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ACTH stimulation test

The ACTH stimulation test, also known as the cosyntropin stimulation test, is a dynamic endocrine evaluation that assesses the adrenal glands' ability to produce in response to synthetic (ACTH), aiding in the diagnosis of (AI). This test is particularly valuable for confirming primary AI (due to adrenal gland damage), secondary AI (due to pituitary dysfunction), or tertiary AI (due to hypothalamic issues), as well as evaluating non-classic in select cases. is a potentially life-threatening condition characterized by inadequate production, which can lead to symptoms such as , , and imbalances if untreated. The standard procedure involves administering 250 micrograms (mcg) of cosyntropin—a synthetic analog of ACTH—via or injection, followed by serial measurements of serum levels. Baseline is drawn immediately before administration, with additional samples collected at 30 and 60 minutes post-injection to capture the peak response; the test is typically performed in the morning (between 6-9 AM) when endogenous levels are highest. Preparation may include withholding certain medications like corticosteroids that could suppress the axis, though recent use is a relative due to risk of false results. The test is generally safe, with rare risks including allergic reactions to cosyntropin or transient facial flushing. Interpretation relies on the post-stimulation : a value of 18 mcg/dL (500 nmol/L) or higher effectively excludes , while a below 14 mcg/dL (390 nmol/L) confirms the , with intermediate values (14-18 mcg/dL) requiring clinical correlation and possibly additional testing like (DHEAS) levels. Plasma ACTH levels measured alongside can differentiate primary (elevated ACTH) from central causes (low or normal ACTH). Although a low-dose variant (1 mcg cosyntropin) has been proposed for detecting subtle secondary , it is not routinely recommended due to issues with availability, precision, and validation. Overall, the ACTH stimulation test serves as the gold standard for when baseline is indeterminate, guiding timely replacement therapy to prevent .

Background and Purpose

Definition and Overview

The ACTH stimulation test is a diagnostic procedure used to evaluate the function of the adrenal glands by measuring serum levels before and after the administration of synthetic (ACTH), typically cosyntropin. This test assesses the integrity of the , which regulates responses and production. It is particularly valuable for diagnosing , a condition where the adrenal glands fail to produce adequate . In the HPA axis, (CRH) from the stimulates the gland to secrete ACTH, a derived from pro-opiomelanocortin (POMC). ACTH binds to melanocortin-2 receptors on the , primarily the , activating and increasing cyclic AMP levels, which promotes synthesis and release. then exerts on the and pituitary to maintain . The stimulation test mimics this physiological pathway by providing exogenous ACTH to provoke a response, thereby distinguishing between primary adrenal failure (high endogenous ACTH, poor response) and secondary or tertiary deficiencies (low endogenous ACTH). A simplified of the HPA axis depicts the at the top releasing CRH to the pituitary, which outputs ACTH to the adrenal glands at the base, with looping back upward for feedback inhibition. The test's historical development began in the with initial descriptions using hog pituitary extracts to assess adrenal response via the fall in eosinophil count. It was refined in the 1950s using purified porcine ACTH and direct measurement of plasma cortisol for greater accuracy. By the , the introduction of synthetic cosyntropin—a 24-amino-acid analog of human ACTH—standardized the procedure and minimized risks like allergic reactions associated with animal-derived preparations, leading to its widespread adoption. This evolution improved reliability and safety in clinical practice.

Clinical Indications

The ACTH stimulation test is primarily indicated for the diagnosis of primary , also known as , where the adrenal glands fail to produce adequate despite stimulation by (ACTH). It serves as the gold standard confirmatory test when clinical suspicion arises from symptoms such as , , , or electrolyte imbalances like , particularly in acutely ill patients. The test is also used to diagnose secondary adrenal insufficiency due to pituitary disorders, where insufficient ACTH production leads to adrenal , and to differentiate it from primary forms by assessing response alongside baseline ACTH levels. Additional applications include postoperative assessment of adrenal function and autonomy after treatment for Cushing's syndrome, such as adrenal tumor resection. The test is employed to assess hypothalamic-pituitary-adrenal (HPA) axis recovery following adrenalectomy for conditions like Cushing's or subclinical hypercortisolism, as well as during glucocorticoid withdrawal after prolonged exogenous steroid use, which can suppress endogenous cortisol production; as per the 2024 joint Endocrine Society and European Society of Endocrinology guideline on glucocorticoid-induced adrenal insufficiency, dynamic testing like the ACTH stimulation test should be used cautiously in selected cases. In critical illness scenarios, such as sepsis, it has been used to screen for critical illness-related corticosteroid insufficiency (CIRCI), though results must be interpreted cautiously due to altered protein binding and assay variability; however, its utility is controversial, and recent guidelines (as of 2022) recommend against using it to identify patients who should receive glucocorticoids in septic shock. Endocrine Society guidelines from 2016 recommend the test for confirmatory diagnosis in suspected primary , with ongoing emphasis in recent updates and reviews (up to 2024) on early application in cases of unexplained or to prevent . As a screening tool, it is not optimal for tertiary adrenal insufficiency (hypothalamic causes), where the axis disruption may not yet manifest as adrenal unresponsiveness, potentially yielding false negatives. It is preferred over the insulin tolerance test due to greater safety, avoiding risks like , while providing a reliable alternative for most patients.

