Davis's Lab & Diagnostic Tests

Adrenocorticotropic Hormone (and Challenge Tests)


Corticotropin, ACTH.

Common Use:
To assist in the investigation of adrenocortical dysfunction using ACTH and cortisol levels in diagnosing disorders such as Addison’s disease, Cushing’s disease, and Cushing’s syndrome.

Plasma from a lavender-top (EDTA) tube for adrenocorticotropic hormone (ACTH) and serum from a red-top tube for cortisol and 11-deoxycortisol. Collect specimens in a prechilled heparinized plastic syringe, and carefully transfer into collection containers by gentle injection to avoid hemolysis. Alternatively, specimens can be collected in prechilled lavender- and red-top tubes. Gold-tiger- and green-top (heparin) tubes are also acceptable for cortisol, but take care to use the same type of collection container for serial measurements. Immediately transport specimen, tightly capped and in an ice slurry, to the laboratory. The specimens should be immediately processed. Plasma for ACTH analysis should be transferred to a plastic container.

ProcedureIndicationsMedication Administered, Adult DosageRecommended Collection Times
ACTH stimulation, rapid testSuspect adrenal insufficiency (Addison’s disease) or congenital adrenal hyperplasia1 mcg (low-dose physiologic protocol) cosyntropin intramuscular (IM) or IV; 250 mcg (standard pharmacologic protocol) cosyntropin IM or intravenous (IV)Three cortisol levels: baseline immediately before bolus, 30 min after bolus, and 60 min (optional) after bolus.Baseline and 30-min levels are adequate for accurate diagnosis using either dosage; low-dose protocol sensitivity is most accurate for 30-min level only
Corticotropin-releasing hormone (CRH) stimulationDifferential diagnosis between ACTH-dependent conditions such as Cushing’s disease (pituitary source) or Cushing’s syndrome (ectopic source) and ACTH-independent conditions such as Cushing’s syndrome (adrenal source)IV dose of 1 mcg/kg human CRHEight cortisol and eight ACTH levels: baseline collected 15 min before injection, 0 min before injection, and then 5, 15, 30, 60, 120, and 180 min after injection
Dexamethasone suppression (overnight)Differential diagnosis between ACTH-dependent conditions such as Cushing’s disease (pituitary source) or Cushing’s syndrome (ectopic source) and ACTH-independent conditions such as Cushing’s syndrome (adrenal source)Oral dose of 1 mg dexamethasone (Decadron) at 2300.Collect cortisol at 0800 on the morning after the dexamethasone dose
Metyrapone stimulation (overnight)Suspect hypothalamic/pituitary disease such as adrenal insufficiency, ACTH-dependent conditions such as Cushing’s disease (pituitary source) or Cushing’s syndrome (ectopic source), and ACTH-independent conditions such as Cushing’s syndrome (adrenal source)Oral dose of 30 mg/kg metyrapone with snack at 2400Collect cortisol, 11-deoxycortisol, and ACTH at 0800 on the morning after the metyrapone dose

Normal Findings:
(Method: Immunochemiluminescent assay for ACTH and cortisol; HPLC/MS-MS for 11-deoxycortisol)


AgeConventional UnitsSI Units (Conventional Units × 0.22)
Cord blood50–570 pg/mL11–125 pmol/L
Newborn10–185 pg/mL2–41 pmol/L
1 wk–9 yr5–46 pg/mL1.1–10.1 pmol/L
10–18 yr6–55 pg/mL1.3–12.1 pmol/L
19 yr–Adult
Male supine (specimen collected in morning)7–69 pg/mL1.5–15.2 pmol/L
Female supine (specimen collected in morning)6–58 pg/mL1.3–12.8 pmol/L
Values may be unchanged or slightly elevated in healthy older adults. Long-term use of corticosteroids, to treat arthritis and autoimmune diseases, may suppress secretion of ACTH.

