Adrenocortical Hormone Tests

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Adrenocortical Hormone Tests


Adrenocortical hormone tests measure levels of aldosterone and cortisol (also known as hydrocortisone) in blood and urine. These hormones are synthesized from cholesterol by the action of several enzymes. The cells responsible are located in the outer part (cortex) of the adrenal glands, two small

Normal findings for aldosterone assay
Source: Pagana, K.D. and T.J. Pagana. Mosby's Diagnostic and Laboratory Test Reference. 3rd ed. St. Louis: Mosby, 1997.
Blood test
Supine3-10 ng/dl (0.08-0.30 nmol/L)
Adult female5-30 ng/dl (0.14-0.80 nmol/L)
Adult male6-22 ng/dl (0.17-0.61 nmol/L)
Newborn5-60 ng/dl
1 week-1 year1-160 ng dl
1-3 years5-60 ng/dl
3-5 years<5-80 ng/dl
5-7 year<5-50 ng/dl
7-11 years5-70 ng/dl
11-15 years<5-50 ng/dl
24-hour urine test2-80 μg/24 hr (5.5-72.0 nmol/24 hr)

organs, one sitting just above each kidney. As steroid hormones, they are an important part of the body's endocrine system and help to regulate body functions. The adrenal cortex also produces many other steroid hormones including androgens, primarily dehydroepiandrosterone (DHEA) and androstenedione, that are converted to testosterone and dihydrotestosterone.

Aldosterone, a mineralcorticoid, helps regulate the amounts of sodium and potassium in the blood and, because fluid follows sodium, helps maintain fluid balance and blood volume, which, in turn, affect blood pressure.

Cortisol, a glucocorticoid, helps regulate the metabolism of proteins, fats, and carbohydrates, especially glucose (sugar). Specifically, cortisol helps convert amino acids (subunits of proteins) into glucose within the liver. It raises blood sugar levels by stimulating the release of glucose from cellular glucose stores and simultaneously acts to inhibit insulin, which moderates glucose transport into the cells. Cortisol also has a number of anti-inflammatory effects, including suppressing the immune system and reducing fever.


Aldosterone measurement is used to detect aldosteronism (hyperaldosteronism), which is the excess secretion of the hormone by the adrenal glands. It is also used to detect those cases of hypoaldosteronism that occur in the absence of a low cortisol.

The cortisol test is performed on patients who are suspected to have malfunctioning adrenal glands. It is considered to be the best indicator of adrenal activity. Blood and urine cortisol measurements, together with the determination of ACTH levels, are the three most important tests in the investigation of Cushing's syndrome (overproduction of cortisol) and Addison's disease (underproduction of cortisol). Additional tests called simulation and suppression tests may be needed to detect disease in difficult cases, or to distinguish between the causes.


Adrenocorticol function tests may be done on serum or urine. The nurse or phletotomist performing venipuncture should observe universal precautions for the prevention of transmission of bloodborne pathogens. Cortisol levels in blood are subject to diurnal variation. Test results must be evaluated with regard to the time of day the blood was collected. Physicians will determine if patients whose fluid balance may already be compromised by illness can undergo 24-hour urine tests. Physicians will also determine if patients can discontinue taking drugs that control sodium and potassium levels and fluid balance prior to testing. These tests are sometimes performed by a method called radioimmunoassay (RIA) that tags test reagents with radioactive iodide. RIA based tests may be contraindicated for patients who have had recent procedures using radiation or who have had radioactive drugs administered, because results may be altered and unreliable.



