Serum Cortisol Levels via Radioimmunoassay vs Liquid Chromatography Mass Spectrophotometry in Healthy Control Subjects and Patients With Adrenal Incidentalomas

Serum Cortisol Levels via Radioimmunoassay vs Liquid Chromatography Mass Spectrophotometry in... Abstract Background Adrenal incidentalomas (AIs) are present in 4% of adults. As many as 30% may secrete cortisol autonomously in the absence of specific signs of overt hypercortisolism, in a phenomenon called subclinical hypercortisolism (SH). Diagnosis of SH is established by serum cortisol resistance to dexamethasone suppression. Methods We compared serum cortisol concentrations, as determined by radioimmunoassay (RIA) and liquid chromatography/tandem mass spectronomy (LC/MS-MS), in 73 patients with AI group (52 with unilateral AI) and 34 control subjects in 3 scenarios: basal; after 1-mg dexamethasone suppression; and after 0.25-mg stimulation with cosyntropin, a synthetic derivative of adrenocorticotropic hormone (ACTH). To bolster evidence for the diagnosis of SH, we also measured salivary cortisol levels at 11 PM and after DST, as well as plasma ACTH and serum dehydroepiandrosterone sulfate (DHEA-S) levels. Results We observed significant positive correlation (r = 0.9345, P <.001) for all 318 pairs of serum cortisol values, as measured by both methods. Conclusions Serum cortisol concentrations in patients with AI and in control subjects were very similar, as measured by RIA and LC/MS-MS. cortisol, adrenal incidentaloma, steroid measurements, LCMS, RIA, subclinical hypercortisolism Subclinical hypercortisolism (SH) is highly prevalent among adrenal incidentalomas (AI): as many as 30% of cases in some series.1-4 Identification of SH is critical because patients chronically exposed to even mild cortisol excess may develop or aggravate metabolic abnormalities, including but not limited to glucose intolerance/diabetes, dyslipidemia, hypertension, and osteopenia/osteoporosis, all of which are associated with increased cardiovascular risk.5,6 Reversal of metabolic abnormalities and their corresponding risks and consequences have been documented after adrenalectomy in patients with AI.7,8 Of note, however, is that characterization of SH is subjective and relies greatly on the response of serum cortisol to suppression by small doses of dexamethasone, usually performed by administration of 1 mg overnight (1-mg dexamethasone suppression testing [DST]). Additional tests can be used to increase specificity because of the presence of low and/or suppressed adrenocorticotropic hormone (ACTH; <10 pg/mL) and dehydroepiandrosterone sulfate (DHEA-S; <30 mcg/dL) levels, elevated late-night salivary cortisol levels, increased 24-hour urinary cortisol excretion, and serum cortisol response to administration of a high dose of dexamethasone or to corticotropin-releasing hormone (CRH), which may aid in the appropriate diagnosis of SH.9-11 Serum cortisol response to ACTH stimulation has seldom been used for this purpose, to our knowledge. Further, the cuttoff to determine serum cortisol resistance do 1-mg DST is not uniform in excluding SH. To increase sensitivity during the screening procedure,12 several groups reported using 1.8 mcg/dL as the upper limit for a normal cortisol response to dexamethasone, as is traditionally used for disclosing overt hypercortisolism Cushing syndrome (sensitivity and specificity, 91% and 88%, respectively).13 Colloquially, others favor 5.0 mcg per dL to increase specificity, thereby reducing the number of tests with false-positive results. It could be argued that an intermediate serum cortisol value of 2.5 or 3.0 mcg per dL may be a reasonable compromise between sensitivity and specificity. Nevertheless, cortisol suppression below 1.8 mcg per dL is a definitively normal response, excluding autonomous production; levels higher than 5 mcg per dL provide strong evidence for autonomous production. Other factors to consider when choosing a cutoff level include the method by which the serum or plasma cortisol is to be determined14,15 and whether the patient has impaired absorption or accelerated metabolic disposal of dexamethasone,16 which may result in an increase in false-positive results. In actuality, although commonly used for differential diagnosis of SH, the reproducibility of 1-mg DST to evaluate SH in AI has not been fully investigated, to our knowledge. In this study, we compared serum cortisol values concomitantly measured by radioimmunoassay (RIA) and liquid chromatography/tandem mass spectronomy (LC/MS-MS) after suppression with 1 mg of dexamethasone and after stimulation with 0.25 mg of cosyntropin, a synthetic derivative of adrenocorticotropic hormone (ACTH), in a series of patients with unilateral or bilateral AI and control subjects. Using the stringent 1.8 mcg per dL value and our previously defined cutoff level of 2.5 mcg per dL after 1-mg DST, we sought to discriminate the AI subpopulation with SH from those with nonfunctioning adrenal adenomas (NFAs). Materials and Methods The study protocol was previously approved by the Committee on Ethical Research in Humans of the Universidade Federal de São Paulo - UNIFESP; informed written consent was obtained from all patients and control subjects before study initiation. We evaluated 73 patients with AI: 52 with unilateral AI (34 women, 18 men, aged 22 to 87 years; median age, 59 years), 21 with bilateral AI (14 women, 7 men, aged 44 to 77 years; median age, 59 years), and 34 control subjects (23 women, 11 men, aged 36 to 74 years; median age, 54 years). All patients were referred to the Adrenal and Hypertension Unit of the Division of Endocrinology and Metabolism at Universidade Federal de São Paulo (UNIFESP; São Paulo, Brazil) for investigation of incidental adrenal abnormalities found via abdominal imaging procedures (computerized tomography [CT] or magnetic resonance imaging [MRI]) requested elsewhere for reasons other than adrenal pathology. Control subjects were volunteers: staff personnel, nurses, and laboratory technicians matched for sex and age. Subjects who were not receiving adequate treatment to control any incidental illness (eg, diabetes mellitus, hypertension) were excluded, as well as those receiving steroids (for at least 6 months previously), estradiol replacement, or any other medications that could potentially interfere with the hypothalamic-pituitary-adrenal (HPA) function or cortisol metabolism. Menstruating female patients and controls were studied during the early follicular phase of their menstrual cycle. Patients and control subjects were examined for clinical signs and symptoms and had blood specimens collected for routine metabolic assessment. After informed consent, all patients and controls underwent abdominal CT/MRI imaging using a specific thin-slice adrenal scanning protocol for improved characterization of the adrenal imaging and/or lesion(s). Images were subsequently examined by 2 independent expert radiologists. Patients with AI were also investigated for hormonal production, including 24-hour urinary free cortisol collection, serum aldosterone and plasma renin activity, and DHEA-S, ACTH, and serum cortisol dynamics. Overt Cushing syndrome, primary aldosteronism, pheochromocytoma, hyperandrogenism, and other clinically manifested endocrine syndromes were excluded in all patients studied. After we obtained baseline serum cortisol levels, all patients underwent ACTH stimulation testing and a week later, they underwent 1-mg DST. For the ACTH test, cosyntropin (Cortrosyn 0.25 mg vials, Amphastar Pharmaceuticals, Inc) was injected as an intravenous (IV) bolus in the morning, with fasting blood specimens drawn before administration and 60 minutes later. For the 1-mg DST, subjects took 1 mg oral dexamethasone (Decadron 0.5 mg tablets, Merck & Co, Inc.) at 11 PM of day 2 and hada fasting blood specimen collected on day 2 between 8:00 AM and 9:00 AM; specimens for salivary cortisol were obtained during the same time periods. Serum specimens were separated within 30 minutes from the time of draw and four 3-mL aliquots were kept frozen at −20°C until assayed. Baseline, p1-mg DST, and post-ACTH (pACTH) serum levels, as well as 11-PM and post-dexamethasone salivary cortisol levels (collected using a commercially available Salivette device [SARSTEDT AG & Co. KG]), were determined via a specific in-house RIA at the Steroids Laboratory (São Paulo, Brazil). Next, 2 frozen serum aliquots were sent to the Mayo Clinic Endocrine Laboratory (Rochester, MN) for liquid chromatography−tandem mass spectrometry (LC-MS/MS) analysis. We measured serum dexamethasone levels by RIA to validate the test (p1 mg DST) were also determined by an in-house RIA (Steroids Laboratory) during all 1-mg DST for test validation.16 Baseline plasma ACTH and serum DHEA-S were measured in all subjects on an in-house basis using commercially available kits (DPC Immulite 2000 immunoassay system; Siemens AG). Renal and liver function were normal in all patients and control individuals. Specimen Preparation The Steroids Laboratory (São Paulo) in-house cortisol RIA uses 100 μL of preextracted (10% methanol) serum in duplicate; antiserum was produced by immunizing rabbits with a cortisol-3-oxime derivative coupled with bovine serum albumin (BSA). Sensitivity (lower limit of quantification) is 0.4 μg per dL and specificity (cross-reactivity with other similar steroids) is less than 8%.17 Intra- and interassay coefficients of variation are both less than 10%. Levels of salivary cortisol were measured by RIA.18 Mayo Endocrine Research Laboratories (Rochester, MN) LC-MS/MS analysis uses an API-5000 triple quadruple mass spectrometer (AB Sciex Pte Ltd) operating in selected reaction monitoring (SRM), positive electrospray ionization (ESI) mode. Specimen introduction to the mass spectrometer was performed using a CTC-PAL autosampler (LEAP Technologies) and a TLX4 online specimen-preparation system (Thermo Fisher Scientific). We added stable isotope internal standard