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Original Studies |
Dipartimento di Scienze Cliniche e Biologiche, Medicina Interna I, A.S.O. San Luigi, Università di Torino (M.T., A.A., G.R., P.P., A.A.), 10043 Torino; and Dipartimento di Medicina Interna, Sezione di Endocrinologia, Università di Brescia (S.B., M.D., G.M., F.M., A.G.), 25125 Brescia, Italy
Address all correspondence and requests for reprints to: A. Giustina, M.D., Endocrine Section, c/o 2° Medicina, Spedali Civili, 25125 Brescia, Italy. E-mail: giustina{at}master.csi.unibs.it
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The current prevalence of unsuspected adrenal masses is approximately 3% in abdominal computed tomography (CT) scan series (1). These adrenal adenomas are detected in patients who do not present with any clear sign of overt hypercortisolism; therefore, they can be labeled as nonfunctioning from a clinical viewpoint. However, autonomous and unregulated cortisol secretion, not fully restrained by pituitary feedback, may be demonstrated in some cases by endocrine work-up. The terms preclinical Cushing’s syndrome (2, 3, 4, 5) or subclinical Cushing’s syndrome (6, 7) have been used sometimes interchangeably in this context. Subclinical Cushing’s syndrome is a heterogeneous condition characterized by various alterations of the hypothalamic-pituitary-adrenal (HPA) axis that may persist over time or proceed with remissions and recurrences (8). The diagnostic criteria of this subtle hypercortisolemic state and its possible (detrimental) effects on other endocrine or nonendocrine systems are presently unclear.
GH synthesis and secretion are mainly regulated by two hypothalamic peptides, GHRH with stimulatory action and somatostatin with inhibitory action (9). Among the several hormonal signals that feed back on GH neuroregulation, glucocorticoids play a significant role. In fact, spontaneous and stimulated GH secretion is blunted in patients with Cushing’s syndrome as well as in patients receiving pharmacological doses of glucocorticoids (10, 11, 12, 13). Several studies have shown that the inhibitory effects of glucocorticoids on GH secretion are due to the in vivo enhancement of hypothalamic somatostatin release (14, 15, 16, 17, 18). At present, no data on the characteristics of GH secretion in patients with adrenal incidentalomas are available.
The aims of the present study were 1) to evaluate the pituitary GH responsivity to GHRH in patients with incidentally discovered adrenal adenomas, and 2) to investigate the effect of pretreatment with arginine, a functional somatostatin antagonist, on the GH response to GHRH in the same patients.
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Subjects and Methods |
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Ten consecutive patients (seven women and three men, aged 48–63 ys) with adrenal incidentalomas who fulfilled the inclusion criteria among the patients referred to our centers from January 1997 to June 1998 were studied. Criteria required for patient enrollment were 1) incidental detection of the adrenal mass by diagnostic procedures performed for extraadrenal complaints, 2) imaging features typical for adrenal adenoma, 3) age less than 65 yr, 4) body mass index (BMI) less than 30 kg/m2, 5) normal fasting glucose levels, and 6) no medication. Patients with hypertension of possible endocrine origin (i.e. paroxysmal hypertension, hypertension associated with hypokalemia, or hypertension resistant to treatment) were excluded. In the hypertensive patients included in the study, abdominal imaging study was requested for some complaint that did not relate to the study of hypertension. However, biochemical screening designed to exclude the presence of pheochromocytoma (measurement of 24-h urinary excretion of catecholamines and vanillylmandelic acid) or aldosterone-producing adenoma (measurement of PRA and aldosterone in orthostatic posture) was always performed. The diagnosis of adrenal adenoma was made after assessment of imaging features on an unenhanced CT scan and pattern of uptake on adrenal scintigraphy. Briefly, CT characteristics considered typical for concluding that the mass was an adenoma were 4.0 cm or less and rounded in shape with smooth margins (19) and an attenuation value of 10 Hounsfield units or less (20); for adrenal scintigraphy a pattern of uptake concordant with the CT picture was requested (21). Scintigrams with [131I]6ß-iodomethyl-19-norcholest-5(10)-en-3ß-ol (NP59) were performed as previously described (8). All CT scans and scintigrams were reviewed by the same radiologists.
All subjects volunteered to participate in the study and gave their
informed consent. The design of the study was approved by the ethical
committee of our hospitals. Thirteen healthy adult nonobese volunteers
matched for age and BMI with the patients and not undergoing any
therapy served as controls (Table 1
).
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The study was performed according to a single blind cross-over design. Arginine and saline were administered in random order. After overnight fasting, an antecubital venous catheter was inserted percutaneously and kept patent by a slow saline infusion. After a 30-min stabilization period, the patients were subjected on 2 separate mornings to the following two tests in random order: 1) an infusion of arginine hydrochloride (Damor, Naples, Italy; 30 g, iv, in 100 mL saline) from -30 to 0 min together with human GHRH-(1–29)NH2 (Geref, Serono, Italy; 100 µg in 1 mL saline, injected as an iv bolus at 0 min), or 2) an infusion of saline (100 mL, iv) over 30 min followed by GHRH administration. Subjects rested in a recumbent position throughout the experiment. Blood samples for GH assay were taken at -45, -30 (time of arginine or saline infusion), 0 (time of GHRH injection), 15, 30, 45, 60, 90, and 120 min. The tests were performed in the Clinical Research Unit, with an interval of at least 3 days between the two studies.