Aldosterone is produced by the outer area of the adrenal cortex called the zona glomerulosa. Aldosteronism, an increased secretion of aldosterone, can be primary (a malfunction in the adrenal glands or in aldosterone secretion itself) or secondary (caused by another condition). Excessive aldosterone production results in the retention of sodium, and subsequently in fluid retention and high blood pressure (hypertension). Because potassium can be lost in this process, muscle weakness is also a frequent symptom. Primary aldosteronism is most often caused by an adrenal tumor (adenoma) a condition referred to as Conn's syndrome. Primary aldosteronism can also be idiopathic (of unknown origin) or congenital. Secondary aldosteronism is more common and occurs as a consequence of non-adrenal conditions characterized by severe imbalances in sodium and potassium with resulting fluid imbalance, such as congestive heart failure, cirrhosis (liver disease) with fluid in the abdominal cavity (ascites), certain kidney diseases, excess potassium (hyperkalemia), a sodium-depleted diet, and the toxemia of pregnancy. To differentiate primary aldosteronism from secondary aldosteronism, a plasma renin test should be performed at the same time as the aldosterone assay. Renin, an enzyme produced in the kidneys, is typically elevated in secondary aldosteronism and reduced in primary aldosteronism.

Hypoaldosteronism is characterized by low serum sodium, dehydration, and high serum potassium (hyperkalemia). Hypoaldosteronism can occur in Addison's disease which usually is caused by autoimmune damage to the adrenal cortex. Addison's disease is marked by decreased glucocorticoids and is detected by tests for deficient cortisol production. However, hypoaldosteronism may also occur in the absence of low glucocorticoids. This condition may result from decreased renin production by the kidney, heparin treatment, and a deficiency of an enzyme needed to produce aldosterone. In such cases, serum aldosterone and plasma renin activity must be measured to establish the diagnosis.


Cortisol and the other glucocorticoid hormones are produced in the zona fasciculata and reticularis of the cortex. Overproduction of glucocorticoids (cortisol) by the adrenal cortex is called Cushing's syndrome. Excessive cortisol production leads to high blood glucose levels, sodium retention, obesity, and excessive hair growth. Because cortisol production can suppress the immune system, excess levels may also cause chronic infection in some patients.

The release of cortisol is controlled by circulating levels of a pituitary hormone, adrenocorticotropic hormone (ACTH). In a complex process, the hypothalamus manufactures corticotropin-releasing hormone (CRH), which, in turn, stimulates the pituitary gland to produce ACTH. While ACTH stimulates the adrenal glands to produce cortisol, rising levels of cortisol act by negative feedback to inhibit further production of CRH and ACTH. Disturbances in this elaborate feedback mechanism can be caused by certain types of stress, such as physical trauma, infection, extreme heat or cold, exhaustion from strenuous exercise, and extreme anxiety. When normal feedback becomes uncoordinated as a result of one of these conditions, excessive amounts of ACTH and, in turn, cortisol will be released. For this reason, cortisol levels in blood can vary considerably with time, a phenomenon called pulse variation.

Cushing's syndrome has four causes. The majority of cases result from the use of cortisol and related compounds for immunosuppression and treatment of inflammatory diseases. This is called iatrogenic hypercortisolism. The next most prevalent cause is Cushing's disease, also called pituitary Cushing's. This results from adenomas of the pituitary gland that secrete ACTH. Less commonly, Cushing's syndrome is caused by adrenal adenoma or carcinoma or by ACTH secreting tumors located outside the pituitary (ectopic ACTH).

Primary Addison's disease results from damage to the adrenal cortex. The most common causes are autoimmune disease and infection. Secondary Addison's disease is caused by a pituitary deficiency of ACTH. These two conditions can often be differentiated by measuring both cortisol and ACTH. In primary Addison's disease the ACTH will usually be increased because the functional pituitary gland is responding to low blood levels of cortisol. In secondary Addison's disease, both cortisol and ACTH levels will be low.


Cortisol is routinely measured by most laboratories. The most common method used is enzyme immunoassay. Aldosterone levels are far lower and require a more sensitive method, typically chemiluminescent immunoassay or radioimmunoassay. Measurement of cortisol intermediates such as 11-dexoycortisol and 17-hydroxyprogesterone are used in conjunction with cortisol for the diagnosis of congenital adrenal hyperplasia. In this condition, an enzyme deficiency in the synthetic pathway of cortisol leads to low cortisol and accumulation of one or more steroid intermediates above the block. These compounds are measured by RIA. ACTH is typically measured by RIA because of its low concentration in plasma. Plasma renin is usually measured by determining the activity of the enzyme (renin activity) rather than by immunoassay of the hormone mass because the latter method measures both the active and inactive forms. In the assay, renin acts on angiotensinogen to produce angiotensin I, which is then measured by RIA.