Procedure

Patient Preparation

Patients undergoing the ACTH stimulation test should undergo specific preparations to minimize factors that could interfere with accurate measurement and ensure safety. Prior to the test, healthcare providers typically review the patient's medication history; withhold the morning dose of short-acting (e.g., ) if clinically feasible, resuming after the test, as recent or ongoing use is a relative due to potential suppression of the hypothalamic-pituitary-adrenal () axis. Similarly, estrogen-containing medications, which increase -binding (CBG) levels and may lead to falsely elevated total readings, should be discontinued 4-6 weeks beforehand if possible. The test is generally scheduled for the morning to align with the natural diurnal rhythm of cortisol secretion, though it can be performed at any time for diagnostic purposes. No is required, but patients are instructed to avoid physical or emotional stress on the day of the test, as stress can transiently elevate levels and confound results. Informed consent is obtained after explaining the test's purpose in evaluating adrenal function, the procedural steps involving an injection of cosyntropin, and potential minor risks such as injection-site reactions. Patients are also screened for allergies to cosyntropin or related compounds to prevent adverse reactions. In pregnant individuals, elevated CBG levels increase total cortisol measurements, requiring adjusted diagnostic cutoffs (e.g., subnormal basal ≤3 mcg/dL is diagnostic) or alternatives like salivary or free assays for accurate interpretation. In cases of recent acute illness, the test may be postponed to allow recovery, as illness can alter axis dynamics and affect responsiveness.

Test Administration

The ACTH stimulation test is typically performed in an outpatient setting and lasts approximately 1 hour, during which are monitored to ensure . The standard protocol begins with the collection of a sample to measure levels, followed immediately by the intravenous () or intramuscular () of 250 micrograms () of cosyntropin, a synthetic analog of (). Additional samples are then drawn at 30 minutes and 60 minutes post-injection to assess the response. Serum cortisol concentrations from these samples are measured using immunoassays, such as assays, or liquid chromatography-tandem (LC-MS/MS) for higher specificity, depending on the laboratory protocol. A low-dose variant of the test, used for more sensitive detection of mild secondary , involves the IV administration of 1 mcg of cosyntropin instead of the standard dose, with cortisol measurements at 20 and 30 minutes post-administration.

Variants of the Test

The standard ACTH stimulation test employs a high-dose (250 mcg) of synthetic cosyntropin intravenously or intramuscularly to screen for primary , as this dose maximally stimulates the in most cases. In contrast, the low-dose variant uses 1 mcg of cosyntropin, which more closely mimics physiological ACTH levels and demonstrates higher sensitivity for detecting subtle secondary , particularly in cases of mild pituitary dysfunction, according to studies evaluating diagnostic accuracy in the early 2020s. The low-dose protocol typically involves baseline measurement followed by post-stimulation samples at 20 and 30 minutes, offering improved specificity over the high-dose test in non-critically ill patients without altering the overall procedural framework. Combined tests integrate ACTH level measurements with responses during stimulation to better assess pituitary-adrenal integrity, especially for evaluating secondary causes where basal ACTH is low. For depot formulations, a prolonged infusion variant administers cosyntropin or tetracosactrin depot intramuscularly over up to 48 hours to provoke a sustained adrenal response, useful in confirming borderline or atrophied adrenal function unresponsive to short-term stimulation. An aldosterone-specific variant of the ACTH stimulation test measures plasma aldosterone alongside after cosyntropin administration to differentiate subtypes, as exaggerated aldosterone responses can indicate versus . This adaptation leverages ACTH's role in modulating aldosterone secretion via . Historically, early variants utilized natural porcine ACTH extracts, but these were discontinued in the 1970s due to frequent anaphylactic reactions stemming from , paving the way for safer synthetic analogs like cosyntropin.

