Clinically inapparent adrenal masses are discovered serendipitously during diagnostic testing or treatment for unrelated disorders and are commonly known as adrenal incidentaloma (AI) [
Previous reports suggested that patients with Cushing’s syndrome had severe atherosclerotic damage, as indicated by reduced caliber, increased stiffness of carotid artery wall, and increased prevalence of atherosclerotic plaques [
The aim of the present study was to investigate the atherosclerotic vascular damage in a consecutive series of patients with AI and to correlate it with midnight salivary cortisol, as an outpatient measure of cortisol excess.
We studied a consecutive, prospective series of 32 patients with incidentally detected adrenocortical adenoma [13 men, 19 women; median age 61.5 years, range: 37–74] referred to our center from January 1, 2013, to February 28, 2014. In all cases, AI were detected by an abdominal CT scan done for unrelated diseases. The diagnosis of cortical adenoma rested on the following CT criteria: size less than 6.0 cm, regular shape with well-defined margins, homogenous texture, and hypodense content. An attenuation value of 10, or less, Hounsfield units on unenhanced CT scan was considered suggestive of an adrenal adenoma. The diagnosis of adenoma was confirmed by a repeat CT scan after 3–6 months showing no significant increase in mass size, or change in mass density, in any patient. The patients with confirmed diagnosis of hyperaldosteronism or pheochromocytoma were excluded.
We performed a 1 : 1 case-control analysis with 32 control subjects matched for age, sex, BMI, smoking status, blood pressure, glucose and lipid profile, and occurrence of previous cardiovascular events. They were collected from the medical staff and their relatives. All of the control subjects had previous imaging exclusion of adrenal masses.
Either patients or control subjects were studied as outpatients and they underwent careful clinical and history examination; none of them displayed specific signs or symptoms of hypercortisolism, such as weakness associated with proximal muscle wasting, skin atrophy, ecchymoses, or purple striae. Moreover, none of them was receiving any drug known to affect the hypothalamic-pituitary-adrenal axis or had current or previous history of alcohol abuse or major mood disorders. Either patients or controls had an apparently normal sleep-wake cycle.
Any subject with BMI greater than 30 kg/m2 was categorized as obese [
The institutional review board approved the study, and all subjects provided written, informed consent.
The patients with incidentally discovered adrenal adenoma underwent a specific endocrine evaluation as outpatients. It included (a) measurement of the 24-hour excretion of urinary free cortisol (UFC), (b) measurement of plasma ACTH at 8:00 a.m., and (c) overnight low-dose dexamethasone suppression test (1 mg, orally, at 11:00 p.m. with measurement of serum cortisol at 8:00 a.m. the following morning).
Either patients or control subjects underwent midnight salivary cortisol (MSC) measurement as outpatients. They were instructed to collect saliva samples at midnight. These were obtained twice and were kept in a refrigerator for up to 1 week before being transferred to our reference laboratory. Saliva was collected using Salivettes (Sarstedt, Newton, NC), a cotton device that is placed in the mouth for 2 min. Before saliva collection, patients were instructed to rinse their mouth without brushing their teeth (to avoid risk of gingival bleeding). Midnight samples were collected after 4-5 h of fasting (last food intake at 1900 or 2000 h). A mean of the two collected samples was calculated. The MSC was determined in our reference laboratory using commercially available kits (RIA, Radim S.p.A., Rome, Italy). The assay sensitivity was 0.5 nmol/L. The intra- and interassay variations were 3 and 9%, respectively. Coefficients of variation in the high and in the low range were, respectively, of 7 and 4%. Levels of salivary cortisol below 8.3 nmol/L were considered in the normal range. Concentrations were defined in a large series of healthy subjects.
Serum and urinary cortisol were measured using commercially available RIAs (Sorin Biomedica, Saluggia, Italy; Radim S.p.A., Pomezia, Rome, Italy; Diagnostic Product Corporation, Los Angeles, CA, USA). Intra-assay and interassay coefficients of variation were 6% and 11.5%, respectively. Plasma ACTH was measured by commercially available immunoradiometric assays (CIS Biointernational, Gif-sur-Yvette, France). Intra-assay coefficients of variation ranged from 2.1 to 5.3% and interassay coefficients of variation ranged from 3.1 to 8.9%, respectively; sensitivity ranged from 0.44 to 1.1 pmol/L.
