP21 mutations may play a role in the pathogenesis of adrenal incidentalomas.

An interesting finding of our present study is the demonstration of an increased response of 17OHP to low-dose ACTH, not only in patients with nonfunctional tumors, but also in APA and CPA. This could be due to an increased cell mass effect, although it is possible that functional adrenal tumors also may harbor an intratumoral defect in the steroidal pathways leading to an increase not only of 17OHP but of the other steroid precursors, too. These results are in agreement with the data furnished by Tóth et al. (30).

In summary, our data show that: 1) low-dose ACTH induces an important stimulation of cortisol, aldosterone, and 17OHP in all patients with functional and nonfunctional adrenal tumors, suggesting that these tumors preserve a high responsiveness to ACTH; 2) this is especially true for the secretion of aldosterone in APA, and this finding could be of primary importance when performing diagnostic tests for hyperaldosteronism; and 3) 17OHP hyperresponsiveness to low dose of ACTH is the most common alteration both in functional and nonfunctional tumors; this is not a specific aspect of NHA as previously thought.

	Acknowledgments

 
We thank Prof. Robert Collu for critical review of the manuscript.

	Footnotes

 
This work was partially supported by grant from Ministero dell’ Istruzione, dell’ Università e della Ricerca (Grant 200106 2719) and Associazione Italiana Ricerca sul Cancro (to F.M.).

Abbreviations: 17OHP, 17OH progesterone; APA, aldosterone-producing adenoma; AT, adrenocortical tumor; C, control; CPA, cortisol-producing adenoma; CT, computed tomography; HPA, hypothalamic-pituitary-adrenal; MRI, magnetic resonance imaging; NHA, nonhypersecreting adenoma; PRA, plasma renin activity.

Received October 23, 2002.

Accepted January 27, 2003.

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