for cortisol to serum specimens before instrumental analysis. Serum specimens with internal standard added were subjected to solid-phase extraction on Strata-X cartridges (Phenomenex Inc). Eluates were dried down and reconstituted before introduction to the LC-MS/MS system. Statistical Analysis We performed statistical analysis using SAS software, version 9.4 (SAS Institute, Inc), and used linear mixed effects models to examine changes in adrenal steroid serum levels over time. Repeated measurements from the same person over time were accounted for by using an autoregressive correlation structure. Data was log-transformed for normalization across concentration ranges. For statistical purposes, undetectable values (below the sensitivity limit of the method) were considered equal to the lower limit of quantification divided by the square root of 2. We compared RIA and LC-MS/MS–determined serum cortisol levels using linear regression. Because individual baseline, pDST, and pACTH cortisol correlations were similar, we combined all values to obtain an integrated correlation equation. We compared mean (SD) (plus median and range [95% confidence interval (CI)]) baseline, pDST, and pACTH serum cortisol values for unilateral and bilateral AI in control subjects. We also compared the percentage change of serum cortisol after ACTH testing and after DST in all groups. Parametric and nonparametric tests were used where appropriate, with P values less than 0.05 considered statistically significant. Results Population demographics are shown in Table 1. A total of 318 pairs of values were used to perform linear regression between RIA and LC/MS-MS for basal, pDST, and pACTH serum cortisol values combined, which yielded linear regression of y = 1.335x + 0.67, r = 0.941 (P <.001) (Figure 1). RIA values are shown on the ordinate and LC/MS-MS values on the abcissa. Three pairs were mismatched and thus were excluded from calculation. Table 1. Baseline Characteristics of Patients and Control Individualsa Variable Patients Controls(n = 34) P Value (n = 34) Unilateral AI(n = 52) Bilateral AI(n = 21) Sex (female/male) 34/18 14/7 23/11 0.3 Age, y, median (range) 59 (22–87) 59 (44–77) 56 (36–74) 0.2 BMI (kg/m2), mean (SD) 30.2 (7.1) 29.4 (5.3) 31.5 (5.8) 0.3 Hypertension, % 70% 92% 52% <.05 Diabetes Mellitus, % 28% 48% 5% <.05 Dyslipidemia, % 56% 60% 5% <.05 Variable Patients Controls(n = 34) P Value (n = 34) Unilateral AI(n = 52) Bilateral AI(n = 21) Sex (female/male) 34/18 14/7 23/11 0.3 Age, y, median (range) 59 (22–87) 59 (44–77) 56 (36–74) 0.2 BMI (kg/m2), mean (SD) 30.2 (7.1) 29.4 (5.3) 31.5 (5.8) 0.3 Hypertension, % 70% 92% 52% <.05 Diabetes Mellitus, % 28% 48% 5% <.05 Dyslipidemia, % 56% 60% 5% <.05 AI, adrenal incidentaloma; BMI, body mass index. aP <.05 for unilateral or bilateral AI group vs controls. View Large Table 1. Baseline Characteristics of Patients and Control Individualsa Variable Patients Controls(n = 34) P Value (n = 34) Unilateral AI(n = 52) Bilateral AI(n = 21) Sex (female/male) 34/18 14/7 23/11 0.3 Age, y, median (range) 59 (22–87) 59 (44–77) 56 (36–74) 0.2 BMI (kg/m2), mean (SD) 30.2 (7.1) 29.4 (5.3) 31.5 (5.8) 0.3 Hypertension, % 70% 92% 52% <.05 Diabetes Mellitus, % 28% 48% 5% <.05 Dyslipidemia, % 56% 60% 5% <.05 Variable Patients Controls(n = 34) P Value (n = 34) Unilateral AI(n = 52) Bilateral AI(n = 21) Sex (female/male) 34/18 14/7 23/11 0.3 Age, y, median (range) 59 (22–87) 59 (44–77) 56 (36–74) 0.2 BMI (kg/m2), mean (SD) 30.2 (7.1) 29.4 (5.3) 31.5 (5.8) 0.3 Hypertension, % 70% 92% 52% <.05 Diabetes Mellitus, % 28% 48% 5% <.05 Dyslipidemia, % 56% 60% 5% <.05 AI, adrenal incidentaloma; BMI, body mass index. aP <.05 for unilateral or bilateral AI group vs controls. View Large Figure 1 View largeDownload slide Correlation of individual baseline, post–adrenocorticotropic hormone (pACTH) testing, and post–1-mg dexamethasone suppression testing (pDST) serum cortisol values, as determined by radioimmunoassay (RIA) and liquid chromatography−tandem mass spectronomy (LC/MS-MS) in control subjects (n = 34) and in patients with unilateral (n = 52) or bilateral (n = 21) adrenal incidentalomas (AIs). Values are shown on a bilogarithmic scale diagram. The lower and upper shadow areas represent the DST and ACTH-stimulation ranges, respectively. The coefficient of correlation is 0.941 (n = 318; P <.001). Unil. indicates unilateral; Bilat., bilateral. Figure 1 View largeDownload slide Correlation of individual baseline, post–adrenocorticotropic hormone (pACTH) testing, and post–1-mg dexamethasone suppression testing (pDST) serum cortisol values, as determined by radioimmunoassay (RIA) and liquid chromatography−tandem mass spectronomy (LC/MS-MS) in control subjects (n = 34) and in patients with unilateral (n = 52) or bilateral (n = 21) adrenal incidentalomas (AIs). Values are shown on a bilogarithmic scale diagram. The lower and upper shadow areas represent the DST and ACTH-stimulation ranges, respectively. The coefficient of correlation is 0.941 (n = 318; P <.001). Unil. indicates unilateral; Bilat., bilateral. Baseline, post–1-mg DST, and pACTH serum cortisol values (mean [SD] and median plus range [95% CI]), determined by LC/MS-MS and RIA for unilateral and bilateral AI and control subjects, are shown in Table 2. Also, percentage changes from baseline of serum cortisol after suppression with 1 mg of dexamethasone, ACTH stimulation, and the percentage of abnormal values (those that were consistent with SH) are also described. Table 2. Cortisol Measurements via RIA and LC/MS-MS, Serum Dexamethasone Testing, Salivary Cortisol Assay, and 11 PM and Post−1-mg DST Basal Serum DHEA-S and ACTH Testing Serum Cortisol Salivary Cortisol LC/MS-MS (mcg/dL) RIA (mcg/dL) RIA (ng/dL) Control Subjects (n = 34) Basal pACTH p1-mg DST Basal pACTH p1-mg DST 11 PM p1-mg DST Mean (SD) 7.5 (3.6) 23.6 (6.5) 1.0 (0.3) 8.3 (3.7) 23.9 (8.6) 1.2 (0.6) 198 (164) 32 (20) Median, range (95% CI) 7.3 (2.8–14.2) 22.8 (13.7–33.4) 0.8 (0.6–1.5) 8.3 (3.8–14.1) 23.9 (11.4–38.2) 1.1 (0.7–3.4) 134 (33–506) 27 (11–69) Change from basal, % median (range) 233 (91–551) −87 (−93 to −63) 176 (68–450) −85 (−94 to −70) Abnormal values, %a 0% 3.0% 24% 3.5% Dexamethasone (ng/dL), mean (SD) 417 (141) DHEA-S (mcg/dL), mean (SD) (range) 67.8; 5 (53.3) (15–173) Below 30 mcg/dL, % 36.3% ACTH (pg/mL), mean (SD), range 16.4 (8.9), (8.5–26) Below 10 pg/mL, % 25.0% Unilateral (n = 52) Mean (SD) 9.7 (3.2) 28 (6.6) 2.1 (1.9) 12.4 (5.7) 34.7 (11.4) 2.1 (1.2) 190.5 (163) 105 (174) Median (range, 95% CI) 9.8 (5.3–15.3) 27 (18–40.2) 1.5 (0.7–5.2) 11.2 (5.4–22.5) 34.1 (20.1–54.7) 1.9 (0.9–3.9) 150 (36–424) 53 (10–366) % change from basal, median (range) 191 (61–528) −83 (−91 to −41) 201 (64–1063) −85 (−94 to −60) % abnormal valuesa 23% 25% 15.3 40.0% Dexamethasone (ng/dL), mean (SD) 435 (199) DHEA-S (mcg/dL), mean (SD) (range) 62.5 (65.7) (15–173) Below 30 mcg/dL, % 36.7% ACTH (pg/mL), mean (SD), (range) 18.5 (13.8) (5.6–46.6) Below 10 pg/mL, % 37.2% Bilateral (n = 21) Mean (SD) 13.1 (4.5) 32.4 (6.7) 3.2 (2.5) 15.5 (5.6) 42.5 (16.2) 3.3 (2.8) 189.5 (98.6) 96 (84) Median, range (95% CI) 12.7 (3.4–22.4) 32 (23.9–40.4) 2.3 (1.2–9.6) 15.8 (7.8–24) 39.3 (18.7–64.4) 2.4 (1.2–6.0) 197.5 (34–333) 86 (9.2–316) Change from basal, %, median (range) 150% (30%–355%) −74.6% (−92% to −49%) 199.3% (45%–506%) −85.2% (−90.7% to −45%) Abnormal values, %a 43% 43% 43% 25% 61.5% Dexamethasone, (ng/dL), mean (SD) 418.6 (202) DHEA-S (mcg/dL), mean (SD) 47.4 (35.9) Below 30 mcg/dL, % 42.1% ACTH (pg/mL), mean (SD) (range) 10.9 (5.9) (3.5–20.3) Below 10 pg/mL, % 47.6% Serum Cortisol Salivary Cortisol LC/MS-MS (mcg/dL) RIA (mcg/dL) RIA (ng/dL) Control Subjects (n = 34) Basal pACTH p1-mg DST Basal pACTH p1-mg DST 11 PM p1-mg DST Mean (SD) 7.5 (3.6) 23.6 (6.5) 1.0 (0.3) 8.3 (3.7) 23.9 (8.6) 1.2 (0.6) 198 (164) 32 (20) Median, range (95% CI) 7.3 (2.8–14.2) 22.8 (13.7–33.4) 0.8 (0.6–1.5) 8.3 (3.8–14.1) 23.9 (11.4–38.2) 1.1 (0.7–3.4) 134 (33–506) 27 (11–69) Change from basal, % median (range) 233 (91–551) −87 (−93 to −63) 176 (68–450) −85 (−94 to −70) Abnormal values, %a 0% 3.0% 24% 3.5% Dexamethasone (ng/dL), mean (SD) 417 (141) DHEA-S (mcg/dL), mean (SD) (range) 67.8; 5 (53.3) (15–173) Below 30 mcg/dL, % 36.3% ACTH (pg/mL), mean (SD), range 16.4 (8.9), (8.5–26) Below 10 pg/mL, % 25.0% Unilateral (n = 52) Mean (SD) 9.7 (3.2) 28 (6.6) 2.1 (1.9) 12.4 (5.7) 34.7 (11.4) 2.1 (1.2) 190.5 (163) 105 (174) Median (range, 95% CI) 9.8 (5.3–15.3) 27 (18–40.2) 1.5 (0.7–5.2) 11.2 (5.4–22.5) 34.1 (20.1–54.7) 1.9 (0.9–3.9) 150 (36–424) 53 (10–366) % change from basal, median (range) 191 (61–528) −83 (−91 to −41) 201 (64–1063) −85 (−94 to −60) % abnormal valuesa 23% 25% 15.3 40.0% Dexamethasone (ng/dL), mean (SD) 435 (199) DHEA-S (mcg/dL), mean (SD) (range) 62.5 (65.7) (15–173) Below 30 mcg/dL, % 36.7% ACTH (pg/mL), mean (SD), (range) 18.5 (13.8) (5.6–46.6) Below 10 pg/mL, % 37.2% Bilateral (n = 21) Mean (SD) 13.1 (4.5) 32.4 (6.7) 3.2 (2.5) 15.5 (5.6) 42.5 (16.2) 3.3 (2.8) 189.5 (98.6) 96 (84) Median, range (95% CI) 12.7 (3.4–22.4) 32 (23.9–40.4) 2.3 (1.2–9.6) 15.8 (7.8–24) 39.3 (18.7–64.4) 2.4 (1.2–6.0) 197.5 (34–333) 86 (9.2–316) Change from basal, %, median (range) 150% (30%–355%) −74.6% (−92% to −49%) 199.3% (45%–506%) −85.2% (−90.7% to −45%) Abnormal values, %a 43% 43% 43% 25% 61.5% Dexamethasone, (ng/dL), mean (SD) 418.6 (202) DHEA-S (mcg/dL), mean (SD) 47.4 (35.9) Below 30 mcg/dL, % 42.1% ACTH (pg/mL), mean (SD) (range) 10.9 (5.9) (3.5–20.3) Below 10 pg/mL, % 47.6% LC/MS-MS, liquid chromatography−tandem mass spectronomy; RIA, radioimmunoassay; pACTH, pre−adrenocorticotropic hormone; p1-mg DST, post−1-mg dexamethasone suppression; CI, confidence interval; DHEA-S, dehydroepiandrosterone sulfate; ACTH, adrenocorticotropic hormone. aPercentage of abnormal values that are consistent with subclinical hypercortisolism (SH): after−1-mg-DST serum cortisol >2.5 mcg/dL; after-1-mg DST salivary cortisol >60 ng/dL; basal serum DHEA-S <30 mcg/dL, and basal plasma ACTH <10 pg/mL. View Large Table 