ACTH Challenge Tests

ACTH (Cosyntropin) Stimulated, Rapid TestConventional UnitsSI Units (Conventional Units × 27.6)
BaselineCortisol greater than 5 mcg/dLGreater than 138 nmol/L
30- or 60-min responseCortisol 18–20 mcg/dL or incremental increase of 7 mcg/dL over baseline value497–552 nmol/L or incremental increase of 193.2 nmol/L over baseline value
Corticotropin-Releasing Hormone StimulatedConventional UnitsSI Units (Conventional Units × 27.6)
Cortisol peaks at greater than 20 mcg/dL within 30–60 minGreater than 552 nmol/L
SI Units (Conventional Units × 0.22)
ACTH increases twofold to fourfold within 30–60 minTwofold to fourfold increase within 30–60 min
Dexamethasone Suppressed Overnight TestConventional UnitsSI Units (Conventional Units × 27.6)
Cortisol less than 1.8 mcg/dL next dayLess than 49.7 nmol/L
Metyrapone Stimulated Overnight TestConventional UnitsSI Units (Conventional Units × 27.6)
Cortisol less than 3 mcg/dL next dayLess than 83 nmol/L
SI Units (Conventional Units × 0.22)
ACTH greater than 75 pg/mLGreater than 16.5 pmol/L
SI Units (Conventional Units × 28.9)
11-deoxycortisol greater than 7 mcg/dLGreater than 202 nmol/L


Hypothalamic-releasing factor stimulates the release of ACTH from the anterior pituitary gland. ACTH stimulates adrenal cortex secretion of glucocorticoids, androgens, and, to a lesser degree, mineralocorticoids. Cortisol is the major glucocorticoid secreted by the adrenal cortex. ACTH and cortisol test results are evaluated together because a change in one normally causes a change in the other. ACTH secretion is stimulated by insulin, metyrapone, and vasopressin It is decreased by dexamethasone. Cortisol excess from any source is termed Cushing’s syndrome. Cortisol excess resulting from ACTH excess produced by the pituitary is termed Cushing’s disease. ACTH levels exhibit a diurnal variation, peaking between 0600 and 0800 and reaching the lowest point between 1800 and 2300. Evening levels are generally one-half to two-thirds lower than morning levels. Cortisol levels also vary diurnally, with the peak values occurring during between 0600 and 0800 and reaching the lowest levels between 2000 and 2400. Specimens are typically collected at 0800 and 1600. This pattern may be reversed in individuals who sleep during daytime hours and are active during nighttime hours. Salivary cortisol levels are known to parallel blood levels and can be used to screen for Cushing’s disease and Cushing’s syndrome.

This procedure is contraindicated for

The metyrapone stimulation test is contraindicated in patients with suspected adrenal insufficiency because it may induce an acute adrenal crisis, a life-threatening condition, in patients whose adrenal function is already compromised.


  • Determine adequacy of replacement therapy in congenital adrenal hyperplasia.
  • Determine adrenocortical dysfunction.
  • Differentiate between increased ACTH release with decreased cortisol levels and decreased ACTH release with increased cortisol levels.

Potential Diagnosis

ACTH Result

Because ACTH and cortisol secretion exhibit diurnal variation with values being highest in the morning, a lack of change in values from morning to evening is clinically significant. Decreased concentrations of hormones secreted by the pituitary gland and its target organs are observed in hypopituitarism. In primary adrenal insufficiency (Addison’s disease), because of adrenal gland destruction by tumor, infectious process, or immune reaction, ACTH levels are elevated while cortisol levels are decreased. Both ACTH and cortisol levels are decreased in secondary adrenal insufficiency (i.e., secondary to pituitary insufficiency). Excess ACTH can be produced ectopically by various lung cancers such as oat-cell carcinoma and large-cell carcinoma of the lung and by benign bronchial carcinoid tumor

Challenge Tests and Results

The ACTH (cosyntropin) stimulated rapid test directly evaluates adrenal gland function and indirectly evaluates pituitary gland and hypothalamus function. Cosyntropin is a synthetic form of ACTH. A baseline cortisol level is collected before the injection of cosyntropin. Specimens are subsequently collected at 30- and 60-min intervals. If the adrenal glands function normally, cortisol levels rise significantly after administration of cosyntropin.