SCS was considered in agreement with the recommendations of the AME Position Statement [
Carotid arteries ultrasound imaging was performed in all subjects by echo-Doppler ultrasonography (US), carried out with a Vingmed Sound CMF 725 (Horten, Norway) using a 7.5 MHz annular phased array transducer. Right and left carotid arteries were scanned longitudinally, 2.5 cm proximal to the bifurcation. The pictures were stored on magnetic media and analyzed later. US imaging studies were performed by the same operator (B.A.), who was blinded to laboratory assessment. Each measurement was repeated three times, and the mean was determined. Wall thickness of both carotid arteries was investigated by measuring the intima media thickness. In all subjects, the presence, location, and size of plaques were also evaluated at the levels of common, internal, and external carotid arteries.
Rates and proportions were calculated for categorical data and means and standard deviations for continuous data. Normality of data was assessed by the Kolmogorov-Smirnov test. For continuous variables, differences were analyzed by means of the two-tailed Student’s
The characteristics of the patients with AI are reported in Table
Demographic, anthropometric, and hormonal characteristics of the patients with AI.
Patients | Age | Sex | BMI | Hypertension | IGT/diabetes | Mass size | MSC | UFC | ACTH | Cortisol after 1 mg DST | SCS | Left IMT | Right IMT |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
(years) | (M/F) | (kg/m2) | (Y/N) | (Y/N) | (cm) | (nmol/L) | (nmol/24 h) | (pmol/L) | (nmol/L) | (Y/N) | (cm) | (cm) | |
#1 | 58 | F | 23.1 | Y | N | 2.2 | 4.7 | 118.6 | 2.4 | 179.4 | Y | 0.07 | 0.07 |
#2 | 56 | F | 23.5 | N | N | 3.5 | 2.7 | 126.9 | 4.6 | 66.2 | N | 0.08 | 0.08 |
#3 | 57 | F | 24.0 | Y | N | 2.4 | 5.8 | 750.7 | 2.4 | 44.1 | Y | 0.05 | 0.05 |
#4 | 56 | M | 27.5 | N | N | 3.0 | 2.7 | 540.9 | 9.0 | 55.2 | Y | 0.06 | 0.08 |
#5 | 58 | F | 24.4 | Y | Y | 2.0 | 22.1 | 110.4 | 2.2 | 44.1 | N | 0.08 | 0.08 |
#6 | 44 | F | 37.7 | Y | Y | 2.0 | 8.5 | 162.8 | 18.9 | 27.6 | N | 0.05 | 0.06 |
#7 | 53 | F | 21.6 | N | N | 2.5 | 7.7 | 52.4 | 2.3 | 74.5 | N | 0.06 | 0.08 |
#8 | 37 | F | 27.6 | N | N | 1.9 | 8.5 | 836.2 | 5.7 | 113.1 | Y | 0.03 | 0.03 |
#9 | 56 | F | 34.0 | Y | Y | 2.4 | 8.5 | 237.3 | 1.7 | 77.2 | Y | 0.08 | 0.07 |
#10 | 53 | F | 20.7 | Y | N | 3.3 | 2.7 | 494.1 | 1.6 | 41.4 | N | 0.06 | 0.06 |