2. Cortisol Measurements via RIA and LC/MS-MS, Serum Dexamethasone Testing, Salivary Cortisol Assay, and 11 PM and Post−1-mg DST Basal Serum DHEA-S and ACTH Testing Serum Cortisol Salivary Cortisol LC/MS-MS (mcg/dL) RIA (mcg/dL) RIA (ng/dL) Control Subjects (n = 34) Basal pACTH p1-mg DST Basal pACTH p1-mg DST 11 PM p1-mg DST Mean (SD) 7.5 (3.6) 23.6 (6.5) 1.0 (0.3) 8.3 (3.7) 23.9 (8.6) 1.2 (0.6) 198 (164) 32 (20) Median, range (95% CI) 7.3 (2.8–14.2) 22.8 (13.7–33.4) 0.8 (0.6–1.5) 8.3 (3.8–14.1) 23.9 (11.4–38.2) 1.1 (0.7–3.4) 134 (33–506) 27 (11–69) Change from basal, % median (range) 233 (91–551) −87 (−93 to −63) 176 (68–450) −85 (−94 to −70) Abnormal values, %a 0% 3.0% 24% 3.5% Dexamethasone (ng/dL), mean (SD) 417 (141) DHEA-S (mcg/dL), mean (SD) (range) 67.8; 5 (53.3) (15–173) Below 30 mcg/dL, % 36.3% ACTH (pg/mL), mean (SD), range 16.4 (8.9), (8.5–26) Below 10 pg/mL, % 25.0% Unilateral (n = 52) Mean (SD) 9.7 (3.2) 28 (6.6) 2.1 (1.9) 12.4 (5.7) 34.7 (11.4) 2.1 (1.2) 190.5 (163) 105 (174) Median (range, 95% CI) 9.8 (5.3–15.3) 27 (18–40.2) 1.5 (0.7–5.2) 11.2 (5.4–22.5) 34.1 (20.1–54.7) 1.9 (0.9–3.9) 150 (36–424) 53 (10–366) % change from basal, median (range) 191 (61–528) −83 (−91 to −41) 201 (64–1063) −85 (−94 to −60) % abnormal valuesa 23% 25% 15.3 40.0% Dexamethasone (ng/dL), mean (SD) 435 (199) DHEA-S (mcg/dL), mean (SD) (range) 62.5 (65.7) (15–173) Below 30 mcg/dL, % 36.7% ACTH (pg/mL), mean (SD), (range) 18.5 (13.8) (5.6–46.6) Below 10 pg/mL, % 37.2% Bilateral (n = 21) Mean (SD) 13.1 (4.5) 32.4 (6.7) 3.2 (2.5) 15.5 (5.6) 42.5 (16.2) 3.3 (2.8) 189.5 (98.6) 96 (84) Median, range (95% CI) 12.7 (3.4–22.4) 32 (23.9–40.4) 2.3 (1.2–9.6) 15.8 (7.8–24) 39.3 (18.7–64.4) 2.4 (1.2–6.0) 197.5 (34–333) 86 (9.2–316) Change from basal, %, median (range) 150% (30%–355%) −74.6% (−92% to −49%) 199.3% (45%–506%) −85.2% (−90.7% to −45%) Abnormal values, %a 43% 43% 43% 25% 61.5% Dexamethasone, (ng/dL), mean (SD) 418.6 (202) DHEA-S (mcg/dL), mean (SD) 47.4 (35.9) Below 30 mcg/dL, % 42.1% ACTH (pg/mL), mean (SD) (range) 10.9 (5.9) (3.5–20.3) Below 10 pg/mL, % 47.6% Serum Cortisol Salivary Cortisol LC/MS-MS (mcg/dL) RIA (mcg/dL) RIA (ng/dL) Control Subjects (n = 34) Basal pACTH p1-mg DST Basal pACTH p1-mg DST 11 PM p1-mg DST Mean (SD) 7.5 (3.6) 23.6 (6.5) 1.0 (0.3) 8.3 (3.7) 23.9 (8.6) 1.2 (0.6) 198 (164) 32 (20) Median, range (95% CI) 7.3 (2.8–14.2) 22.8 (13.7–33.4) 0.8 (0.6–1.5) 8.3 (3.8–14.1) 23.9 (11.4–38.2) 1.1 (0.7–3.4) 134 (33–506) 27 (11–69) Change from basal, % median (range) 233 (91–551) −87 (−93 to −63) 176 (68–450) −85 (−94 to −70) Abnormal values, %a 0% 3.0% 24% 3.5% Dexamethasone (ng/dL), mean (SD) 417 (141) DHEA-S (mcg/dL), mean (SD) (range) 67.8; 5 (53.3) (15–173) Below 30 mcg/dL, % 36.3% ACTH (pg/mL), mean (SD), range 16.4 (8.9), (8.5–26) Below 10 pg/mL, % 25.0% Unilateral (n = 52) Mean (SD) 9.7 (3.2) 28 (6.6) 2.1 (1.9) 12.4 (5.7) 34.7 (11.4) 2.1 (1.2) 190.5 (163) 105 (174) Median (range, 95% CI) 9.8 (5.3–15.3) 27 (18–40.2) 1.5 (0.7–5.2) 11.2 (5.4–22.5) 34.1 (20.1–54.7) 1.9 (0.9–3.9) 150 (36–424) 53 (10–366) % change from basal, median (range) 191 (61–528) −83 (−91 to −41) 201 (64–1063) −85 (−94 to −60) % abnormal valuesa 23% 25% 15.3 40.0% Dexamethasone (ng/dL), mean (SD) 435 (199) DHEA-S (mcg/dL), mean (SD) (range) 62.5 (65.7) (15–173) Below 30 mcg/dL, % 36.7% ACTH (pg/mL), mean (SD), (range) 18.5 (13.8) (5.6–46.6) Below 10 pg/mL, % 37.2% Bilateral (n = 21) Mean (SD) 13.1 (4.5) 32.4 (6.7) 3.2 (2.5) 15.5 (5.6) 42.5 (16.2) 3.3 (2.8) 189.5 (98.6) 96 (84) Median, range (95% CI) 12.7 (3.4–22.4) 32 (23.9–40.4) 2.3 (1.2–9.6) 15.8 (7.8–24) 39.3 (18.7–64.4) 2.4 (1.2–6.0) 197.5 (34–333) 86 (9.2–316) Change from basal, %, median (range) 150% (30%–355%) −74.6% (−92% to −49%) 199.3% (45%–506%) −85.2% (−90.7% to −45%) Abnormal values, %a 43% 43% 43% 25% 61.5% Dexamethasone, (ng/dL), mean (SD) 418.6 (202) DHEA-S (mcg/dL), mean (SD) 47.4 (35.9) Below 30 mcg/dL, % 42.1% ACTH (pg/mL), mean (SD) (range) 10.9 (5.9) (3.5–20.3) Below 10 pg/mL, % 47.6% LC/MS-MS, liquid chromatography−tandem mass spectronomy; RIA, radioimmunoassay; pACTH, pre−adrenocorticotropic hormone; p1-mg DST, post−1-mg dexamethasone suppression; CI, confidence interval; DHEA-S, dehydroepiandrosterone sulfate; ACTH, adrenocorticotropic hormone. aPercentage of abnormal values that are consistent with subclinical hypercortisolism (SH): after−1-mg-DST serum cortisol >2.5 mcg/dL; after-1-mg DST salivary cortisol >60 ng/dL; basal serum DHEA-S <30 mcg/dL, and basal plasma ACTH <10 pg/mL. View Large Figure 2 depicts mean (SE) values of basal, after-dexamethasone, and after-ACTH serum cortisol levels in control subjects and individuals with unilateral and bilateral AI, as measured by RIA and LC/MS-MS. Overall, the average cortisol values determined by LC/MS-MS were 15% lower than those obtained by RIA. However, 32% of all 318 pairs of values disclosed LC/MS-MS cortisol levels above the identity line. In other words, LC/MS-MS cortisol values were higher than concurrent RIA levels, with only 3 values (0.9%) identical in both methods. Figure 2 View largeDownload slide Histogram showing mean (SE) values of serum cortisol at baseline, post−adrenocorticotropic hormone (pACTH) testing, and post-dexamethasone suppression testing (aDST) from control subjects (n = 34) and patients with unilateral (n = 52) or bilateral (n = 21) adrenal incidentalomas (AIs), as determined by liquid chromatography−tandem mass spectronomy (LC/MS-MS) and radioimmunoassay (RIA). Note that LC/MS-MS values are, on average, 15% to 20% lower than their RIA counterparts. Baseline, p-DST, and p-ACTH values were significantly higher (P <.01) in patients with AIs vs controls and were significantly higher in bilateral vs unilateral AI. Figure 2 View largeDownload slide Histogram showing mean (SE) values of serum cortisol at baseline, post−adrenocorticotropic hormone (pACTH) testing, and post-dexamethasone suppression testing (aDST) from control subjects (n = 34) and patients with unilateral (n = 52) or bilateral (n = 21) adrenal incidentalomas (AIs), as determined by liquid chromatography−tandem mass spectronomy (LC/MS-MS) and radioimmunoassay (RIA). Note that LC/MS-MS values are, on average, 15% to 20% lower than their RIA counterparts. Baseline, p-DST, and p-ACTH values were significantly higher (P <.01) in patients with AIs vs controls and were significantly higher in bilateral vs unilateral AI. The average baseline and pACTH cortisol levels were significantly higher in patients with unilateral AI than in control subjects and even higher in patients with bilateral AI. Likewise, cortisol levels were significantly more resistant to dexamethasone suppression in unilateral AI than in controls and even more resistant in bilateral AI (Figure 2). Using a previously defined cutoff level of 2.5 mcg per dL for post–1-mg DST serum cortisol (valid test results, with serum dexamethasone levels ≥ 140 ng/dL16), we identified 12 of 52 cases of unilateral (23%) and 9 of 21 of bilateral AI (42.8%), for which concurring RIA and LC/MS-MS values were above that level and consistent with SH. Cortisol levels suppressed below 2.5 mcg per dL (both methods) in 40 of the 52 patients with unilateral AI (76.9%) and 12 of the 21 patients with bilateral AI (57.1%) suggested NFA. Specimens from the remaining 3 of 52 patients with unilateral AI and 2 of 21 patients with bilateral AI yielded discordant results in both methods and were disregarded as statistical outliers. If a lower and more sensitive post-dexamethasone serum cortisol cutoff level of 1.8 mcg per dL is adopted to exclude SH, 32 of the patients with 52 unilateral AI (61.5%) and 8 of the 21 patients with bilateral AI (38%) had concurring lower values, consistent with NFA. Conversely, when a more stringent (specific) cutoff level of 5.0 mcg per dL is used, only 5.7% of the cases of unilateral AI and 19% of the cases of bilateral AI would be designated as SH. As depicted in Table 2, reduced levels of plasma ACTH (<10 pg/mL) and serum DHEA-S (<30 mcg/dL) were present in 37.2% and 36.7% of the cases of unilateral AI and in 47.6% and 42.1% of the cases of bilateral AI but also in 25% and 36.3% of the controls, respectively. These numbers were considerably more consistent in the SH subpopulations with unilateral AI (70% and 50%, respectively) and with bilateral AI (77.8% and 66.7%, respectively). Similarly, post-dexamethasone salivary cortisol levels were elevated (>60 ng/dL) in 40% and 61.5% of the patients with unilateral and bilateral AI, respectively, but only in 3.5% of controls. Discussion The methodology for measuring steroids has changed over time, moving from the RIA of the 1960s to other immuno- and chemiluminescent assays across the subsequent decades, reaching the most accurate criterion-standard technology of today, namely, LC-MS/MS.19 Because several of these methods/assays are still currently in use, comparison of the results they deliver is an important issue for clinicians who do not have access to LC-MS/MS technology. Recently, the Endocrine Society recommended new instructions to authors on the reporting of steroid hormone assay measurement.20 However, questions regarding costs and availability remain a major problem in most places. We believe that it is interesting but not entirely unexpected that the correlation for most steroids does not reach 100%. Similar studies, measuring steroids or other hormones in dupicate (comparison of two methodologies) showed a better accuracy for LC-MS/S.21-24 Conversely, cortisol is produced in substantial amounts, and so measurement of its concentrations in biological fluids by different methods may not be a critical issue.25 Routine screening or tests performed for other reasons using advanced imaging techniques often show incidentalomas, most of which are NFAs; however, a significant percentage has a relatively autonomous cortisol pattern but without the typical manifestations of Cushing syndrome. For those incidentalomas, the term subclinical hypercortisolism has been applied.1 