Interpretation of Results

Normal Cortisol Response

In healthy individuals, the ACTH stimulation test evaluates the adrenal glands' capacity to produce in response to synthetic ACTH (cosyntropin). Baseline serum levels, typically measured in the morning between 8:00 and 9:00 AM, range from 5 to 25 mcg/dL (138 to 690 nmol/L) in adults, reflecting the normal diurnal rhythm with peak secretion occurring shortly after awakening. This range can vary slightly by laboratory assay method, such as or , which may influence absolute values but not the relative response dynamics. Following intravenous or intramuscular administration of 250 mcg cosyntropin, a normal response involves a cortisol level of at least 15 mcg/dL (414 nmol/L) or higher at 30 or 60 minutes post-stimulation, depending on the method (traditional 18 mcg/dL or 500 nmol/L; modern assays often use 14-15 mcg/dL to exclude ). This threshold, established through clinical guidelines and validated in large cohorts, confirms intact adrenal reserve and hypothalamic-pituitary-adrenal axis function. are assay-dependent; recent guidelines (as of 2021) recommend 14-15 mcg/dL for many modern assays to reduce false positives. The 30-minute sample often captures the maximal response, though both time points are assessed to ensure reliability. Reference ranges for normal responses are lab-specific and may require adjustments for age and pediatric populations, where cortisol peaks can be higher—up to 20-25 mcg/dL in younger children due to developmental differences in adrenal sensitivity. Testing is preferably conducted in the morning to align with circadian peaks and avoid potential blunting of the response in the afternoon, when baseline is naturally lower (3-10 mcg/dL). Factors such as recent , medications, or can subtly affect interpretation, emphasizing the need for standardized protocols.

Abnormal Responses and Diagnoses

In primary adrenal insufficiency, also known as , the ACTH stimulation test typically reveals a flat cortisol response, with a peak level below 15 mcg/dL (414 nmol/L) at 30 or 60 minutes post-stimulation (traditional below 18 mcg/dL). This subnormal response confirms damage or dysfunction, often due to autoimmune destruction (as in autoimmune adrenalitis), infections such as , or other causes like hemorrhage or metastases, and is accompanied by elevated baseline plasma ACTH levels greater than 100 pg/mL to distinguish it from central causes. Secondary adrenal insufficiency, resulting from pituitary gland failure to produce adequate ACTH, shows a subnormal cortisol response with peak below 15 mcg/dL (414 nmol/L) in long-standing cases (traditional below 18 mcg/dL), indicating adrenal unresponsiveness due to chronic ACTH deficiency, with low or inappropriately normal baseline ACTH levels (often <10 pg/mL). In recent cases, baseline cortisol may be normal, but the peak response remains inadequate. Common etiologies include pituitary tumors, surgical resection, radiation, or Sheehan's syndrome (postpartum pituitary necrosis). Tertiary adrenal insufficiency, arising from hypothalamic dysfunction leading to reduced corticotropin-releasing hormone (CRH) and subsequent low ACTH, produces a response pattern similar to secondary insufficiency, with subnormal cortisol peak and low ACTH levels. Differentiation from secondary causes requires additional testing, such as the CRH stimulation test, which may elicit a modest ACTH response in tertiary cases but is not performed during the standard ACTH test. An exaggerated cortisol response, with a peak exceeding normal ranges (e.g., >25 mcg/dL or greater than 50% increment), can occur in conditions like early or subclinical due to adrenal or autonomous cortisol secretion, or in states of acute that enhance adrenal sensitivity. Such hyperresponsiveness prompts integration with other diagnostics, including dexamethasone suppression testing or , to guide of hypercortisolism versus insufficiency.