Adrenocortical hormone tests are typically performed on blood plasma or 24-hour urine samples. When performed on blood, physicians may request that tests be performed on two samples, each drawn at a different time, to account for fluctuations in hormone levels at different times during the day (diurnal variation).

Tests for aldosterone are performed on blood plasma or on a 24-hour urine specimen. Levels of aldosterone peak in early morning and are at half that level by afternoon, making the time of drawing a blood sample significant. To help ensure a more reliable evaluation, two samples may be drawn for testing, one in early morning (8 am) and one midafternoon (4 pm). Diet and posture (upright or lying down) may also cause aldosterone levels to fluctuate, so that a single blood sample may miss increased or decreased levels and may not accurately reflect hormone production. Because a 24-hour urine specimen reflects hormone production over an entire day, it will usually provide a more reliable aldosterone measurement. Elevated blood levels should ideally be confirmed with a 24-hour urine test.

Since posture and body position affect aldosterone production, hospitalized patients should remain in an upright position (at least sitting) for two hours before blood is drawn. Occasionally blood will be drawn before and after the patient gets out of bed. Nonhospitalized patients (outpatients) should arrive at the laboratory in time to maintain an upright position for at least two hours.

Cortisol can be measured in both blood plasma and urine. Blood levels of cortisol are most reliably measured in the morning at 8 am, in the afternoon at 4 pm, or in the evening at 10 pm. Levels of cortisol normally peak in the morning and decline gradually during the day, reaching the lowest level in the early hours of the next day. It is important to note, however, that when the normal cycle of activity and sleep (circadian rhythm) is reversed, as in night-shift work, cortisol levels will reverse also. Physicians often order blood plasma cortisol rather than a 24-hour urine collection because the earliest sign of adrenal malfunction is sometimes the loss of diurnal variation, even though cortisol levels are not yet elevated. For example, individuals with Cushing's syndrome often exhibit peak plasma cortisol levels in the morning with no decline as the day progresses. Cortisol levels may also be elevated during pregnancy, in the presence of physical and emotional stress, and with the administration of certain drugs. A 24-hour urine test for cortisol will reflect cortisol production over the entire period of collection.

Cortisol is bound in the plasma by cortisol binding protein (transcortin) and other proteins such as albumin. Very little of the plasma cortisol is free, but when cortisol is produced in excess, the binding proteins become saturated, and excess free cortisol is excreted in the urine. Measurement of 24-hour urinary free cortisol is not influenced by serum binding protein levels and is not subject to the diurnal variation (pulse variation) seen with serum total cortisol measurements. Therefore, it is somewhat more sensitive than measurement of total serum cortisol in detecting persons with Cushing's syndrome.

Both aldosterone and cortisol (and other hormones, such as adrenomedullary hormones) are sometimes measured with blood samples from the adrenal veins, particularly when the existence of a tumor is suspected and surgery may be an option. This procedure, called adrenal venography, is performed by an angiographer in the radiology department, although the cortisol test is still performed in the clinical laboratory. Testing adrenal vein samples helps confirm the presence of adrenal tumors by allowing blood to be drawn through a catheter from the right and left adrenal glands. High levels on one side will confirm the presence of a tumor on that side. Normal levels on both sides indicates a non-adrenal source for the patient's condition. This is a specialized rather than a standard sample collection procedure and preparation for the procedure is not discussed below.

Adrenocorticotropic hormone (ACTH) test

Although difficult to measure, ACTH aids in the differential diagnosis of the cause of Cushing's syndrome and in distinguishing primary from secondary Addison's disease. Approximately half of persons with Cushing's disease (pituitary Cushing's) have a normal ACTH level and half will have an elevated level. Most persons with adrenal tumors will have low (less than 10 picograms/L) or undetectable ACTH in the plasma owing suppression by cortisol. Most persons with ectopic ACTH secreting tumors will have elevated levels in excess of 200 pg/L. Persons with primary Addison's disease will usually have high ACTH levels (greater than 150 picrograms/L) caused by negative feedback (low cortisol) while those with secondary Addison's disease will have low or normal ACTH levels owing to pituitary failure or hypothalmic suppression.