Identification of this particular subset of patients seems important because even mild cortisol excess may be responsible for the development or worsening of a range of metabolic abnormalities, such as glucose intolerance, diabetes mellitus, dyslipidemia, arterial hypertension, and osteopenia/osteoporosis, which are also prevalent among the general population.26,27 Recently, several prospective studies, including one by Chiodini et al,28 documented the improvement of metabolic manifestations on removal of adrenal adenomas. However, a high number of these cases present evidence of postoperatory adrenal insufficiency, indicating prolonged contralateral adrenocortical atrophy.7 A definitive diagnosis of SH remains challenging. Several isolated or combined tests have been used for this purpose (resistance of serum cortisol to low-dose dexamethasone suppression, elevated late-night serum levels, or salivary cortisol levels [elevated 24-hour urinary free cortisol excretion and other tests4]). Detection of reduced or suppressed levels of ACTH and/or DHEA-S are additional markers of chronic autonomous cortisol excess. However, the most-used test is 1-mg DST, in which it is determined whether serum and/or salivary cortisol levels remain above a certain limit the morning after a 1-mg oral dose of dexamethasone is administered at 11 PM. Nonsuppressible post-dexamethasone cortisol levels are suggestive of autonomous production or altered HPA axis, the latter of which acts directly on corticotrophic cells. As a result, the conceptual definition of subclinical hypercortisolism varies among groups regarding which tests and how many tests are necessary for diagnosis and which cutoff points are chosen to define excess or nonsuppressibility.12 Also, the methodology used to measure serum, plasma, and salivary and urinary cortisol also plays an important role in this diagnosis when the HPA axis is evaluated.29,30 The results of a comparison of different methodologies suggest that LC/MS-MS is the most reliable method.31-33 When the widely accepted cutoff value for serum cortisol level (<1.8 mcg/dL) after dexamethasone suppression is used to define SH, studies find that 15% to 30% of the patients with AI have SH.12 We found good correlation between serum cortisol values measured by RIA and LC/MS-MS in the normal basal state or after dexamethasone suppression. However, after ACTH stimulation, there was considerable discordance between the 2 methods. A reasonable explanation is that there is cortisol-binding globulin (CBG) saturation, which is more evident in RIA methods, due to a more significant cross-reactivity with other steroids or metabolites.34,35 These factors suggest that other steroids produced after ACTH stimulation bind to the antibody in the RIA test and render the test less specific. Using a cutoff level of 2.5 mcg per dL after dexamethasone suppression, we identify 40% and 55% of SH among the patients with unilateral and bilateral AI, respectively.36 The more-sensitive cutoff of 1.8 mcg per dL probably best defines SH among AIs when valid 1-mg DST (dexamethasone serum levels ≥140 ng/dL) is used for evaluation. Also, 81.8% and 88.9% of the unilateral and bilateral AIs also satisfy 2 of 3 additional criteria for autonomous cortisol production (suppressed basal plasma ACTH [<10 pg/mL], suppressed basal serum DHEA-S [<30 mcg/dL], and/or nonsuppressed post–1-mg DST salivary cortisol levels [>60 ng/dL]), further reinforcing the diagnosis of SH.1 Recently, serum free cortisol was evaluated in patients with primary and secondary adrenal insufficiency and patients with cirrhosis; it was found that serum free cortisol (SFF) had higher specificity than total cortisol that is 85% to 95% bound to CBG and albumin.37 Others performed adrenalectomies in patients with SH and observed a reduction of cortisol levels and improvement of the metabolic parameters soon after the procedure (as determined by lower fasting glucose, glycated hemoglobin, and low-density lipoprotein [LDL]−cholesterol levels and amelioration of blood pressure38). Also, the percentage of adrenal insufficiency after unilateral adrenalectomy for an AI with diagnosed SH increased as the number of tests used to define SH in these patients also increased.38,39 Urinary free cortisol excretion and salivary cortisol tests are not sensitive enough and do not show any limitation in detecting SH and mild Cushing syndrome.40 Therefore, in our study, we found higher salivary cortisol levels after dexamethasone in the patient group than in controls but did no found any difference at the midnight salivary cortisol among the groups. In conclusion, serum cortisol measurement by RIA and LC/MS-MS in patients with unilateral and bilateral AI, as well as in control subjects, results in similar concentrations for low values (basal or after suppression). However, after ACTH stimulation, we observed some discordance between the 2 methods. Baseline serum cortisol, as well as post-dexamethasone and post-ACTH values, are significantly higher than controls in patients with unilateral AI and higher yet in those with bilateral AI. In general, AIs appear to produce higher amounts of basal and stimulated cortisol simultaneously, showing less suppression after dexamethasone. In this sense, bilateral AI, perhaps due to a mass effect, shows more evidence of SH than unilateral AI. Concordant (between RIA and LC-MS/MS methods) serum cortisol values higher than 2.5 mcg per dL, in addition to the results from 2 other tests (from reduced serum DHEA-S, reduced plasma ACTH, and/or elevated pDST salivary cortisol), best establish SH diagnosis.The subpopulation with these factors comprises 22% of the patients with unilateral AI and 40% with bilateral AI. It is expected that surgical removal or radiofrequency ablation of adrenal lesions will be beneficial in restoring metabolic abnormalities and preventing further cardiovascular and osteometabolic risks. Abbreviations SH subclinical hypercortisolism AIs adrenal incidentalomas DST dexamethasone suppression testing ACTH adrenocorticotropic hormone DHEA-S dehydroepiandrosterone sulfate CRH corticotropin-releasing hormone RIA radioimmunoassay LC/MS-MS liquid chromatography/tandem mass spectronomy NFAs nonfunctioning adrenal adenomas UNIFESP Universidade Federal de São Paulo CT computerized tomography MRI magnetic resonance imaging HPA hypothalamic-pituitary-adrenal IV intravenous BSA bovine serum albumin SRM selected reaction monitoring ESI electrospray ionization HPA hypothalamic-pituitary-adrenal CBG cortisol-binding globulin SFF serum free cortisol LDL low-density lipoprotein NS no significant BMI body mass index CI confidence interval Unil. unilateral Bilat. bilateral. Acknowledgments We are thankful for the laboratory expertise of Lilian F. Hayashi, MSc, and Kelly C. Oliveira, MSc, for performing cortisol RIA in São Paulo, Brazil. References 1. Reincke M . Subclinical Cushing’s syndrome . Endocrinol Metab Clin North Am . 2000 ; 29 ( 1 ): 43 – 56 . 2. Rossi R , Tauchmanova L , Luciano A , et al. Subclinical Cushing’s syndrome in patients with adrenal incidentaloma: clinical and biochemical features . J Clin Endocrinol Metab . 2000 ; 85 ( 4 ): 1440 – 1448 . 3. Valli N , Catargi B , Ronci N , et al. Biochemical screening for subclinical cortisol-secreting adenomas amongst adrenal incidentalomas . Eur J Endocrinol . 2001 ; 144 ( 4 ): 401 – 408 . 4. Di Dalmazi G , Pasquali R , Beuschlein F , Reincke M . Subclinical hypercortisolism: a state, a syndrome, or a disease ? Eur J Endocrinol . 2015 ; 173 ( 4 ): M61 – M71 . 5. Di Dalmazi G , Vicennati V , Garelli S , et al. 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A radioimmunoassay method for measurement of serum cortisol [in Portuguese] . Rev Bras Patol Clin . 1979 ; 15 ( 3 ): 125 – 130 . 18. Vieira JGH , Noguti KO , Hidal JT , Russo EMK , Maciel RMB . Measurement of saliva cortisol as a method for the evaluation of serum free fraction . Arq Bras Endocrinol Metabol . 1984 ; 28 ( 1 ): 8 – 10 . 19. Shackleton C . Clinical steroid mass spectrometry: a 45-year history culminating in HPLC-MS/MS becoming an essential tool for patient diagnosis . J Steroid Biochem Mol Biol . 2010 ; 121 ( 3-5 ): 481 – 490 . 20. Wierman ME , Auchus RJ , Haisenleder DJ , et al. Editorial: The new instructions to authors for the reporting of steroid hormone measurements . J Clin Endocrinol Metab . 2014 ; 99 ( 12 ): 4375 . 21. Vieira JG , Nakamura OH , Ferrer CM , Tachibana TT , Endo MH , Carvalho VM . The importance of methodology in serum testosterone measurement: comparison between a direct immunoassay and a method based on high performance liquid chromatography and tandem mass spectrometry (HPLC/MS-MS) . Arq Bras Endocrinol Metabol . 2008 ; 52 ( 6 ): 1050 – 1055 . 22. Key TJ , Appleby PN , Reeves GK , et al. ; Endogenous Hormones and Breast Cancer Collaborative Group . Steroid hormone measurements from different types of assays in relation to body mass index and breast cancer risk in postmenopausal women: reanalysis of eighteen prospective studies . Steroids . 2015 ; 99 ( pt A ): 49 – 55 . 23. Madeira JL , Bussmann LZ , Lima-Valassi HP , Mendonça BB . Analysis of an iodide radioimmunoassay for 11-deoxicortisol measurement . Arq Bras Endocrinol Metabol . 2014 ; 58 ( 3 ): 232 – 236 . 24. Lee SM , Lee MN , Oh HJ , et al. 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Beneficial metabolic effects of prompt surgical treatment in patients with an adrenal incidentaloma causing biochemical hypercortisolism . J Clin Endocrinol Metab . 2010 ; 95 ( 6 ): 2736 – 2745 . 29. Guo T , Taylor RL , Singh RJ , Soldin SJ . Simultaneous determination of 12 steroids by isotope dilution liquid chromatography-photospray ionization tandem mass spectrometry . Clin Chim Acta . 2006 ; 372 ( 1-2 ): 76 – 82 . 30. Kushnir MM , Rockwood AL , Bergquist J . Liquid chromatography-tandem mass spectrometry applications in endocrinology . Mass Spectrom Rev . 2010 ; 29 ( 3 ): 480 – 502 . 