The Biochemical Investigation of Cushing Syndrome

Marie Simard, M.D.


Neurosurg Focus. 2004;16(4) 

In This Article

Differential Diagnosis of Cushing Syndrome

Once the diagnosis of Cushing syndrome has been established, the clinician must seek to identify the cause of excess cortisol secretion. Dynamic biochemical tests help distinguish corticotropin-secreting adenomas, which retain their responsiveness to both suppression by corticosteroids and stimulation by CRH from ectopic ACTH-secreting tumors, which usually function autonomously.

The CRH stimulation Test

Approximately 85% of patients with Cushing disease respond to ovine CRH with an increase in plasma levels of ACTH and cortisol. Only 5% of patients with ectopic ACTH-secreting tumors respond.[20] The CRH (1 µg/kg or 100 µg) is intravenously administered in the morning; this illicits an increase in plasma ACTH or cortisol levels in patients with Cushing disease, but no response in patients with ectopic ACTH secretion.[30] A rise in plasma ACTH values greater than 35% (measured at 15 and 30 minutes postinjection) compared with baseline values yields a 100% rate of specificity and a 93% rate of sensitivity. An increase of at least 20% in the cortisol level measured 30 and 45 minutes after CRH administration yields a specificity of 88% and a sensitivity of 91% in the diagnosis of Cushing disease.[30] When the CRH stimulation test is performed in conjunction with the DST, nondiagnostic results from both tests rule out a diagnosis of Cushing disease with a diagnostic accuracy greater than 98%.[29]

Dexamethasone Suppression–CRH Stimulation Test

The dexamethasone–CRH test distinguishes patients with pseudo-Cushing syndrome from those with Cushing syndrome. Integrating the low-dose DST with the CRH test (described below) significantly increases its diagnostic accuracy.[28] This test is performed by oral administration of 0.5 mg, dexamethasone every 6 hours, providing eight doses beginning at noon and ending at 6 a.m. Corticotropin- releasing hormone (Acthrel; Ferring Pharmaceuticals, Inc., Tarrytown, NY), 1 µg/kg body weight, is given intravenously 2 hours after the last dose, and the level of cortisol is measured just before CRH administration and 15 minutes later. A plasma level of dexamethasone should be recorded before the CRH test is given to confirm the patient's normal metabolism. A plasma cortisol level of 1.4 µg/dl (38.6 nmol/L) or greater supports the diagnosis of Cushing syndrome.[28] Furthermore, Isidori and colleagues[15] have demonstrated that more than a 30% suppression of serum cortisol during the low-dose DST and/ or more than a 20% increase in cortisol during the CRH test had significantly higher rates of sensitivity (97%) and specificity (94%) than either the high-dose DST or the CRH test alone in the differential diagnosis of ACTH-dependent Cushing syndrome. Thus, the differential diagnosis between Cushing disease and ectopic ACTH secretion can be performed with a high accuracy by combining the results of the formal 2 mg/day 48-hour low-dose DST and the CRH test for serum cortisol.[15]

Plasma ACTH

The advent of a sensitive and specific two-site immunometric assay for plasma ACTH has facilitated the diagnosis of Cushing disease.[7] Adrenocorticotropic hormone has a short plasma halflife, necessitating that samples be kept in an ice water bath, centrifuged, separated into aliquots, and frozen within a few hours to avoid obtaining spuriously low results.[27] Simultaneous plasma cortisol levels should be determined.[1] Using an immunometric assay, plasma ACTH levels measuring more than 10 pg/ml (2.2 pmol/L) and ACTH levels higher than 20 pg/ml (4.5 pmol/L) are indicative of an ACTH-secreting neoplasm.[27] Patients with ectopic ACTH syndrome generally have very high plasma ACTH values, although these values may overlap with those seen in patients with Cushing disease.[10] In patients with Cushing disease, 50% have a 9 a.m. plasma ACTH level within the normal reference range of 9 to 54 pg/ml (2–12 pmol/L) and the remaining patients have a slightly elevated ACTH level.[36] Due to the loss of circadian rhythm, however, nighttime ACTH secretion is abnormal. A midnight plasma ACTH level greater than 23 pg/dl (5 pmol/L) confirms the presence of an ACTH excess.[37] Plasma ACTH levels are suppressed when the source of the hypercortisolism is an adrenal cortisol-secreting tumor or a micronodular or macronodular adrenal disease.[20] Subnormal daytime plasma ACTH levels that are lower than 5 pg/ml (1.1 pmol/L) are usually present in patients with ACTH-independent Cushing syndrome.[10]