#11 | 43 | F | 20.7 | N | Y | 3.5 | 2.7 | 460.9 | 3.5 | 46.9 | N | 0.06 | 0.06 |
#12 | 60 | F | 36.0 | N | N | 1.5 | 2.7 | 242.8 | 4.6 | 63.4 | N | 0.07 | 0.08 |
#13 | 43 | M | 26.6 | N | N | 3.0 | 5.5 | 184.9 | 3.3 | 41.4 | N | 0.07 | 0.08 |
#14 | 58 | F | 36.9 | N | Y | 2.7 | 2.7 | 314.6 | 4.6 | 184.9 | Y | 0.05 | 0.05 |
#15 | 46 | F | 21.0 | N | N | 2.0 | 2.7 | 474.7 | 3.5 | 41.4 | N | 0.05 | 0.05 |
#16 | 71 | M | 24.5 | N | N | 2.0 | 2.7 | 411.2 | 3.4 | 66.2 | N | 0.08 | 0.08 |
#17 | 70 | F | 23.4 | Y | N | 4.0 | 7.4 | 322.9 | 1.5 | 55.2 | Y | 0.08 | 0.08 |
#18 | 63 | M | 28.0 | Y | N | 2.0 | 6.6 | 673.4 | 1.1 | 41.4 | N | 0.06 | 0.05 |
#19 | 67 | M | 27.7 | Y | N | 2.5 | 2.7 | 298.1 | 4.1 | 82.8 | N | 0.07 | 0.07 |
#20 | 72 | M | 29.6 | Y | Y | 3.0 | 2.7 | 146.2 | 4.6 | 77.2 | N | 0.11 | 0.12 |
#21 | 64 | F | 20.3 | N | Y | 3.0 | 3.8 | 140.7 | 4.8 | 71.7 | N | 0.06 | 0.07 |
#22 | 70 | F | 19.0 | Y | N | 3.0 | 9.9 | 146.2 | 1.1 | 146.8 | Y | 0.09 | 0.10 |
#23 | 62 | M | 29.4 | Y | Y | 2.0 | 2.7 | 342.2 | 1.1 | 44.1 | N | 0.09 | 0.08 |
#24 | 64 | F | 26.9 | Y | Y | 2.0 | 4.7 | 226.3 | 0.7 | 113.1 | Y | 0.08 | 0.07 |
#25 | 64 | M | 27.3 | Y | Y | 3.7 | 6.9 | 447.1 | 3.3 | 110.4 | Y | 0.06 | 0.07 |
#26 | 62 | M | 26.8 | Y | Y | 3.0 | 2.7 | 113.1 | 2.6 | 66.2 | N | 0.08 | 0.08 |
#27 | 62 | M | 27.2 | N | N | 3.5 | 2.7 | 419.5 | 3.3 | 55.2 | Y | 0.06 | 0.07 |
#28 | 74 | M | 29.7 | Y | Y | 6.0 | 2.7 | 171.1 | 3.3 | 124.2 | N | 0.08 | 0.08 |
#29 | 70 | F | 27.9 | N | Y | 2.5 | 13.2 | 794.8 | 2.8 | 38.6 | N | 0.05 | 0.05 |
#30 | 70 | M | 28.1 | N | Y | 2.7 | 11.0 | 126.9 | 4.6 | 27.6 | N | 0.05 | 0.05 |
#31 | 61 | F | 28.0 | Y | Y | 3.0 | 8.5 | 187.6 | 2.2 | 38.6 | N | 0.10 | 0.10 |
#32 | 68 | M | 31.8 | Y | Y | 2.5 | 8.0 | 187.6 | 1.1 | 129.7 | Y | 0.09 | 0.10 |
SCS = subclinical Cushing syndrome, AI = adrenal incidentaloma, IMT = intima media thickness, BMI = body mass index, IGT = impaired glucose tolerance, Y = yes, N = no, MSC = midnight salivary cortisol, UFC = urinary free cortisol, and ACTH = adrenocorticotropin hormone.
The whole group of patients with AI had higher IMT values than control subjects (left IMT: 0.069 ± 0.017 cm versus 0.060 ± 0.010 cm,
Comparison of IMT values between SCS and nonsecreting AI with the matched controls (a). Comparison of clinical parameters between patients with SCS and nonsecreting AI (b).