Stimulation and suppression tests

Because measurement of cortisol may not be definitive for the diagnosis of Cushing's syndrome or Addison's disease, several stimulation and suppression tests are available. Some of these tests are used to establish a diagnosis while others are used to help distinguish the cause of the disease. The most commonly used suppression test for Cushing's syndrome is the dexamethasone suppression test. Dexamethasone is a powerful synthetic analog of cortisol and will normally inhibit the pituitary release of ACTH, and thus suppress cortisol secretion. In the low dose overnight dexamethasone suppression test 1 mg of dexamethasone is given orally at midnight and the 8 am cortisol is measured. The cortisol level is suppressed in normals and will be less than 5 micrograms/dL. Persons with Cushing's syndrome will usually show no suppression and the plasma cortisol will be 10 micrograms/dL or higher. In the high dose overnight dexamethasone suppression test the patient is given 8 mg (other doses are sometimes used) of dexamethasone at midnight. A blood sample is collected at 8 am and cortisol is measured. Persons with Cushing's disease show a suppression in cortisol production of at least 50% of their baseline (i.e., the initial cortisol level without the drug). Persons with adenoma or ectopic ACTH tumors remain unsuppressed. In addition to dexamethasone suppression tests, metyrapone stimulation tests and corticotropin releasing hormone (CRH) stimulation tests are sometimes used for difficult cases. The former measures the ACTH reserve of the pituitary gland and the latter measures the ACTH response to hypothalmic stimulation. Patients with Cushing's disease (pituitary Cushing's) usually have an exaggerated response (increased ACTH) to both tests. Persons with adenoma or ectopic ACTH tumors do not respond with increases over the baseline in either test.

A sensitive screening test for Addison's disease is the rapid Cosyntropin stimulation test. The patient's 8 am cortisol is measured and followed by intravenous administration of 250 micrograms of synthetic ACTH. Plasma cortisol is measured at 30 and 60 minutes after administration of the drug. Normal individuals produce a twofold increase in the baseline ACTH level while persons with either primary or secondary Addison's disease show a subnormal response.


Fasting is not required for aldosterone blood or urine tests. The patient should maintain a normal sodium diet (approximately 3 g/day) for at least two weeks before either test. The testing physician should decide if drugs that alter sodium, potassium, and fluid balance (e.g., diuretics, antihypertensives, steroids, oral contraceptives) should be withheld. The test will be more accurate if these are discontinued for a period of at least two weeks before the test. Renin inhibitors (e.g., propranolol) should not be taken one week before the test, unless permitted by the physician. The patient should avoid licorice for at least two weeks before the test, because of its aldosterone-like effect. Because strenuous exercise and stress can increase aldosterone levels, these should be avoided as well. Two blood samples are often drawn for aldosterone evaluation, one in the early morning and one mid-afternoon.

Fasting is not required for cortisol blood or urine tests, although cortisol levels may increase after a meal and patients should not eat for at least a two-hour period prior to testing. For cortisol tests on blood or urine, the testing physician may discontinue the patient's regular medications for up to two weeks prior to testing. Drugs that may increase levels of cortisol include estrogen, oral contraceptives, amphetamines, cortisone, and spironolactone (Aldactone). Drugs that may decrease cortisol levels include androgens, aminoglutethimide, betamethasone, and other steroid medications, as well as danazol, lithium, levodopa, metyrapone and phenytoin (Dilantin). The patient may eat a normal diet but must not eat for at least two hours before blood samples are drawn, since cortisol levels may rise after meals. Two blood samples are usually drawn for cortisol evaluation, one in the early morning and one mid-afternoon.

The clinical laboratory will provide instructions and containers for patients collecting their own 24-hour urine samples for the measurement of aldosterone or cortisol. Patients should be advised to avoid touching the preservative, if any, in the collection container. If the preservative comes in contact with the skin, the area should be rinsed with water immediately. Collection should begin in the morning on any day except Friday or Saturday to ensure that samples will be delivered to the laboratory on a work day. Urine samples must be refrigerated continuously in the patient's home or in the nursing facility. The specimen should be delivered to the laboratory promptly at the end of the 24-hour period.


