Adrenal Function Testing: Cortisol and ACTH

Adrenal function testing measures the output of cortisol and adrenocorticotropic hormone (ACTH) to identify disorders affecting the hypothalamic-pituitary-adrenal (HPA) axis. These tests support the diagnosis of conditions ranging from adrenal insufficiency (Addison's disease) to Cushing's syndrome, both of which carry significant morbidity if undetected. Understanding how these assays work, when they are ordered, and how results are interpreted is essential for anyone navigating an endocrinology workup. The broader regulatory context for endocrinology that governs laboratory testing and clinical standards shapes how these protocols are applied in practice.

Definition and Scope

Cortisol is a glucocorticoid hormone produced by the adrenal cortex in response to ACTH secreted by the pituitary gland. ACTH itself is regulated upstream by corticotropin-releasing hormone (CRH) from the hypothalamus. This three-tier feedback loop — hypothalamus → pituitary → adrenal cortex — is the central axis that adrenal function testing evaluates.

The Endocrine Society, which publishes clinical practice guidelines on adrenal disorders, defines adrenal insufficiency as a failure of the adrenal glands to produce sufficient steroid hormones, a condition affecting approximately 1 in 8,000 people in the United States (Endocrine Society Clinical Practice Guideline on Adrenal Insufficiency, 2016). Cushing's syndrome, representing the opposite extreme of cortisol excess, has an estimated incidence of 10–15 cases per million people per year (Endocrine Society).

Tests within this domain fall into two broad categories:

The Clinical and Laboratory Standards Institute (CLSI) and College of American Pathologists (CAP) set analytical performance standards that laboratories must meet when running cortisol immunoassays and ACTH measurements, including requirements for assay sensitivity, precision, and reference interval validation.

How It Works

Adrenal function testing follows a structured sequence depending on the clinical question. The primary protocols are:

  1. Morning serum cortisol: Cortisol peaks between 6:00 a.m. and 8:00 a.m. due to circadian rhythm. A serum cortisol below 3 µg/dL at this time strongly suggests adrenal insufficiency; a value above 18 µg/dL makes it unlikely (Endocrine Society, 2016 guideline).

  2. ACTH stimulation test (Cosyntropin test): 250 µg of synthetic ACTH (cosyntropin) is administered intravenously or intramuscularly. Cortisol is measured at baseline and at 30 and 60 minutes. A peak cortisol of at least 18 µg/dL at 30–60 minutes is the widely used threshold for a normal adrenal response, though laboratory-specific reference intervals apply.

  3. 24-hour urinary free cortisol (UFC): Urine is collected over a full 24-hour period and total cortisol is quantified. Values exceeding the upper limit of the normal reference range on at least two independent collections support a diagnosis of Cushing's syndrome. Late-night salivary cortisol (LNSC) testing exploits the nadir of the circadian cycle — normally the lowest cortisol point — to detect pathological cortisol excess.

  4. Low-dose dexamethasone suppression test (DST): 1 mg of dexamethasone is taken orally at 11:00 p.m., and serum cortisol is measured the following morning at 8:00 a.m. Failure to suppress cortisol below 1.8 µg/dL is a positive screening result for Cushing's syndrome (Endocrine Society Cushing's Syndrome Guideline, 2008, updated 2015).

  5. Plasma ACTH measurement: ACTH levels distinguish ACTH-dependent causes (pituitary or ectopic tumors) from ACTH-independent causes (primary adrenal tumors or hyperplasia). A plasma ACTH below 5 pg/mL in the context of cortisol excess points toward a primary adrenal source; levels above 20 pg/mL suggest a pituitary or ectopic origin.

ACTH is a highly labile peptide that degrades rapidly at room temperature. The National Academy of Clinical Biochemistry (NACB) specifies that plasma for ACTH measurement must be collected in EDTA tubes, immediately placed on ice, and centrifuged within 15 minutes to prevent pre-analytical degradation.

Common Scenarios

Adrenal function testing is ordered across a defined set of clinical presentations. The blood tests for endocrine conditions page provides broader context for how these fit into general endocrine panels.

Suspected adrenal insufficiency: Patients presenting with fatigue, hypotension, hyponatremia, hyperkalemia, and hyperpigmentation warrant HPA axis evaluation. The ACTH stimulation test is the primary confirmatory tool. Primary adrenal insufficiency (Addison's disease) produces elevated ACTH alongside low cortisol; secondary insufficiency (pituitary failure) produces low or inappropriately normal ACTH with low cortisol.

Incidentally discovered adrenal mass: Adrenal incidentalomas — discovered on imaging performed for unrelated indications — require biochemical exclusion of autonomous cortisol secretion in approximately 30–40% of cases, depending on lesion size and imaging characteristics (per Endocrine Society 2016 adrenal incidentaloma guideline). The 1 mg overnight DST is the first-line screen.

Cushing's syndrome workup: Patients with central obesity, proximal muscle weakness, wide purple striae, easy bruising, and hypertension undergo screening with at least two of the three first-line tests: 24-hour UFC, late-night salivary cortisol, or 1 mg overnight DST. A confirmed biochemical result then triggers pituitary hormone panels and MRI to localize the source.

Monitoring treated adrenal disease: Patients on glucocorticoid replacement therapy for adrenal insufficiency or post-surgical Cushing's syndrome require periodic cortisol profiling to confirm adequacy of replacement or remission.

Decision Boundaries

Interpreting adrenal function tests requires understanding the structured thresholds that separate normal from abnormal findings, as well as the conditions that confound results.

Cortisol assay interference: Synthetic glucocorticoids such as prednisolone and methylprednisolone cross-react with some cortisol immunoassays, producing falsely elevated readings. Mass spectrometry-based methods (LC-MS/MS) avoid this interference and are increasingly used in reference laboratories, per CLSI guideline EP7.

Conditions that elevate cortisol binding globulin (CBG): Oral contraceptives and pregnancy raise CBG, increasing total cortisol but not free cortisol. Total serum cortisol thresholds are unreliable in these populations; salivary or urinary free cortisol measurements are preferred.

Comparison — primary vs. secondary adrenal insufficiency:

Feature Primary (Addison's) Secondary (Pituitary)
Cortisol Low Low
ACTH High (>100 pg/mL typical) Low or inappropriately normal
Mineralocorticoid deficiency Present Absent
Hyperpigmentation Present Absent
Cause Autoimmune, infection, hemorrhage Pituitary tumor, surgery, radiation

Low-dose vs. high-dose DST: The 8 mg high-dose dexamethasone suppression test differentiates pituitary-dependent Cushing's disease from ectopic ACTH secretion. Pituitary adenomas retain partial sensitivity to glucocorticoid feedback and suppress cortisol by more than 50% in approximately 70–80% of cases; ectopic tumors typically do not suppress (Endocrine Society guideline). This distinction guides whether surgical planning focuses on the pituitary or requires further imaging for ectopic sources.

Metyrapone and CRH stimulation tests: These second-line dynamic tests are used in specialized centers when standard testing yields equivocal results. Metyrapone blocks cortisol synthesis and tests ACTH reserve; CRH infusion distinguishes pituitary from ectopic ACTH by assessing pituitary responsiveness. Both require endocrinology specialist oversight given risk of acute adrenal insufficiency.

The endocrinology authority index provides navigational access to the full scope of diagnostic and therapeutic topics covered within this resource.

References

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