31. Masserini B , Morelli V , Bergamaschi S , et al. The limited role of midnight salivary cortisol levels in the diagnosis of subclinical hypercortisolism in patients with adrenal incidentaloma . Eur J Endocrinol . 2009 ; 160 ( 1 ): 87 – 92 . 32. Eisenhofer G , Dekkers T , Peitzsch M , et al. 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Performance of the overnight 1mg dexamethasone suppression test coupled with serum dexamethasone measurement in identifying subclinical hypercortisolism among patients with an adrenal incidentaloma . Paper presented at: ENDO 2015: The Endocrine Society’s 97th Annual Meeting ; March 5 –8, 2015 ; San Diego, CA . Abstract no. SAT-393. 37. Rauschecker M , Abraham SB , Abel BS , et al. Cosyntropin-stimulated serum free cortisol in healthy, adrenally insufficient, and mildly cirrhotic populations . J Clin Endocrinol Metab . 2016 ; 101 ( 3 ): 1075 – 1081 . 38. Kim HK , Yoon JH , Jeong YA , Kang HC . The recovery of hypothalamic-pituitary-adrenal axis is rapid in subclinical cushing syndrome . Endocrinol Metab (Seoul) . 2016 ; 31 ( 4 ): 592 – 597 . 39. Raffaelli M , De Crea C , D’Amato G , Gallucci P , Lombardi CP , Bellantone R . Outcome of adrenalectomy for subclinical hypercortisolism and Cushing syndrome . Surgery . 2017 ; 161 ( 1 ): 264 – 271 . 40. Masserini B , Morelli V , Bergamaschi S , et al. The limited role of midnight salivary cortisol levels in the diagnosis of subclinical hypercortisolism in patients with adrenal incidentaloma . Eur J Endocrinol . 2009 ; 160 ( 1 ): 87 – 92 . © American Society for Clinical Pathology 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Laboratory Medicine Oxford University Press

Serum Cortisol Levels via Radioimmunoassay vs Liquid Chromatography Mass Spectrophotometry in Healthy Control Subjects and Patients With Adrenal Incidentalomas

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© American Society for Clinical Pathology 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
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0007-5027
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Abstract

Abstract Background Adrenal incidentalomas (AIs) are present in 4% of adults. As many as 30% may secrete cortisol autonomously in the absence of specific signs of overt hypercortisolism, in a phenomenon called subclinical hypercortisolism (SH). Diagnosis of SH is established by serum cortisol resistance to dexamethasone suppression. Methods We compared serum cortisol concentrations, as determined by radioimmunoassay (RIA) and liquid chromatography/tandem mass spectronomy (LC/MS-MS), in 73 patients with AI group (52 with unilateral AI) and 34 control subjects in 3 scenarios: basal; after 1-mg dexamethasone suppression; and after 0.25-mg stimulation with cosyntropin, a synthetic derivative of adrenocorticotropic hormone (ACTH). To bolster evidence for the diagnosis of SH, we also measured salivary cortisol levels at 11 PM and after DST, as well as plasma ACTH and serum dehydroepiandrosterone sulfate (DHEA-S) levels. Results We observed significant positive correlation (r = 0.9345, P <.001) for all 318 pairs of serum cortisol values, as measured by both methods. Conclusions Serum cortisol concentrations in patients with AI and in control subjects were very similar, as measured by RIA and LC/MS-MS. cortisol, adrenal incidentaloma, steroid measurements, LCMS, RIA, subclinical hypercortisolism Subclinical hypercortisolism (SH) is highly prevalent among adrenal incidentalomas (AI): as many as 30% of cases in some series.1-4 Identification of SH is critical because patients chronically exposed to even mild cortisol excess may develop or aggravate metabolic abnormalities, including but not limited to glucose intolerance/diabetes, dyslipidemia, hypertension, and osteopenia/osteoporosis, all of which are associated with increased cardiovascular risk.5,6 Reversal of metabolic abnormalities and their corresponding risks and consequences have been documented after adrenalectomy in patients with AI.7,8 Of note, however, is that characterization of SH is subjective and relies greatly on the response of serum cortisol to suppression by small doses of dexamethasone, usually performed by administration of 1 mg overnight (1-mg dexamethasone suppression testing [DST]). Additional tests can be used to increase specificity because of the presence of low and/or suppressed adrenocorticotropic hormone (ACTH; <10 pg/mL) and dehydroepiandrosterone sulfate (DHEA-S; <30 mcg/dL) levels, elevated late-night salivary cortisol levels, increased 24-hour urinary cortisol excretion, and serum cortisol response to administration of a high dose of dexamethasone or to corticotropin-releasing hormone (CRH), which may aid in the appropriate diagnosis of SH.9-11 Serum cortisol response to ACTH stimulation has seldom been used for this purpose, to our knowledge. Further, the cuttoff to determine serum cortisol resistance do 1-mg DST is not uniform in excluding SH. To increase sensitivity during the screening procedure,12 several groups reported using 1.8 mcg/dL as the upper limit for a normal cortisol response to dexamethasone, as is traditionally used for disclosing overt hypercortisolism Cushing syndrome (sensitivity and specificity, 91% and 88%, respectively).13 Colloquially, others favor 5.0 mcg per dL to increase specificity, thereby reducing the number of tests with false-positive results. It could be argued that an intermediate serum cortisol value of 2.5 or 3.0 mcg per dL may be a reasonable compromise between sensitivity and specificity. Nevertheless, cortisol suppression below 1.8 mcg per dL is a definitively normal response, excluding autonomous production; levels higher than 5 mcg per dL provide strong evidence for autonomous production. Other factors to consider when choosing a cutoff level include the method by which the serum or plasma cortisol is to be determined14,15 and whether the patient has impaired absorption or accelerated metabolic disposal of dexamethasone,16 which may result in an increase in false-positive results. In actuality, although commonly used for differential diagnosis of SH, the reproducibility of 1-mg DST to evaluate SH in AI has not been fully investigated, to our knowledge. In this study, we compared serum cortisol values concomitantly measured by radioimmunoassay (RIA) and liquid chromatography/tandem mass spectronomy (LC/MS-MS) after suppression with 1 mg of dexamethasone and after stimulation with 0.25 mg of cosyntropin, a synthetic derivative of adrenocorticotropic hormone (ACTH), in a series of patients with unilateral or bilateral AI and control subjects. Using the stringent 1.8 mcg per dL value and our previously defined cutoff level of 2.5 mcg per dL after 1-mg DST, we sought to discriminate the AI subpopulation with SH from those with nonfunctioning adrenal adenomas (NFAs). Materials and Methods The study protocol was previously approved by the Committee on Ethical Research in Humans of the Universidade Federal de São Paulo - UNIFESP; informed written consent was obtained from all patients and control subjects before study initiation. We evaluated 73 patients with AI: 52 with unilateral AI (34 women, 18 men, aged 22 to 87 years; median age, 59 years), 21 with bilateral AI (14 women, 7 men, aged 44 to 77 years; median age, 59 years), and 34 control subjects (23 women, 11 men, aged 36 to 74 years; median age, 54 years). All patients were referred to the Adrenal and Hypertension Unit of the Division of Endocrinology and Metabolism at Universidade Federal de São Paulo (UNIFESP; São Paulo, Brazil) for investigation of incidental adrenal abnormalities found via abdominal imaging procedures (computerized tomography [CT] or magnetic resonance imaging [MRI]) requested elsewhere for reasons other than adrenal pathology. Control subjects were volunteers: staff personnel, nurses, and laboratory technicians matched for sex and age. Subjects who were not receiving adequate treatment to control any incidental illness (eg, diabetes mellitus, hypertension) were excluded, as well as those receiving steroids (for at least 6 months previously), estradiol replacement, or any other medications that could potentially interfere with the hypothalamic-pituitary-adrenal (HPA) function or cortisol metabolism. Menstruating female patients and controls were studied during the early follicular phase of their menstrual cycle. Patients and control subjects were examined for clinical signs and symptoms and had blood specimens collected for routine metabolic assessment. After informed consent, all patients and controls underwent abdominal CT/MRI imaging using a specific thin-slice adrenal scanning protocol for improved characterization of the adrenal imaging and/or lesion(s). Images were subsequently examined by 2 independent expert radiologists. Patients with AI were also investigated for hormonal production, including 24-hour urinary free cortisol collection, serum aldosterone and plasma renin activity, and DHEA-S, ACTH, and serum cortisol dynamics. Overt Cushing syndrome, primary aldosteronism, pheochromocytoma, hyperandrogenism, and other clinically manifested endocrine syndromes were excluded in all patients studied. After we obtained baseline serum cortisol levels, all patients underwent ACTH stimulation testing and a week later, they underwent 1-mg DST. For the ACTH test, cosyntropin (Cortrosyn 0.25 mg vials, Amphastar Pharmaceuticals, Inc) was injected as an intravenous (IV) bolus in the morning, with fasting blood specimens drawn before administration and 60 minutes later. For the 1-mg DST, subjects took 1 mg oral dexamethasone (Decadron 0.5 mg tablets, Merck & Co, Inc.) at 11 PM of day 2 and hada fasting blood specimen collected on day 2 between 8:00 AM and 9:00 AM; specimens for salivary cortisol were obtained during the same time periods. Serum specimens were separated within 30 minutes from the time of draw and four 3-mL aliquots were kept frozen at −20°C until assayed. Baseline, p1-mg DST, and post-ACTH (pACTH) serum levels, as