Following venipuncture for blood plasma hormone tests, the nurse or phlebotomist drawing the sample should inspect the venipuncture site to make sure that the wound has closed and no bleeding is present. The site should be covered with an adhesive bandage. There is no notable aftercare for patients undergoing 24-hour urine hormone tests. Patients can be reminded to resume foods and medications (according to the physician's orders) that were restricted prior to testing.


Complications for blood hormone tests are minimal, but may include slight bleeding from the venipuncture site, fainting or feeling lightheaded after venipuncture, or the development of a hematoma (blood accumulating under the puncture site). There are no complications for the urine test, although patients should be instructed to drink clear fluids regularly to maintain fluid balance while collecting the 24-hour urine specimen. Adrenal venography used to obtain adrenal vein samples has many potential complications, including allergic reactions to iodinated dye used in the procedure, adrenal hemorrhage, blood clots, and infection. This procedure is usually reserved for pre-surgical patients with suspected adrenal tumors and is not discussed here in detail.



Normal results for the aldosterone assay are laboratory-specific and vary according to test methodology used and the source of the specimen. Blood plasma levels, urine levels, and adrenal vein levels will be different. Results will also vary between patients depending upon average sodium intake, time of day, source of specimen, age, sex, and posture. Reference ranges are shown below for radioimmunoassay, the most common assay method for aldosterone.

Reference ranges for blood plasma levels:

  • Supine (lying down): 3-10 ng/dL.
  • Upright (sitting for at least two hours): Female: 5-30ng/dL; Male: 6-22 ng/dL.

Reference ranges for urine: 2-80 micrograms/24 hr.

Increased levels of aldosterone are found in Conn's disease (aldosterone-producing adrenal tumor), and in Bartter's syndrome (overexcretion of potassium, sodium, and chloride by the kidneys resulting in low blood levels of potassium and high blood levels of aldosterone and renin). Elevated levels are also seen in secondary aldosteronism (in primary conditions such as congestive heart failure, cirrhosis of the liver, certain kidney diseases, hyperkalemia, a sodiumdepleted diet, and toxemia of pregnancy), stress, and malignant hypertension.

Decreased levels of aldosterone are found in aldosterone deficiency, steroid therapy, high-sodium diets, certain antihypertensive therapies, and Addison's disease (an autoimmune disorder also involving abnormal cortisol and ACTH levels).


Normal results for the cortisol assay are laboratory-specific and vary according to the test methodology used and the source of the specimen. Blood serum levels, urine levels, and adrenal vein levels will be different.

Reference ranges for blood serum levels:

  • Adults (8 a.m.): 6-28 micrograms/dL; adults (4 p.m.): 2-12 mcg/dL.
  • Child 1-6 years (8 a.m.): 3-21 mcg/dL; child 1-6 years (4 p.m.): 3-10 mcg/dL.
  • Newborn: 1/24 mcg/dL.

Reference ranges for urine (free cortisol):

  • Adult: 10-100 micrograms/24 hr.
  • Adolescent: 5-55 mcg/24 hr.
  • Child: 2-27 mcg/24 hr.

Increased levels of cortisol are found in Cushing's syndrome, hyperthyroidism (excess thyroid activity), obesity, ACTH-producing tumors, pregnancy, and high levels of physical or emotional stress.

Decreased levels of cortisol are found in Addison's disease, conditions of hypothyroidism (reduced thyroid activity), and hypopituitarism (diminished pituitary activity).

Drugs that may increase the levels of cortisol include estrogen, oral contraceptives, amphetamines, cortisone, and spironolactone (Aldactone). Drugs that may decrease the levels of cortisol include androgens, aminoglutethimide, betamethasone, other steroid medications, danazol, lithium, levodopa, metyrapone and phenytoin (Dilantin).