|
Mean | SD |
|
|
---|---|---|---|---|
Left IMT (cm) | ||||
SCS | 11 | 0.068 | 0.018 | 0.01 |
Controls | 11 | 0.060 | 0.007 | |
Right IMT (cm) | ||||
SCS | 11 | 0.071 | 0.019 | 0.04 |
Controls | 11 | 0.063 | 0.009 | |
Left IMT (cm) | ||||
Nonsecreting | 21 | 0.069 | 0.017 | 0.03 |
Controls | 21 | 0.059 | 0.011 | |
Right IMT (cm) | ||||
Nonsecreting | 21 | 0.071 | 0.018 | 0.01 |
Controls | 21 | 0.059 | 0.011 |
|
Mean | SD | % |
|
|
---|---|---|---|---|---|
Left IMT (cm) | |||||
SCS | 11 | 0.068 | 0.018 | NS | |
Nonsecreting | 21 | 0.069 | 0.017 | ||
Right IMT (cm) | |||||
SCS | 11 | 0.071 | 0.019 | NS | |
Nonsecreting | 21 | 0.071 | 0.018 | ||
Age (yrs) | |||||
SCS | 11 | 60.2 | 9.3 | NS | |
Nonsecreting | 21 | 59.4 | 9.7 | ||
BMI (kg/m2) | |||||
SCS | 11 | 27.7 | 5.1 | NS | |
Nonsecreting | 21 | 26.4 | 4.6 | ||
Mass size (cm) | |||||
SCS | 11 | 2.8 | 0.7 | NS | |
Nonsecreting | 21 | 2.7 | 0.9 | ||
Hypertension (%) | |||||
SCS | 11 | 63.6 | NS | ||
Nonsecreting | 21 | 52.4 | |||
IGT/diabetes (%) | |||||
SCS | 11 | 45.4 | NS | ||
Nonsecreting | 21 | 47.6 |
SCS = subclinical Cushing syndrome, AI = adrenal incidentaloma, IMT = intima media thickness, BMI = body mass index, IGT = impaired glucose tolerance,
The patients with AI had higher midnight salivary cortisol levels than control subjects (5.90 ± 4.17 nmol/L versus 1.90 ± 0.77 nmol/L,
Comparison of clinical parameters between patients with MSC > 8.3 nmol/L (Group A) and ≤8.3 nmol/L (Group B) (a). Comparison of IMT values between Group A and Group B with the matched controls (b).
|
Mean | SD | % |
|
|
---|---|---|---|---|---|
Left IMT (cm) | |||||
Group A | 8 | 0.068 | 0.024 | NS | |
Group B | 24 | 0.070 | 0.014 | ||
Right IMT (cm) | |||||
Group A | 8 | 0.069 | 0.024 | NS | |
Group B | 24 | 0.071 | 0.016 | ||
Age (yrs) | |||||
Group A | 8 | 58.2 | 12.4 | NS | |
Group B | 24 | 60.2 | 8.5 | ||
BMI (kg/m2) | |||||
Group A | 8 | 28.3 | 5.6 | NS | |
Group B | 24 | 26.4 | 4.4 | ||
Mass size (cm) | |||||
Group A | 8 | 2.5 | 0.4 | NS | |
Group B | 24 | 2.8 | 0.9 | ||
Hypertension (%) | |||||
Group A | 8 | 62.5 | NS | ||
Group B | 24 | 54.2 | |||
IGT/diabetes (%) | |||||
Group A | 8 | 75.0 | NS | ||
Group B | 24 | 71.4 |
|
Mean | SD |
|
|
---|---|---|---|---|
Left IMT (cm) | ||||
Group A | 8 | 0.066 | 0.024 | 0.01 |
Controls | 8 | 0.0588 | 0.009 | |
Right IMT (cm) | ||||
Group A | 8 | 0.067 | 0.024 | 0.03 |
Controls | 8 | 0.057 | 0.011 | |
Left IMT (cm) | ||||
Group B | 24 | 0.070 | 0.014 | 0.01 |
Controls | 24 | 0.060 | 0.010 | |
Right IMT (cm) | ||||
Group B | 24 | 0.073 | 0.016 | 0.006 |
Controls | 24 | 0.062 | 0.010 |
IMT = intima media thickness, BMI = body mass index, IGT = impaired glucose tolerance,
In univariate analysis we have found a correlation at the limit of the statistical significance between IMT and hypertension (
In a multivariate analysis performed in patients with AI that included hypertension, smoking status, age, and cortisol levels after 1 mg DST, age was the best predictor for both left IMT and right IMT (
Comparison between patients and controls stratified by age.