The GH secretory responses to GHRH plus saline or GHRH plus arginine were expressed as peak values (micrograms per L) or as the area under the curve (AUC; micrograms/L·min) calculated by trapezoidal integration. The enhancement of the GH response to GHRH induced by arginine was expressed as the percent ratio between the GH peak after GHRH plus arginine treatment and the GH peak after GHRH plus saline treatment, respectively.
All patients also underwent the following endocrine evaluation aimed to study the HPA axis: 1) measurement of serum cortisol at 0800 h, 2) measurement of the 24-h excretion of urinary free cortisol (UFC), 3) measurement of plasma ACTH at 0800 h (mean of at least two samples on different days), 4) overnight low dose dexamethasone suppression test (1 mg, orally, at 2300 h with measurement of serum cortisol at 0800 h the following morning). Premenopausal women were studied in the early follicular phase of the menstrual cycle. The same endocrine work-up with the exception of dexamethasone suppression test was performed in control subjects. Adequate dexamethasone suppression was demonstrated when cortisol fell below 5 µg/dL the morning after dexamethasone administration (22).
Assays
Hormonal variables were measured by RIA or IRMA methods, using commercially available kits: serum GH, IRMA (Allegro hGH, Nichols Institute Diagnostics, San Juan Capistrano, CA); plasma IGF-I, RIA after acid-ethanol extraction (Nichols Institute Diagnostics), serum and urinary cortisol, RIA (Sorin Biomedica, Saluggia, Italy); and plasma ACTH, IRMA (Nichols Institute Diagnostics). Normal ranges of plasma ACTH and serum and urinary cortisol are as follows: ACTH, 6–61 pg/mL; cortisol, 12–26 µg/dL; and UFC, 15–150 µg/24 h. All of the hormone assays were performed in the same laboratory. Intraassay coefficients of variation were 2.3% for serum GH, 4.2% for plasma IGF-I, 5.4% for serum cortisol, 8.9% for urinary cortisol, and 3.4% for ACTH, respectively. Interassay coefficients of variation were 5.4%, 6.3%, 9.6%, 11.1%, and 7.8%, respectively. All samples for an individual subject were determined in a single assay in duplicate.
Statistical analysis
All analyses were performed using the Statistica software package (Microsoft Corp., Tulsa, OK). Before statistical analysis, normal distribution was tested with the Kolmogorov-Smirnov’s test. Statistical comparisons of GH peaks and GH AUCs after GHRH plus saline and GHRH plus arginine were made using the two-tailed Hotelling’s test for unpaired data. Single and multiple regression analyses were carried out to determine which variables may influence GH secretory response. Levels of statistical significance were set at P < 0.05. The results are expressed as the mean ± 1 SD.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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and 2. The kinetics of the GH responses to
GHRH and GHRH plus arginine are illustrated in Fig. 1. Baseline GH levels were not
significantly different between the two groups. Arginine did not
significantly increase baseline GH levels in either patients or
controls.
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). After arginine pretreatment, a significant increase in
the GH responses to GHRH compared to saline pretreatment was found in
both healthy subjects (GH peak, 33.7 ± 17.5 µg/L;
P = 0.0005; Fig. 1) and patients (GH peak, 33.5 ±
20.3 µg/L; P = 0.001; Fig. 1). The GH response to
GHRH plus arginine did not differ between patients and controls. The
ratio between GH peaks after GHRH plus arginine and GHRH plus saline,
expressed as the percent variation, was significantly greater in
patients than in controls (751 ± 531% vs. 81 ±
45%; P = 0.0001). Similar information were obtained
when comparing GH AUC after GHRH plus saline or GHRH plus arginine
between the two groups (Table 1 and Fig. 2).
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The patients showed higher UFC levels than controls (115.5 ± 61.3
vs. 60 ± 13.5 µg/24 h; P = 0.01) and
lower ACTH concentrations (17.3 ± 11.9 vs. 37.5
± 6.4 pg/mL; P = 0.0001). GH responses to GHRH were
not significantly different in the five patients with biochemical signs
of altered function of the HPA axis compared with patients with
apparently normal HPA axis (GH peak, 7.4 ± 7.0 vs.
4.0 ± 2.2 µg/L; P = NS; Fig. 3).
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). The models accounted for 32% and
31% (r2) of the total variation, respectively.