Health care team roles

The testing physician will typically instruct the patient about preparatory steps for adrenocortical hormone tests, making recommendations for diet and for discontinuing medications. A nurse or phlebotomist will perform a venipuncture to obtain blood samples. The phlebotomist or nurse should mark the laboratory slip with the time each blood sample was drawn and indicate any medications the patient may be taking. Laboratories will provide containers for urine collection and should help ensure compliance by providing written instructions for 24-hour urine collection. Laboratory technologists or nursing staff interacting with the patient may gently question the patient at the time of the blood test or urine collection to make sure they have discontinued medications prior to the test according to their doctor's instructions. When the 24-hour urine collection is completed, health care providers may remind the patient to resume medications if the testing physician has instructed them to do so. Measurements of adrenal hormones are performed by clinical laboratory scientists/medical technologists. Results are interpreted by a physician. In most cases, an endocrinologist is usually consulted.

Patient education

Patients must be instructed regarding changes in medications or diet, sources of sodium in the diet, and maintaining an upright position prior to testing. Outpatients who will be collecting their urine specimens at home should have written instructions for the 24-hour urine collection. Health care providers should also encourage the patient to drink clear fluids regularly throughout the urine collection process.


Laboratory technologists performing adrenocortical hormone tests will have studied biochemistry, enabling them to understand the role of these hormones in the body and the chemical basis for expression of the hormones in body fluids. Hands-on clinical laboratory training will prepare technologists to perform radioimmunoassay or other methods of measuring aldosterone and cortisol in blood and urine. Nursing personnel responsible for patients undergoing hormone testing for will understand the critical nature of preparation for these tests and help to encourage patient compliance. They will note any areas of noncompliance and inform the testing physician. Laboratory and nursing staff will be responsible for noting abnormal results and calling them to the attention of physicians.


Adrenal glands— The adrenal glands are paired triangular structures located immediately above and loosely attached to each kidney. The inner portion is called the adrenal medulla and the outer portion is called the adrenal cortex.

Adrenocortical hormones— Adrenocortical hormones are steroid hormones produced and released by the adrenal cortex. They are important in regulating certain body functions, especially fluid balance, blood pressure, and fat metabolism.

Adrenocorticotrophic hormone (ACTH)— ACTH is a pituitary hormone that directly controls the rate of release of cortisol and is also needed to maintain production of aldosterone by the adrenal glands.

Aldosterone— Aldosterone is a steroid hormone released by the adrenal cortex. It helps balance fluid levels and blood pressure by regulating the amounts of sodium and potassium in the blood.

Corticotropin-releasing hormone (CRH)— CRH is produced by the hypothalamus and, in a complex process, serves to stimulate the pituitary gland to produce ACTH.

Cortisol— Cortisol is a steroid hormone released by the adrenal cortex in response to stimulation by ACTH. It helps regulate the release of amino acids from muscles and convert them to glucose for use as energy. It releases fatty acids from body tissue for use as energy by muscles, allowing circulating glucose to be reserved for use by the brain.

Glucocorticoid— A glucocorticoid (cortisol) is released by the adrenal cortex to help regulate the release and utilization of glucose.

Hyperaldosteronism— Hyperaldosteronism, known also as aldosteronism, is the excess secretion of the hormone aldosterone by the adrenal glands. It may be caused by an adrenal tumor or may occur secondary to non-adrenal conditions such as congestive heart failure, cirrhosis of the liver, certain kidney diseases, excess potassium, a sodium-depleted diet, and toxemia of pregnancy.

Mineralcorticoid— A mineralcorticoid (aldosterone) is released by the adrenal cortex to help regulate mineral levels in the blood, especially sodium and potasium.



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Pagana, Kathleen D. and Timothy J. Manual of Diagnostic and Laboratory Tests. St. Louis: Mosby, Inc., 1998.


American Nurses Association. 600 Maryland Ave. SW, Ste. 100 West, Washington, DC 20024. (800)274-4ANA. 〈〉.

American Society of Clinical Pathologists. 2100 West Harrison St., Chicago, IL 60612 (312) 738-1336. 〈〉.


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"Adrenal Cortical Hypofunction." Merck Manual Online, 17th ed. Whitehouse Station, NJ.: Merck & Co., Inc. 2001. 〈〉.