The CRH stimulation test works as well as the dexamethasone suppression test (DST) in distinguishing Cushing’s disease from conditions in which ACTH is secreted ectopically (e.g., tumors not located in the pituitary gland that secrete ACTH). Patients with pituitary tumors tend to respond to CRH stimulation, whereas those with ectopic tumors do not. Patients with adrenal insufficiency demonstrate one of three patterns depending on the underlying cause:

  • Primary adrenal insufficiency—high baseline ACTH (in response to IV-administered ACTH) and low cortisol levels pre- and post-IV ACTH.
  • Secondary adrenal insufficiency (pituitary)—low baseline ACTH that does not respond to ACTH stimulation. Cortisol levels do not increase after stimulation.
  • Tertiary adrenal insufficiency (hypothalamic)—low baseline ACTH with an exaggerated and prolonged response to stimulation. Cortisol levels usually do not reach 20 mcg/dL (SI = 552 nmol/L).

(The DST is useful in differentiating the causes of increased cortisol levels. Dexamethasone is a synthetic glucocorticoid that is significantly more potent than cortisol. It works by negative feedback. It suppresses the release of ACTH in patients with a normal hypothalamus. A cortisol level less than 1.8 mcg/dL (SI = 49.7 nmol/L) usually excludes Cushing’s syndrome. With the DST, a baseline morning cortisol level is collected, and the patient is given a 1-mg dose of dexamethasone at bedtime. A second specimen is collected the following morning. If cortisol levels have not been suppressed, adrenal adenoma is suspected. The DST also produces abnormal results in the presence of certain psychiatric illnesses [e.g., endogenous depression]).

The metyrapone stimulation test is used to distinguish corticotropin-dependent causes (pituitary Cushing’s disease and ectopic Cushing’s disease) from corticotropin-independent causes (e.g., carcinoma of the lung or thyroid) of increased cortisol levels. Metyrapone inhibits the conversion of 11-deoxycortisol to cortisol. Cortisol levels should decrease to less than 3 mcg/dL if normal pituitary stimulation by ACTH occurs after an oral dose of metyrapone. Specimen collection and administration of the medication are performed as with the overnight dexamethasone test.

Increased In:

Overproduction of ACTH can occur as a direct result of either disease (e.g., primary or ectopic tumor that secretes ACTH) or stimulation by physical or emotional stress, or it can be an indirect response to abnormalities in the complex feedback mechanisms involving the pituitary gland, hypothalamus, or adrenal glands.

ACTH Increased in

  • Addison’s disease (primary adrenocortical hypofunction)
  • Carcinoid syndrome
  • Congenital adrenal hyperplasia
  • Cushing’s disease (pituitary-dependent adrenal hyperplasia)
  • Cushing’s syndrome (ectopic secretion of ACTH)
  • Depression
  • Ectopic ACTH-producing tumors
  • Menstruation
  • Nelson’s syndrome (ACTH-producing pituitary tumors)
  • Pregnancy
  • Sepsis
  • Septic shock
  • Type 2 diabetes

Decreased In:

Secondary adrenal insufficiency due to hypopituitarism (inadequate production by the pituitary) can result in decreased levels of ACTH. Conditions that result in overproduction or availability of high levels of cortisol can also result in decreased levels of ACTH.

ACTH Decreased in

  • Adrenal adenoma
  • Adrenal cancer
  • Cushing’s syndrome
  • Exogenous steroid therapy

Summary of the Relationship Between Cortisol and ACTH Levels in Conditions Affecting the Adrenal and Pituitary Glands
DiseaseCortisol LevelACTH Level
Addison’s disease (adrenal insufficiency)DecreasedIncreased
Cushing’s disease (pituitary adenoma)IncreasedIncreased
Cushing’s syndrome related to ectopic source of ACTHIncreasedIncreased
Cushing’s syndrome (ACTH independent; adrenal cancer or adenoma)IncreasedDecreased
Congenital adrenal hyperplasiaDecreasedIncreased

Critical Findings


Interfering Factors

  • Drugs and other substances that may increase ACTH levels include insulin, metoclopramide, metyrapone, mifepristone (RU 486), spironolactone, and vasopressin
  • Drugs and other substances that may decrease ACTH levels include corticosteroids (e.g., dexamethasone) and pravastatin
  • Test results are affected by the time the test is done because ACTH levels vary diurnally, with the highest values occurring between 0600 and 0800 and the lowest values occurring at night. Samples should be collected at the same time of day, between 0600 and 0800.
  • Excessive physical activity can produce elevated levels.
  • Metyrapone may cause gastrointestinal distress and/or confusion. Administer oral dose of metyrapone with milk and snack.
  • Rapid clearance of metyrapone, resulting in falsely increased cortisol levels, may occur if the patient is taking drugs that enhance steroid metabolism (e.g., phenytoin, rifampin, phenobarbital, mitotane, and corticosteroids). The requesting health-care provider (HCP) should be consulted prior to a metyrapone stimulation test regarding a decision to withhold these medications.