well as 11-PM and post-dexamethasone salivary cortisol levels (collected using a commercially available Salivette device [SARSTEDT AG & Co. KG]), were determined via a specific in-house RIA at the Steroids Laboratory (São Paulo, Brazil). Next, 2 frozen serum aliquots were sent to the Mayo Clinic Endocrine Laboratory (Rochester, MN) for liquid chromatography−tandem mass spectrometry (LC-MS/MS) analysis. We measured serum dexamethasone levels by RIA to validate the test (p1 mg DST) were also determined by an in-house RIA (Steroids Laboratory) during all 1-mg DST for test validation.16 Baseline plasma ACTH and serum DHEA-S were measured in all subjects on an in-house basis using commercially available kits (DPC Immulite 2000 immunoassay system; Siemens AG). Renal and liver function were normal in all patients and control individuals. Specimen Preparation The Steroids Laboratory (São Paulo) in-house cortisol RIA uses 100 μL of preextracted (10% methanol) serum in duplicate; antiserum was produced by immunizing rabbits with a cortisol-3-oxime derivative coupled with bovine serum albumin (BSA). Sensitivity (lower limit of quantification) is 0.4 μg per dL and specificity (cross-reactivity with other similar steroids) is less than 8%.17 Intra- and interassay coefficients of variation are both less than 10%. Levels of salivary cortisol were measured by RIA.18 Mayo Endocrine Research Laboratories (Rochester, MN) LC-MS/MS analysis uses an API-5000 triple quadruple mass spectrometer (AB Sciex Pte Ltd) operating in selected reaction monitoring (SRM), positive electrospray ionization (ESI) mode. Specimen introduction to the mass spectrometer was performed using a CTC-PAL autosampler (LEAP Technologies) and a TLX4 online specimen-preparation system (Thermo Fisher Scientific). We added stable isotope internal standard for cortisol to serum specimens before instrumental analysis. Serum specimens with internal standard added were subjected to solid-phase extraction on Strata-X cartridges (Phenomenex Inc). Eluates were dried down and reconstituted before introduction to the LC-MS/MS system. Statistical Analysis We performed statistical analysis using SAS software, version 9.4 (SAS Institute, Inc), and used linear mixed effects models to examine changes in adrenal steroid serum levels over time. Repeated measurements from the same person over time were accounted for by using an autoregressive correlation structure. Data was log-transformed for normalization across concentration ranges. For statistical purposes, undetectable values (below the sensitivity limit of the method) were considered equal to the lower limit of quantification divided by the square root of 2. We compared RIA and LC-MS/MS–determined serum cortisol levels using linear regression. Because individual baseline, pDST, and pACTH cortisol correlations were similar, we combined all values to obtain an integrated correlation equation. We compared mean (SD) (plus median and range [95% confidence interval (CI)]) baseline, pDST, and pACTH serum cortisol values for unilateral and bilateral AI in control subjects. We also compared the percentage change of serum cortisol after ACTH testing and after DST in all groups. Parametric and nonparametric tests were used where appropriate, with P values less than 0.05 considered statistically significant. Results Population demographics are shown in Table 1. A total of 318 pairs of values were used to perform linear regression between RIA and LC/MS-MS for basal, pDST, and pACTH serum cortisol values combined, which yielded linear regression of y = 1.335x + 0.67, r = 0.941 (P <.001) (Figure 1). RIA values are shown on the ordinate and LC/MS-MS values on the abcissa. Three pairs were mismatched and thus were excluded from calculation. Table 1. Baseline Characteristics of Patients and Control Individualsa Variable Patients Controls(n = 34) P Value (n = 34) Unilateral AI(n = 52) Bilateral AI(n = 21) Sex (female/male) 34/18 14/7 23/11 0.3 Age, y, median (range) 59 (22–87) 59 (44–77) 56 (36–74) 0.2 BMI (kg/m2), mean (SD) 30.2 (7.1) 29.4 (5.3) 31.5 (5.8) 0.3 Hypertension, % 70% 92% 52% <.05 Diabetes Mellitus, % 28% 48% 5% <.05 Dyslipidemia, % 56% 60% 5% <.05 Variable Patients Controls(n = 34) P Value (n = 34) Unilateral AI(n = 52) Bilateral AI(n = 21) Sex (female/male) 34/18 14/7 23/11 0.3 Age, y, median (range) 59 (22–87) 59 (44–77) 56 (36–74) 0.2 BMI (kg/m2), mean (SD) 30.2 (7.1) 29.4 (5.3) 31.5 (5.8) 0.3 Hypertension, % 70% 92% 52% <.05 Diabetes Mellitus, % 28% 48% 5% <.05 Dyslipidemia, % 56% 60% 5% <.05 AI, adrenal incidentaloma; BMI, body mass index. aP <.05 for unilateral or bilateral AI group vs controls. View Large Table 1. Baseline Characteristics of Patients and Control Individualsa Variable Patients Controls(n = 34) P Value (n = 34) Unilateral AI(n = 52) Bilateral AI(n = 21) Sex (female/male) 34/18 14/7 23/11 0.3 Age, y, median (range) 59 (22–87) 59 (44–77) 56 (36–74) 0.2 BMI (kg/m2), mean (SD) 30.2 (7.1) 29.4 (5.3) 31.5 (5.8) 0.3 Hypertension, % 70% 92% 52% <.05 Diabetes Mellitus, % 28% 48% 5% <.05 Dyslipidemia, % 56% 60% 5% <.05 Variable Patients Controls(n = 34) P Value (n = 34) Unilateral AI(n = 52) Bilateral AI(n = 21) Sex (female/male) 34/18 14/7 23/11 0.3 Age, y, median (range) 59 (22–87) 59 (44–77) 56 (36–74) 0.2 BMI (kg/m2), mean (SD) 30.2 (7.1) 29.4 (5.3) 31.5 (5.8) 0.3 Hypertension, % 70% 92% 52% <.05 Diabetes Mellitus, % 28% 48% 5% <.05 Dyslipidemia, % 56% 60% 5% <.05 AI, adrenal incidentaloma; BMI, body mass index. aP <.05 for unilateral or bilateral AI group vs controls. View Large Figure 1 View largeDownload slide Correlation of individual baseline, post–adrenocorticotropic hormone (pACTH) testing, and post–1-mg dexamethasone suppression testing (pDST) serum cortisol values, as determined by radioimmunoassay (RIA) and liquid chromatography−tandem mass spectronomy (LC/MS-MS) in control subjects (n = 34) and in patients with unilateral (n = 52) or bilateral (n = 21) adrenal incidentalomas (AIs). Values are shown on a bilogarithmic scale diagram. The lower and upper shadow areas represent the DST and ACTH-stimulation ranges, respectively. The coefficient of correlation is 0.941 (n = 318; P <.001). Unil. indicates unilateral; Bilat., bilateral. Figure 1 View largeDownload slide Correlation of individual baseline, post–adrenocorticotropic hormone (pACTH) testing, and post–1-mg dexamethasone suppression testing (pDST) serum cortisol values, as determined by radioimmunoassay (RIA) and liquid chromatography−tandem mass spectronomy (LC/MS-MS) in control subjects (n = 34) and in patients with unilateral (n = 52) or bilateral (n = 21) adrenal incidentalomas (AIs). Values are shown on a bilogarithmic scale diagram. The lower and upper shadow areas represent the DST and ACTH-stimulation ranges, respectively. The coefficient of correlation is 0.941 (n = 318; P <.001). Unil. indicates unilateral; Bilat., bilateral. Baseline, post–1-mg DST, and pACTH serum cortisol values (mean [SD] and median plus range [95% CI]), determined by LC/MS-MS and RIA for unilateral and bilateral AI and control subjects, are shown in Table 2. Also, percentage changes from baseline of serum cortisol after suppression with 1 mg of dexamethasone, ACTH stimulation, and the percentage of abnormal values (those that were consistent with SH) are also described. Table 2. Cortisol Measurements via RIA and LC/MS-MS, Serum Dexamethasone Testing, Salivary Cortisol Assay, and 11 PM and Post−1-mg DST Basal Serum DHEA-S and ACTH Testing Serum Cortisol Salivary Cortisol LC/MS-MS (mcg/dL) RIA (mcg/dL) RIA (ng/dL) Control Subjects (n = 34) Basal pACTH p1-mg DST Basal pACTH p1-mg DST 11 PM p1-mg DST Mean (SD) 7.5 (3.6) 23.6 (6.5) 1.0 (0.3) 8.3 (3.7) 23.9 (8.6) 1.2 (0.6) 198 (164) 32 (20) Median, range (95% CI) 7.3 (2.8–14.2) 22.8 (13.7–33.4) 0.8 (0.6–1.5) 8.3 (3.8–14.1) 23.9 (11.4–38.2) 1.1 (0.7–3.4) 134 (33–506) 27 (11–69) Change from basal, % median (range) 233 (91–551) −87 (−93 to −63) 176 (68–450) −85 (−94 to −70) Abnormal values, %a 0% 3.0% 24% 3.5% Dexamethasone (ng/dL), mean (SD) 417 (141) DHEA-S (mcg/dL), mean (SD) (range) 67.8; 5 (53.3) (15–173) Below 30 mcg/dL, % 36.3% ACTH (pg/mL), mean (SD), range 16.4 (8.9), (8.5–26) Below 10 pg/mL, % 25.0% Unilateral (n = 52) Mean (SD) 9.7 (3.2) 28 (6.6) 2.1 (1.9) 12.4 (5.7) 34.7 (11.4) 2.1 (1.2) 190.5 (163) 105 (174) Median (range, 95% CI) 9.8 (5.3–15.3) 27 (18–40.2) 1.5 (0.7–5.2) 11.2 (5.4–22.5) 34.1 (20.1–54.7) 1.9 (0.9–3.9) 150 (36–424) 53 (10–366) % change from basal, median (range) 191 (61–528) −83 (−91 to −41) 201 (64–1063) −85 (−94 to −60) % abnormal valuesa 23% 25% 15.3 40.0% Dexamethasone (ng/dL), mean (SD) 435 (199) DHEA-S (mcg/dL), mean (SD) (range) 62.5 (65.7) (15–173) Below 30 mcg/dL, % 36.7% ACTH (pg/mL), mean (SD), (range) 18.5 (13.8) (5.6–46.6) Below 10 pg/mL, % 37.2% Bilateral (n = 21) Mean (SD) 13.1 (4.5) 32.4 (6.7) 3.2 (2.5) 15.5 (5.6) 42.5 (16.2) 3.3 (2.8) 189.5 (98.6) 96 (84) Median, range (95% CI) 12.7 (3.4–22.4) 32 (23.9–40.4) 2.3 (1.2–9.6) 15.8 (7.8–24) 39.3 (18.7–64.4) 2.4 (1.2–6.0) 197.5 (34–333) 86 (9.2–316) Change from basal, %, median (range) 150% (30%–355%) −74.6% (−92% to −49%) 199.3% (45%–506%) −85.2% (−90.7% to −45%) Abnormal values, %a 43% 43% 43% 25% 61.5% Dexamethasone, (ng/dL), mean (SD) 418.6 (202) DHEA-S (mcg/dL), mean (SD) 47.4 (35.9) Below 30 mcg/dL, % 42.1% ACTH (pg/mL), mean (SD) (range) 10.9 (5.9) (3.5–20.3) Below 10 pg/mL, % 47.6% Serum Cortisol Salivary Cortisol LC/MS-MS (mcg/dL) RIA (mcg/dL) RIA (ng/dL) Control Subjects (n = 34) Basal pACTH p1-mg DST Basal pACTH p1-mg DST 11 PM p1-mg DST Mean (SD) 7.5 (3.6) 23.6 (6.5) 1.0 (0.3) 8.3 (3.7) 23.9 (8.6) 1.2 (0.6) 198 (164) 32 (20) Median, range (95% CI) 7.3 (2.8–14.2) 22.8 (13.7–33.4) 0.8 (0.6–1.5) 8.3 (3.8–14.1) 23.9 (11.4–38.2) 1.1 (0.7–3.4) 134 (33–506) 27 (11–69) Change from basal, % median (range) 233 (91–551) −87 (−93 to −63) 176 (68–450) −85 (−94 to −70) Abnormal values, %a 0% 3.0% 24% 3.5% Dexamethasone (ng/dL), mean (SD) 417 (141) DHEA-S (mcg/dL), mean (SD) (range) 67.8; 5 (53.3) (15–173) Below 30 mcg/dL, % 36.3% ACTH (pg/mL), mean (SD), range 16.4 (8.9), (8.5–26) Below 10 pg/mL, % 25.0% Unilateral (n = 52) Mean (SD) 9.7 (3.2) 28 (6.6) 2.1 (1.9) 12.4 (5.7) 34.7 (11.4) 2.1 (1.2) 190.5 (163) 105 (174) Median (range, 95% CI) 9.8 (5.3–15.3) 27 (18–40.2) 1.5 (0.7–5.2) 11.2 (5.4–22.5) 34.1 (20.1–54.7) 1.9 (0.9–3.9) 150 (36–424) 53 (10–366) % change from basal, median (range) 191 (61–528) −83 (−91 to −41) 201 (64–1063) −85 (−94 to −60) % abnormal valuesa 23% 25% 15.3 40.0% Dexamethasone (ng/dL), mean (SD) 435 (199) DHEA-S (mcg/dL), mean (SD) (range) 62.5 (65.7) (15–173) Below 30 mcg/dL, % 36.7% ACTH (pg/mL), mean (SD), (range) 18.5 (13.8) (5.6–46.6) Below 10 pg/mL, % 37.2% Bilateral (n = 21) Mean (SD) 13.1 (4.5) 32.4 (6.7) 3.2 (2.5) 15.5 (5.6) 42.5 (16.2) 3.3 (2.8) 189.5 (98.6) 96 (84) Median, range (95% CI) 12.7 (3.4–22.4) 32 (23.9–40.4) 2.3 (1.2–9.6) 15.8 (7.8–24) 39.3 (18.7–64.4) 2.4 (1.2–6.0) 197.5 (34–333) 86 (9.2–316) Change from basal, %, median (range) 150% (30%–355%) −74.6% (−92% to −49%) 199.3% (45%–506%) −85.2% (−90.7% to −45%) Abnormal values, %a 43% 43% 43% 25% 61.5% Dexamethasone, (ng/dL), mean (SD) 418.6 (202) DHEA-S (mcg/dL), mean (SD) 47.4 (35.9) Below 30 mcg/dL, % 42.1% ACTH (pg/mL), mean (SD) (range) 10.9 (5.9) (3.5–20.3) Below 10 pg/mL, % 47.6% LC/MS-MS, liquid chromatography−tandem mass spectronomy; RIA, radioimmunoassay; pACTH, pre−adrenocorticotropic hormone; p1-mg DST, post−1-mg dexamethasone suppression; CI, confidence interval; DHEA-S, dehydroepiandrosterone sulfate; ACTH, adrenocorticotropic hormone. aPercentage of abnormal values that are consistent with subclinical hypercortisolism (SH): after−1-mg-DST serum cortisol >2.5 mcg/dL; after-1-mg DST salivary cortisol >60 ng/dL; basal serum DHEA-S <30 mcg/dL, and basal plasma ACTH <10 pg/mL. View Large Table 2. Cortisol Measurements via RIA and LC/MS-MS, Serum Dexamethasone Testing, Salivary Cortisol Assay, and 11 PM and Post−1-mg DST Basal Serum DHEA-S and ACTH Testing Serum Cortisol Salivary Cortisol LC/MS-MS (mcg/dL) RIA (mcg/dL) RIA (ng/dL) Control Subjects (n = 34) Basal pACTH p1-mg DST Basal pACTH p1-mg DST 11 PM p1-mg DST Mean (SD) 7.5 (3.6) 23.6 (6.5) 1.0 (0.3) 8.3 (3.7) 23.9 (8.6) 1.2 (0.6) 198 (164) 32 (20) Median, range (95% CI) 7.3 (2.8–14.2) 22.8 (13.7–33.4) 0.8 (0.6–1.5) 8.3 (3.8–14.1) 23.9 (11.4–38.2) 1.1 (0.7–3.4) 134 (33–506) 27 (11–69) Change from basal, % median (range) 233 (91–551) −87 (−93 to −63) 176 (68–450) −85 (−94 to −70) Abnormal values, %a 0% 3.0% 24% 3.5% Dexamethasone (ng/dL), mean (SD) 417 (141) DHEA-S (mcg/dL), mean (SD) (range) 67.8; 5 (53.3) (15–173) Below 30 mcg/dL, % 36.3% ACTH (pg/mL), mean (SD), range 16.4 (8.9), (8.5–26) Below 10 pg/mL, % 25.0% Unilateral (n = 52) Mean (SD) 9.7 (3.2) 28 (6.6) 2.1 (1.9) 12.4 (5.7) 34.7 (11.4) 2.1 (1.2) 190.5 (163) 105 (174) Median (range, 95% CI) 9.8 (5.3–15.3) 27 (18–40.2) 1.5 (0.7–5.2) 11.2 (5.4–22.5) 34.1 (20.1–54.7) 1.9 (0.9–3.9) 150 (36–424) 53 (10–366) % change from basal, median (range) 191 (61–528) −83 (−91 to −41) 201 (64–1063) −85 (−94 to −60) % abnormal valuesa 23% 25% 15.3 40.0% Dexamethasone (ng/dL), mean (SD) 435 (199) DHEA-S (mcg/dL), mean (SD) (range) 62.5 (65.7) (15–173) Below 30 mcg/dL, % 36.7% ACTH (pg/mL), mean (SD), (range) 18.5 (13.8) (5.6–46.6) Below 10 pg/mL, % 37.2% Bilateral (n = 21) Mean (SD) 13.1 (4.5) 32.4 (6.7) 3.2 (2.5) 15.5 (5.6) 42.5 (16.2) 3.3 (2.8) 189.5 (98.6) 96 (84) Median, range (95% CI) 12.7 (3.4–22.4) 32 (23.9–40.4) 2.3 (1.2–9.6) 15.8 (7.8–24) 39.3 (18.7–64.4) 2.4 (1.2–6.0) 197.5 (34–333) 86 (9.2–316) Change from basal, %, median (range) 150% (30%–355%) −74.6% (−92% to −49%) 199.3% (45%–506%) −85.2% (−90.7% to −45%) Abnormal values, %a 43% 43% 43% 25% 61.5% Dexamethasone, (ng/dL), mean (SD) 418.6 (202) DHEA-S (mcg/dL), mean (SD) 47.4 (35.9) Below 30 mcg/dL, % 42.1% ACTH (pg/mL), mean (SD) (range) 10.9 (5.9) (3.5–20.3) Below 10 pg/mL, % 47.6% Serum Cortisol Salivary Cortisol LC/MS-MS (mcg/dL) RIA (mcg/dL) RIA (ng/dL) Control Subjects (n = 34) Basal pACTH p1-mg DST Basal pACTH p1-mg DST 11 PM p1-mg DST Mean (SD) 7.5 (3.6) 23.6 (6.5) 1.0 (0.3) 8.3 (3.7) 23.9 (8.6) 1.2 (0.6) 198 (164) 32 (20) Median, range (95% CI) 7.3 (2.8–14.2) 22.8 (13.7–33.4) 0.8 (0.6–1.5) 8.3 (3.8–14.1) 23.9 (11.4–38.2) 1.1 (0.7–3.4) 134 (33–506) 27 (11–69) Change from basal, % median (range) 233 (91–551) −87 (−93 to −63) 176 (68–450) −85 (−94 to −70) Abnormal values, %a 0% 3.0% 24% 3.5% Dexamethasone (ng/dL), mean (SD) 417 (141) DHEA-S (mcg/dL), mean (SD) (range) 67.8; 5 (53.3) (15–173) Below 30 mcg/dL, % 36.3% ACTH (pg/mL), mean (SD), range 16.4 (8.9), (8.5–26) Below 10 pg/mL, % 25.0% Unilateral (n = 52) Mean (SD) 9.7 (3.2) 28 (6.6) 2.1 (1.9) 12.4 (5.7) 34.7 (11.4) 2.1 (1.2) 190.5 (163) 105 (174) Median (range, 95% CI) 9.8 (5.3–15.3) 27 (18–40.2) 1.5 (0.7–5.2) 11.2 (5.4–22.5) 34.1 (20.1–54.7) 1.9 (0.9–3.9) 150 (36–424) 53 (10–366) % change from basal, median (range) 191 (61–528) −83 (−91 to −41) 201 (64–1063) −85 (−94 to −60) % abnormal valuesa 23% 25% 15.3 40.0% Dexamethasone (ng/dL), mean (SD) 435 (199) DHEA-S (mcg/dL), mean (SD) (range) 62.5 (65.7) (15–173) Below 30 mcg/dL, % 36.7% ACTH (pg/mL), mean (SD), (range) 18.5 (13.8) (5.6–46.6) Below 10 pg/mL, % 37.2% Bilateral (n = 21) Mean (SD) 13.1 (4.5) 32.4 (6.7) 3.2 (2.5) 15.5 (5.6) 42.5 (16.2) 3.3 (2.8) 189.5 (98.6) 96 (84) Median, range (95% CI) 12.7 (3.4–22.4) 32 (23.9–40.4) 2.3 (1.2–9.6) 15.8 (7.8–24) 39.3 (18.7–64.4) 2.4 (1.2–6.0) 197.5 (34–333) 86 (9.2–316) Change from basal, %, median (range) 150% (30%–355%) −74.6% (−92% to −49%) 199.3% (45%–506%) −85.2% (−90.7% to −45%) Abnormal values, %a 43% 43% 43% 25% 61.5% Dexamethasone, (ng/dL), mean (SD) 418.6 (202) DHEA-S (mcg/dL), mean (SD) 47.4 (35.9) Below 30 mcg/dL, % 42.1% ACTH (pg/mL), mean (SD) (range) 10.9 (5.9) (3.5–20.3) Below 10 pg/mL, % 47.6% LC/MS-MS, liquid chromatography−tandem mass spectronomy; RIA, radioimmunoassay; pACTH, pre−adrenocorticotropic hormone; p1-mg DST, post−1-mg dexamethasone suppression; CI, confidence interval; DHEA-S, dehydroepiandrosterone sulfate; ACTH, adrenocorticotropic hormone. aPercentage of abnormal values that are consistent with subclinical hypercortisolism (SH): after−1-mg-DST serum cortisol >2.5 mcg/dL; after-1-mg DST salivary cortisol >60 ng/dL; basal serum DHEA-S <30 mcg/dL, and basal plasma ACTH <10 pg/mL. View Large Figure 2 depicts mean (SE) values of basal, after-dexamethasone, and after-ACTH serum cortisol levels in control subjects and individuals with unilateral and bilateral AI, as measured by RIA and LC/MS-MS. Overall, the average cortisol values determined by LC/MS-MS were 15% lower than those obtained by RIA. However, 32% of all 318 pairs of values disclosed LC/MS-MS cortisol levels above the identity line. In other words, LC/MS-MS cortisol values were higher than concurrent RIA levels, with only 3 values (0.9%) identical in both methods. Figure 2 View largeDownload slide Histogram showing mean (SE) values of serum cortisol at baseline, post−adrenocorticotropic hormone (pACTH) testing, and post-dexamethasone suppression testing (aDST) from control subjects (n = 34) and patients with unilateral (n = 52) or bilateral (n = 21) adrenal incidentalomas (AIs), as determined by liquid chromatography−tandem mass spectronomy (LC/MS-MS) and radioimmunoassay (RIA). Note that LC/MS-MS values are, on average, 15% to 20% lower than their RIA counterparts. Baseline, p-DST, and p-ACTH values were significantly higher (P <.01) in patients with AIs vs controls and were significantly higher in bilateral vs unilateral AI. Figure 2 View largeDownload slide Histogram showing mean (SE) values of serum cortisol at baseline, post−adrenocorticotropic hormone (pACTH) testing, and post-dexamethasone suppression testing (aDST) from control subjects (n = 34) and patients with unilateral (n = 52) or bilateral (n = 21) adrenal incidentalomas (AIs), as determined by liquid chromatography−tandem mass spectronomy (LC/MS-MS) and radioimmunoassay (RIA). Note that LC/MS-MS values are, on average, 15% to 20% lower than their RIA counterparts. Baseline, p-DST, and p-ACTH values were significantly higher (P <.01) in patients with AIs vs controls and were significantly higher in bilateral vs unilateral AI. The average baseline and pACTH cortisol levels were significantly higher in patients with unilateral AI than in control subjects and even higher in patients with bilateral AI. Likewise, cortisol levels were significantly more resistant to dexamethasone suppression in unilateral AI than in controls and even more resistant in bilateral AI (Figure 2). Using a previously defined cutoff level of 2.5 mcg per dL for post–1-mg DST serum cortisol (valid test results, with serum dexamethasone levels ≥ 140 ng/dL16), we identified 12 of 52 cases of unilateral (23%) and 9 of 21 of bilateral AI (42.8%), for which concurring RIA and LC/MS-MS values were above that level and consistent with SH. Cortisol levels suppressed below 2.5 mcg per dL (both methods) in 40 of the 52 patients with unilateral AI (76.9%) and 12 of the 21 patients with bilateral AI (57.1%) suggested NFA. Specimens from the remaining 3 of 52 patients with unilateral AI and 2 of 21 patients with bilateral AI yielded discordant results in both methods and were disregarded as statistical outliers. If a lower and more sensitive post-dexamethasone serum cortisol cutoff level of 1.8 mcg per dL is adopted to exclude SH, 32 of the patients with 52 unilateral AI (61.5%) and 8 of the 21 patients with bilateral AI (38%) had concurring lower values, consistent with NFA. Conversely, when a more stringent (specific) cutoff level of 5.0 mcg per dL is used, only 5.7% of the cases of unilateral AI and 19% of the cases of bilateral AI would be designated as SH. As depicted in Table 2, reduced levels of plasma ACTH (<10 pg/mL) and serum DHEA-S (<30 mcg/dL) were present in 37.2% and 36.7% of the cases of unilateral AI and in 47.6% and 42.1% of the cases of bilateral AI but also in 25% and 36.3% of the controls, respectively. These numbers