High-Dose DST

When plasma ACTH levels are higher than 10 pg/ml, the source of ACTH secretion—pituitary or ectopic—must be localized. Secretion of ACTH by corticotropinomas is usually inhibited by high-dose glucocorticoid therapy. The high-dose DST is performed by collecting a 24-hour baseline urine sample of free cortisol and 17-hydroxysteroid, administering 2 mg of dexamethasone orally every 6 hours for 2 days (in children 80–120 µg/kg/day divided into four doses every 6 hours or a maximum of 2 mg every 6 hours for 2 days),[20,36] and repeating the 24-hour urine collection during the last 24 hours of the test. The criterion of 69% suppression from the baseline value of 24-hour UFC is required to yield a specificity of 100% in the diagnosis of Cushing disease.[23,27] Urinary levels of 17-hydroxysteroid are similarly suppressed in 85% of patients with Cushing disease.[20] Paradoxical responses to dexamethasone indicate the presence of either micronodular adrenal disease or ACTH-independent Cushing syndrome.[21]

Eight-Milligram Overnight DST

The 8-mg overnight DST is widely used because of its convenience. It consists of measuring a baseline plasma level of cortisol followed by oral administration of 8 mg dexamethasone at 11 p.m. A second specimen of plasma is obtained 9 hours later, at 8 a.m., and the cortisol level is measured. A decrease in the plasma level of cortisol that is 50% or greater—the criterion for Cushing disease—yields a diagnostic accuracy comparable to that provided by the high-dose DST.[23,27]

Bilateral Simultaneous Inferior Petrosal Sinus Sampling

Inferior petrosal sinus sampling for ACTH has emerged as the most accurate and reliable means of distinguishing pituitary from nonpituitary ACTH-dependent Cushing syndrome.[4,8,10,14,17,19] The IPSS should be reserved for patients with classic clinical and biochemical Cushing disease in whom magnetic resonance imaging findings are nondiagnostic or equivocal, for patients with equivocal results from suppression and stimulation tests,[39] and for patients whose clinical presentation is consistent with ectopic ACTH secretion.[9] In experienced hands, the diagnostic accuracy of IPSS approaches 80 to 100%.[17] The procedure must be performed when cortisol levels in the peripheral circulation are elevated to suppress the normal corticotroph population of the anterior pituitary. The midnight plasma cortisol level or the amount of UFC excretion should thus be measured immediately before IPSS.[27] Peripheral CRH levels should be measured routinely to exclude the possibility of a nonpituitary CRH-secreting neoplasm as the source of hypercortisolism.[9] Concentrations of ACTH are greater in inferior petrosal sinus samples obtained from patients with Cushing disease and increase after CRH administration. Corticotropin-reducing hormone significantly reduces the number of false-negative basal results.[17] An IPS/P greater than 3 following administration of CRH is considered consistent with Cushing disease. When ACTH secretion is ectopic, values of this hormone in inferior petrosal sinus and peripheral specimens are similar and do not increase after CRH is given.[27] Most patients with ectopic ACTH syndrome have an IPS/P less than 2 and, rarely, certain patients have ratios between 2 and 3. Bilateral simultaneous sampling is essential because the maximal basal nondominant IPS/P is less than 2 in more than 50% of patients with Cushing disease and remains less than 2 after administration of ovine CRH in 33% of cases.[8] Lateralization of the pituitary microadenoma is defined by an ACTH IPS gradient greater than 1.4, before and after CRH stimulation, with positive predictive values of 74 and of 83%, respectively.[10,17] Midline adenomas may cause misleading lateralization gradients.[17] The rate of correlation of the ACTH IPS gradient with operative outcome ranges from 47 to 75%.[4,17] The IPSS has been associated with morbid and even fatal complications, including deep vein thrombosis, pulmonary emboli, and brainstem vascular damage.[3,4,17] The use of intravenous heparin during the procedure is advocated to help prevent thrombosis.[17]

Sampling of the cavernous sinus has yielded a 20% false-negative rate[5] and has a higher incidence of occlusive events.[27] Jugular venous sampling is easier to perform and has a sensitivity of 88% and a specificity of 100% when the interpretation criteria is the same as those for IPSS. This approach may be used as an initial procedure with a referral for IPSS when results are nondiagnostic.[6]