Nursing Implications Procedure

Related Studies

  • Related tests include cortisol and challenge tests, CT abdomen, CT pituitary, MRI abdomen, MRI pituitary, TSH, thyroxine, and US abdomen.
  • See the Endocrine System table online at DavisPlus for related tests by body system.

Potential Nursing Problems

ProblemSigns & SymptomsInterventions
Fluid volume (water) (related to loss of water secondary to vomiting; diarrhea)Deficient: Hypotension; decreased cardiac output; decreased urinary output; dry skin/mucous membranes; poor skin turgor; sunken eyeballs; increased urine-specific gravity; hemoconcentrationMonitor intake and output; assess for symptoms of dehydration (dry skin, dry mucous membranes, poor skin turgor, sunken eyeballs); monitor and trend vital signs; monitor for symptoms of poor cardiac output (rapid, weak, thready pulse); monitor and trend daily weight; collaborate with HCP regarding administration of IV fluids to support hydration; monitor laboratory values that reflect alterations in fluid status (potassium, blood urea nitrogen, creatinine, calcium, hemoglobin, and hematocrit, sodium); manage underlying cause of fluid alteration; monitor urine characteristics and respiratory status; establish baseline assessment data; collaborate with HCP to adjust oral and IV fluids to provide optimal hydration status; administer replacement electrolytes, as ordered; adjust diuretics, as appropriate
Infection risk (related to impaired immune response secondary to elevated cortisol level)Delayed wound healing; inhibited collagen formation; impaired blood flow to edematous tissues; symptoms of infection (temperature; increased heart rate; increased blood pressure; shaking; chills; mottled skin; lethargy; fatigue; swelling; edema; pain; localized pressure; diaphoresis; night sweats; confusion; vomiting; nausea; headache)Decrease exposure to environment by placing the patient in a private room; monitor and trend vital signs; monitor and trend laboratory values that would indicate an infection (WBC, CRP); promote good hygiene; assist with hygiene, as needed; administer prescribed antibiotics, antipyretics; use cooling measures; administer prescribed IV fluids; monitor vital signs and trend temperatures; encourage oral fluids; adhere to standard or universal precautions; isolate as appropriate; obtain cultures, as ordered; encourage lightweight clothing and bedding
Injury risk (related to poor wound healing; decreased bone density; capillary fragility)Easy bruising; blood in stool; skin breakdown; fracture; poor wound healingAssess for bruising; assess stool for occult blood; assess for skin breakdown; assess wound for healing progress; facilitate ordered bone density screening


  • Positively identify the patient using at least two person-specific identifiers before services, treatments, or procedures are performed.
  • Patient Teaching: Inform the patient this test can assist in evaluating the amount of hormone produced by the pituitary gland located at the base of the brain.
  • Obtain a history of the patient’s health concerns, symptoms, surgical procedures, and results of previously performed laboratory and diagnostic studies. Include a list of known allergens, especially allergies or sensitivities to latex.
  • Note any recent procedures that can interfere with test results.
  • Obtain a list of the patient’s current medications, including over-the-counter medications and dietary supplements (see Effects of Dietary Supplements online at DavisPlus).
  • Weigh patient and report weight to pharmacy for dosing of metyrapone (30 mg/kg body weight to a maximum dose of 3 g).
  • Review the procedure with the patient. When ACTH hypersecretion is suspected, a second sample may be requested between 0600 and 0800 to determine if changes are the result of diurnal variation in ACTH levels. Inform the patient that more than one sample may be necessary to ensure accurate results, and samples are obtained at specific times to determine high and low levels of ACTH. Inform the patient that each specimen collection takes approximately 5 to 10 min. Address concerns about pain, and explain that there may be some discomfort during the venipuncture.
  • Sensitivity to social and cultural issues, as well as concern for modesty, is important in providing psychological support before, during, and after the procedure.
  • Note that there are no food, fluid, or medication restrictions unless by medical direction.
  • Drugs that enhance steroid metabolism may be withheld by medical direction prior to metyrapone stimulation testing.
  • Instruct the patient to refrain from strenuous exercise for 12 hr before the test and to remain in bed or at rest for 1 hr immediately before the test. Avoid smoking and alcohol use.
  • Prepare an ice slurry in a cup or plastic bag to have on hand for immediate transport of the specimen to the laboratory.