were considerably more consistent in the SH subpopulations with unilateral AI (70% and 50%, respectively) and with bilateral AI (77.8% and 66.7%, respectively). Similarly, post-dexamethasone salivary cortisol levels were elevated (>60 ng/dL) in 40% and 61.5% of the patients with unilateral and bilateral AI, respectively, but only in 3.5% of controls. Discussion The methodology for measuring steroids has changed over time, moving from the RIA of the 1960s to other immuno- and chemiluminescent assays across the subsequent decades, reaching the most accurate criterion-standard technology of today, namely, LC-MS/MS.19 Because several of these methods/assays are still currently in use, comparison of the results they deliver is an important issue for clinicians who do not have access to LC-MS/MS technology. Recently, the Endocrine Society recommended new instructions to authors on the reporting of steroid hormone assay measurement.20 However, questions regarding costs and availability remain a major problem in most places. We believe that it is interesting but not entirely unexpected that the correlation for most steroids does not reach 100%. Similar studies, measuring steroids or other hormones in dupicate (comparison of two methodologies) showed a better accuracy for LC-MS/S.21-24 Conversely, cortisol is produced in substantial amounts, and so measurement of its concentrations in biological fluids by different methods may not be a critical issue.25 Routine screening or tests performed for other reasons using advanced imaging techniques often show incidentalomas, most of which are NFAs; however, a significant percentage has a relatively autonomous cortisol pattern but without the typical manifestations of Cushing syndrome. For those incidentalomas, the term subclinical hypercortisolism has been applied.1 Identification of this particular subset of patients seems important because even mild cortisol excess may be responsible for the development or worsening of a range of metabolic abnormalities, such as glucose intolerance, diabetes mellitus, dyslipidemia, arterial hypertension, and osteopenia/osteoporosis, which are also prevalent among the general population.26,27 Recently, several prospective studies, including one by Chiodini et al,28 documented the improvement of metabolic manifestations on removal of adrenal adenomas. However, a high number of these cases present evidence of postoperatory adrenal insufficiency, indicating prolonged contralateral adrenocortical atrophy.7 A definitive diagnosis of SH remains challenging. Several isolated or combined tests have been used for this purpose (resistance of serum cortisol to low-dose dexamethasone suppression, elevated late-night serum levels, or salivary cortisol levels [elevated 24-hour urinary free cortisol excretion and other tests4]). Detection of reduced or suppressed levels of ACTH and/or DHEA-S are additional markers of chronic autonomous cortisol excess. However, the most-used test is 1-mg DST, in which it is determined whether serum and/or salivary cortisol levels remain above a certain limit the morning after a 1-mg oral dose of dexamethasone is administered at 11 PM. Nonsuppressible post-dexamethasone cortisol levels are suggestive of autonomous production or altered HPA axis, the latter of which acts directly on corticotrophic cells. As a result, the conceptual definition of subclinical hypercortisolism varies among groups regarding which tests and how many tests are necessary for diagnosis and which cutoff points are chosen to define excess or nonsuppressibility.12 Also, the methodology used to measure serum, plasma, and salivary and urinary cortisol also plays an important role in this diagnosis when the HPA axis is evaluated.29,30 The results of a comparison of different methodologies suggest that LC/MS-MS is the most reliable method.31-33 When the widely accepted cutoff value for serum cortisol level (<1.8 mcg/dL) after dexamethasone suppression is used to define SH, studies find that 15% to 30% of the patients with AI have SH.12 We found good correlation between serum cortisol values measured by RIA and LC/MS-MS in the normal basal state or after dexamethasone suppression. However, after ACTH stimulation, there was considerable discordance between the 2 methods. A reasonable explanation is that there is cortisol-binding globulin (CBG) saturation, which is more evident in RIA methods, due to a more significant cross-reactivity with other steroids or metabolites.34,35 These factors suggest that other steroids produced after ACTH stimulation bind to the antibody in the RIA test and render the test less specific. Using a cutoff level of 2.5 mcg per dL after dexamethasone suppression, we identify 40% and 55% of SH among the patients with unilateral and bilateral AI, respectively.36 The more-sensitive cutoff of 1.8 mcg per dL probably best defines SH among AIs when valid 1-mg DST (dexamethasone serum levels ≥140 ng/dL) is used for evaluation. Also, 81.8% and 88.9% of the unilateral and bilateral AIs also satisfy 2 of 3 additional criteria for autonomous cortisol production (suppressed basal plasma ACTH [<10 pg/mL], suppressed basal serum DHEA-S [<30 mcg/dL], and/or nonsuppressed post–1-mg DST salivary cortisol levels [>60 ng/dL]), further reinforcing the diagnosis of SH.1 Recently, serum free cortisol was evaluated in patients with primary and secondary adrenal insufficiency and patients with cirrhosis; it was found that serum free cortisol (SFF) had higher specificity than total cortisol that is 85% to 95% bound to CBG and albumin.37 Others performed adrenalectomies in patients with SH and observed a reduction of cortisol levels and improvement of the metabolic parameters soon after the procedure (as determined by lower fasting glucose, glycated hemoglobin, and low-density lipoprotein [LDL]−cholesterol levels and amelioration of blood pressure38). Also, the percentage of adrenal insufficiency after unilateral adrenalectomy for an AI with diagnosed SH increased as the number of tests used to define SH in these patients also increased.38,39 Urinary free cortisol excretion and salivary cortisol tests are not sensitive enough and do not show any limitation in detecting SH and mild Cushing syndrome.40 Therefore, in our study, we found higher salivary cortisol levels after dexamethasone in the patient group than in controls but did no found any difference at the midnight salivary cortisol among the groups. In conclusion, serum cortisol measurement by RIA and LC/MS-MS in patients with unilateral and bilateral AI, as well as in control subjects, results in similar concentrations for low values (basal or after suppression). However, after ACTH stimulation, we observed some discordance between the 2 methods. Baseline serum cortisol, as well as post-dexamethasone and post-ACTH values, are significantly higher than controls in patients with unilateral AI and higher yet in those with bilateral AI. In general, AIs appear to produce higher amounts of basal and stimulated cortisol simultaneously, showing less suppression after dexamethasone. In this sense, bilateral AI, perhaps due to a mass effect, shows more evidence of SH than unilateral AI. Concordant (between RIA and LC-MS/MS methods) serum cortisol values higher than 2.5 mcg per dL, in addition to the results from 2 other tests (from reduced serum DHEA-S, reduced plasma ACTH, and/or elevated pDST salivary cortisol), best establish SH diagnosis.The subpopulation with these factors comprises 22% of the patients with unilateral AI and 40% with bilateral AI. It is expected that surgical removal or radiofrequency ablation of adrenal lesions will be beneficial in restoring metabolic abnormalities and preventing further cardiovascular and osteometabolic risks. Abbreviations SH subclinical hypercortisolism AIs adrenal incidentalomas DST dexamethasone suppression testing ACTH adrenocorticotropic hormone DHEA-S dehydroepiandrosterone sulfate CRH corticotropin-releasing hormone RIA radioimmunoassay LC/MS-MS liquid chromatography/tandem mass spectronomy NFAs nonfunctioning adrenal adenomas UNIFESP Universidade Federal de São Paulo CT computerized tomography MRI magnetic resonance imaging HPA hypothalamic-pituitary-adrenal IV intravenous BSA bovine serum albumin SRM selected reaction monitoring ESI electrospray ionization HPA hypothalamic-pituitary-adrenal CBG cortisol-binding globulin SFF serum free cortisol LDL low-density lipoprotein NS no significant BMI body mass index CI confidence interval Unil. unilateral Bilat. bilateral. Acknowledgments We are thankful for the laboratory expertise of Lilian F. Hayashi, MSc, and Kelly C. Oliveira, MSc, for performing cortisol RIA in São Paulo, Brazil. References 1. Reincke M . Subclinical Cushing’s syndrome . Endocrinol Metab Clin North Am . 2000 ; 29 ( 1 ): 43 – 56 . 2. Rossi R , Tauchmanova L , Luciano A , et al. Subclinical Cushing’s syndrome in patients with adrenal incidentaloma: clinical and biochemical features . J Clin Endocrinol Metab . 2000 ; 85 ( 4 ): 1440 – 1448 . 3. Valli N , Catargi B , Ronci N , et al. Biochemical screening for subclinical cortisol-secreting adenomas amongst adrenal incidentalomas . Eur J Endocrinol . 2001 ; 144 ( 4 ): 401 – 408 . 4. Di Dalmazi G , Pasquali R , Beuschlein F , Reincke M . Subclinical hypercortisolism: a state, a syndrome, or a disease ? Eur J Endocrinol . 2015 ; 173 ( 4 ): M61 – M71 . 5. Di Dalmazi G , Vicennati V , Garelli S , et al. 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Masserini B , Morelli V , Bergamaschi S , et al. The limited role of midnight salivary cortisol levels in the diagnosis of subclinical hypercortisolism in patients with adrenal incidentaloma . Eur J Endocrinol . 2009 ; 160 ( 1 ): 87 – 92 . © American Society for Clinical Pathology 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

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Laboratory MedicineOxford University Press

Published: Mar 28, 2018

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