Left IMT (cm) | Right IMT (cm) | Midnight salivary cortisol (nmol/L) | |||||||
---|---|---|---|---|---|---|---|---|---|
Patients (#16) | Controls (#16) |
|
Patients (#16) | Controls (#16) |
|
Patients (#16) | Controls (#16) |
| |
Age ≥ 60 yrs | 0.075 ± 0.017 | 0.060 ± 0.012 | 0.005 | 0.077 ± 0.019 | 0.060 ± 0.012 | 0.006 | 5.85 ± 3.39 | 2.12 ± 0.66 | 0.001 |
Age < 60 yrs | 0.061 ± 0.014 | 0.060 ± 0.008 | NS | 0.065 ± 0.015 | 0.060 ± 0.009 | NS | 5.98 ± 4.99 | 1.68 ± 0.77 | 0.002 |
The present data demonstrate that patients with AI and of 60 years or more have higher carotid artery IMT values than control subjects matched for age, sex, BMI, smoking status, blood pressure, glycemic state, lipid profile, and occurrence of previous cardiovascular events. Moreover, the overall group of patients shows higher MSC levels in comparison with control subjects. However, MSC was not a predictor of increased IMT.
The link between cortisol and atherosclerosis is suggested by fragments of information indicating that prolonged corticosteroid therapy accelerates the development of atherosclerosis. In animal experiments, ACTH and cortisone were shown to produce vascular injury and to enhance experimentally induced atherosclerosis [
Several data from cross-sectional studies point to an association between clinically inapparent cortisol excess and some manifestations of the metabolic syndrome (arterial hypertension, hyperglycemia, and overweight) and predisposition to thrombosis and cardiovascular disease in patients with adrenal incidentaloma [
Carotid IMT is generally recognized as a marker of early atherosclerosis. In fact, ultrasound examination of the carotid arteries with measurement of the IMT and detection of plaques has repeatedly been shown to predict occurrence of both stroke and myocardial infarction. The relation between carotid artery IMT and cardiovascular events is continuous, but a threshold ≥0.9 mm can be considered as a conservative estimate of significant alteration [
In 2002 Tauchmanovà et al. [
MSC is already an established test to screen patients with suspected overt Cushing’s syndrome [
Our findings on IMT are in agreement with previous studies confirming increased IMT and more wall plaque in AI (secreting and nonsecreting) compared to controls. Moreover, we have demonstrated that the whole group of AI patients presents higher MSC levels than controls, and 25% of them had MSC levels higher than the upper limit of normality. However, we have not confirmed a correlation between IMT and hormonal and metabolic factors, whereas the only predictor was age. We acknowledge the limit of a reduced number of patients that may have hampered the demonstration of a correlation between IMT and hormonal variables. Moreover, the hormonal evaluations of patients with AI remain controversial, since we use parameters that are not representative of the possible persistent exposure to cortisol excess. Thus, also patients with AI and formally normal hormonal value should have metabolic alterations and an increased cardiovascular risk. On the other hand the strength has been to have designed our study to evaluate IMT in patients with AI carefully matched to control subjects for age and known cardiovascular risk factors. Nonetheless, our data support the hypothesis that patients with AI may present a worsened cardiovascular risk profile when compared to the general population, as a possible consequence of being exposed to a chronic cortisol excess, albeit of minimal intensity. Patients with AI showed higher MSC than controls, as an additional marker of an altered HPA axis. Additionally, our data point out the importance of assessing IMT in patients older than 60 years.
In conclusion, the present findings confirm that patients with AI show signs of accelerated atherosclerosis compared to matched controls. Patients older than 60 years seem more compromised. Levels of MSC are not a strong predictor of accelerated atherosclerosis, although further studies are needed to fully evaluate whether MSC may be exploited as a marker of subclinical cortisol excess that may potentially raise cardiovascular risk.
Author Massimo Terzolo has received research grants and speaker honorarium from Company HRA Pharma. He is also consultant for the Company Atterocor.