It was not possible to find out any significant model when GH peak and
AUC after GHRH plus arginine were considered.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Some incidental adrenal adenomas show a functional autonomy even if the entity of hypercortisolism is relatively mild, because the patients do not present with a Cushingoid habit (5, 7, 8, 27). However, assessment of the HPA axis in such patients may provide a wide spectrum of results, from nonfunction to autonomous secretion, so that it is difficult to identify the individuals with subclinical Cushing’s syndrome (5, 7). NP-59 uptake on the side of the mass with nonvisualization of the contralateral adrenal gland (concordant uptake) may occur despite overall normal endocrine tests, and it was assumed as the more precocious sign of functional autonomy, in analogy with hot, pretoxic, thyroid nodules (21). In the literature, the frequency of subclinical Cushing’s syndrome among patients with adrenal incidentalomas ranged from 5–12% as a result of different endocrine protocols and disparate diagnostic criteria (5, 7, 24, 27, 28, 29).
As GH secretion is impaired by the chronic effects of glucocorticoids, either in patients with endogenous hypercortisolism (Cushing’s syndrome) or in patients undergoing chronic glucocorticoid treatment (10, 12), the aim of the present study was to assess GH secretory responses in patients who may be exposed to a slight degree of cortisol excess. The present data demonstrate that patients with incidental adenomas have blunted GH responses to GHRH compared to healthy controls. Arginine pretreatment is able to normalize the GH response to GHRH in these patients.
These findings are consistent with several reports on the glucocorticoid-induced inhibition of GH secretion in animal models and in humans (15, 18, 30). The increases in serum GH concentrations in response to commonly used pharmacological stimuli are typically inhibited in patients with significant endogenous hypercortisolism (10, 31). Recent clinical experiments on the neuroendocrine mechanisms involved in GH axis suppression demonstrated an increased hypothalamic somatostatin tone in Cushing’s disease (14, 15, 16, 17, 18). Other relevant contributing factors in Cushing’s disease could include elevated blood glucose and free fatty acid concentrations and/or obesity, all of which are known to suppress GH secretion (9).
Arginine is an amino acid that is thought to increase GH secretion, acting as a functional somatostatin antagonist (9). It has been previously shown that arginine pretreatment is able to normalize the GH response to GHRH in adults receiving chronic immunosuppressive therapy with glucocorticoids (16). In our patients with adrenal incidentaloma, arginine was able to restore fully the GH response to GHRH. The degree of glucocorticoid excess to which steroid-treated patients are exposed is remarkably greater than that observed in subjects with incidental adrenal adenomas, and this difference may explain the greater efficacy of arginine. In fact, only half of the patients displayed some biochemical abnormalities of the HPA axis, pointing to functional adrenal autonomy. An interesting finding of our study is that the blunting of the GH response to GHRH was not restricted to these subjects, but GH responses were indeed very similar between the two groups of patients, with or without demonstrable hypercortisolism.
It can be hypothesized that in patients with adrenal incidentalomas the slight hypercortisolism that characterizes subclinical Cushing’s syndrome may be intermittent, with phases of active secretion and quiescence. We have indeed previously found that cortisol secretion may fluctuate over time in subclinical Cushing’s syndrome (8), as occurs in the full-blown syndrome (22, 32). However, this transient or intermittent silent hypercortisolism may cause sustained metabolic alterations, such as glucose intolerance (33, 34) or osteoporosis (35), even if development of clinical Cushing’s syndrome is quite rare (1, 2, 3, 4, 5, 6, 7, 8, 24, 27, 28, 29). The somatostati- nergic tone may represent a very sensitive marker of subclinical Cushing’s syndrome, being enhanced also in patients without any sign of actual hypercortisolism.
Thus, that arginine is able to reverse the blunted GH response to GHRH in patients with adrenal incidentalomas suggests that these patients may have increased somatostatin tone. Interestingly, our data support the concept that cortisol-mediated GH inhibition is not linked to a circulating threshold value of cortisol, but is probably linked to dysregulated (autonomous) cortisol secretion. In fact, the parameters used to evaluate the GH response to GHRH were significantly correlated to plasma ACTH concentrations, which reflect the autonomy of the adrenal adenoma (7), whereas no statistical correlation was found between ACTH and the GH response after GHRH plus arginine. This finding is in keeping with the hypothesis of increased somatostatinergic tone in patients with functional adrenal autonomy. It is pertinent to remember that all patients had unilateral uptake of tracer on the side of the mass with suppression of the contralateral normal adrenal gland.
In summary, the present data suggest that in patients with incidental adrenal adenomas the GH response to GHRH is blunted due to increased somatostatinergic tone, as it can be restored to normal by pretreatment with the functional somatostatin antagonist arginine. We hypothesize that the observed pattern of GH-stimulated secretion is a consequence of subclinical Cushing’s syndrome. The blunted GH release to GHRH may be a very sensitive and early sign of functional autonomy of the adrenal adenoma, but the role of the GHRH test in the endocrine evaluation of patients with suspected subclinical Cushing’s syndrome remains investigational. As this is the first evidence of such an endocrine feature in patients with incidental adrenal adenomas, the potential clinical utility of GH testing should be clarified in prospective studies assessing the biochemical and clinical outcome of such patients in whom suppressed GH response to GHRH has been observed.
Received May 28, 1999.
Revised September 23, 1999.
Accepted December 20, 1999.
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