Potential Complications:

Adverse reactions to metyrapone include nausea and vomiting (N/V), abdominal pain, headache, dizziness, sedation, allergic rash, decreased white blood cell (WBC) count, and bone marrow depression. Signs and symptoms of overdose or acute adrenocortical insufficiency include cardiac dysrhythmias, hypotension, dehydration, anxiety, confusion, weakness, impairment of consciousness, N/V, epigastric pain, diarrhea, hyponatremia, and hyperkalemia.

  • Ensure that strenuous exercise was avoided for 12 hr before the test and that 1 hr of bed rest was taken immediately before the test. Samples should be collected between 0600 and 0800.
  • Have emergency equipment readily available in case of adverse reaction to metyrapone.
  • Avoid the use of equipment containing latex if the patient has a history of allergic reaction to latex.
  • Instruct the patient to cooperate fully and to follow directions. Direct the patient to breathe normally and to avoid unnecessary movement.
  • Observe standard precautions, and follow the general guidelines in Patient Preparation and Specimen Collection online at DavisPlus. Positively identify the patient, and label the appropriate tubes with the corresponding patient demographics, date, and time of collection. Perform a venipuncture; collect the specimen in a prechilled plastic heparinized syringe or in prechilled collection containers, as listed under the “Specimen” subheading.
  • Remove the needle and apply direct pressure with dry gauze to stop bleeding. Observe/assess venipuncture site for bleeding or hematoma formation, and secure gauze with adhesive bandage.
  • Promptly transport the specimen to the laboratory for processing and analysis. The tightly capped sample should be placed in an ice slurry immediately after collection. Information on the specimen label should be protected from water in the ice slurry by first placing the specimen in a protective plastic bag.

Post Test

  • Inform the patient that a report of the results will be made available to the requesting HCP, who will discuss the results with the patient.
  • Recognize anxiety related to test results, and offer support.
  • Observe/assess the patient who has been administered metyrapone for signs and symptoms of an acute adrenal (addisonian) crisis, which may include abdominal pain, N/V, hypotension, tachycardia, tachypnea, dehydration, excessively increased perspiration of the face and hands, sudden and significant fatigue or weakness, confusion, loss of consciousness, shock, coma. Potential interventions include immediate corticosteroid replacement (IV or IM), airway protection and maintenance, administration of dextrose for hypoglycemia, correction of electrolyte imbalance, and rehydration with IV fluids.
  • Depending on the results of this procedure, additional testing may be performed to evaluate or monitor progression of the disease process and determine the need for a change in therapy. If a diagnosis of Cushing’s disease is made, pituitary computed tomography (CT) or magnetic resonance imaging (MRI) may be indicated prior to surgery. If a diagnosis of ectopic corticotropin syndrome is made, abdominal CT or MRI may be indicated prior to surgery. Evaluate test results in relation to the patient’s symptoms and other tests performed.

Patient Education:

  • Instruct the patient to resume normal activity as directed by the HCP.
  • Provide contact information, if desired, for the Cushing’s Support and Research Foundation (www.csrf.net).
  • Reinforce information given by the patient’s HCP regarding further testing, treatment, or referral to another HCP.
  • Answer any questions or address any concerns voiced by the patient or family.
  • Teach the patient and family the effects of the disease process and associated treatments.

Expected Patient Outcomes:


  • The patient and family state the importance of compliance with the recommended therapeutic regime to health maintenance.
  • The patient and family state the necessity of altering the medication regime during times of illness and stress.

  • The patient and family demonstrate proficiency in the administration of prescribed steroids.
  • The patient complies with the request to stand slowly to prevent orthostatic hypotension.

  • The patient complies with the HCP’s request to wear a medic alert bracelet indicating adrenal insufficiency and steroid use.
  • The patient and family comply with the HCP’s request to increase oral fluid intake with a diet high in sodium and low in potassium (Addison’s disease).

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