Additional Hormone Measurements

In the ACTH stimulation test, measurement of endogenous (ACTH) levels at provides critical diagnostic differentiation between primary and secondary . In primary , endogenous ACTH is typically elevated above 100 pg/mL due to lack of from low production by the adrenal glands. Conversely, in secondary resulting from pituitary dysfunction, endogenous ACTH levels are low or inappropriately normal, often below 10 pg/mL, reflecting reduced pituitary secretion. These measurements are obtained via simultaneous blood sampling prior to exogenous ACTH administration, using immunoassays with reference ranges varying by laboratory but generally 10-60 pg/mL for normal morning levels. Aldosterone levels may also be assessed in specific cases during the ACTH stimulation test, particularly to evaluate adrenal function in conditions like primary hyperaldosteronism. A normal aldosterone response involves a rise of more than 10 ng/dL from following ACTH , helping to confirm autonomous aldosterone when combined with other confirmatory tests such as saline suppression variants. aldosterone reference values typically range from 4 to 31 ng/dL in adults under standard conditions (upright posture, normal sodium intake), measured concurrently with via or liquid chromatography-tandem for accuracy. In primary , post- aldosterone may remain low (<5 ng/dL), distinguishing it from secondary forms where function is preserved. Additional analytes, such as (DHEA-S) and 17-hydroxyprogesterone, extend the test's utility for assessing adrenal production and steroidogenesis defects. DHEA-S, a marker of adrenal activity, is measured at baseline and post-stimulation; levels below 25 μg/dL suggest , particularly when cortisol response is indeterminate, as DHEA-S reflects zona reticularis integrity. In screening, 17-hydroxyprogesterone levels are evaluated before and 60 minutes after ACTH injection; an exaggerated rise exceeding 1000 ng/dL (30 nmol/L) indicates deficiency, guiding diagnosis of nonclassic forms. These measurements employ or for precision, with simultaneous sampling protocols ensuring synchronized assessment of multiple hormones to avoid diurnal variations.

Risks and Considerations

Potential Side Effects

The ACTH stimulation test is generally well-tolerated, with most adverse reactions being mild and self-limiting. Common mild effects include facial flushing, , and anxiety, which are reported in some patients and typically resolve within hours without intervention. Allergic reactions to synthetic cosyntropin are rare, occurring in less than 1% of cases based on postmarketing reports and case studies, though severe manifestations such as can occur and require immediate . Historically, natural ACTH preparations derived from animal sources (e.g., porcine) were associated with a higher incidence of compared to modern synthetic forms. Cardiovascular effects, such as transient or , may arise shortly after administration and are more relevant in patients with underlying cardiac conditions, necessitating close during the procedure. To manage potential adverse reactions, epinephrine and other treatments for must be readily available at the testing site, and patients should be observed for at least 30 minutes post-injection to detect and address any delayed symptoms.

Contraindications and Precautions

The ACTH stimulation test is generally safe with minimal absolute contraindications. The primary absolute is a known to cosyntropin (synthetic ACTH) or its components, as this can precipitate severe allergic reactions including . Relative contraindications include recent exogenous administration (e.g., , ), which suppresses the axis and may cause false-normal results; such medications should be withheld for an appropriate period prior to testing when possible. Another relative is recent pituitary (within 4-6 weeks), where the test may yield falsely normal responses due to incomplete adrenal . In , particularly the first , the test is not absolutely contraindicated but requires cautious interpretation, as elevated cortisol-binding globulin levels can inflate total measurements without reflecting true adrenal reserve. Precautions are warranted in patients with cardiovascular conditions, given rare reports of transient or following cosyntropin administration; monitoring during the test is recommended, though no specific dose adjustments are indicated. In elderly patients, while the standard dose (250 mcg) remains appropriate, heightened vigilance for adverse effects is advised due to potential comorbidities affecting axis dynamics. Historically, natural pituitary-derived ACTH was contraindicated in systemic fungal infections owing to risks, but this does not apply to modern synthetic cosyntropin formulations. For scenarios where the ACTH stimulation test is unsuitable (e.g., to evaluate central axis function beyond adrenal response), alternatives like the metyrapone test may be employed, though it involves greater risks such as and requires close monitoring. Updated clinical guidelines underscore the importance of , informing patients of potential incidental findings (e.g., subclinical adrenal abnormalities) that could necessitate additional investigations.

Applications and Future Directions

Use in Veterinary Medicine

The ACTH stimulation test is widely employed in to evaluate adrenal function in companion animals, most notably for diagnosing and cats, which parallels in humans. In dogs, the test is particularly valuable for confirming inadequate production, with protocols emphasizing synthetic ACTH analogs like cosyntropin to assess adrenocortical reserve. Standard administration in dogs involves collecting a baseline serum sample followed by intravenous or intramuscular injection of cosyntropin at 5 μg/kg, with a second sample drawn 1 hour post-injection to measure cortisol response. A normal response typically shows post-stimulation cortisol levels of 8.0–20 μg/dL, with an increment of more than 2 μg/dL from baseline indicating adequate adrenal function; values below 2 μg/dL post-stimulation are diagnostic for hypoadrenocorticism. For cats, the protocol uses 5 μg/kg or a fixed dose of 0.125 mg cosyntropin intramuscularly, with baseline and 1-hour post-samples; normal responses exhibit post-stimulation cortisol exceeding 5 μg/dL, while levels below 2 μg/dL suggest adrenal insufficiency. ACTH gel formulations, administered at 2.2 U/kg intramuscularly, serve as an alternative when cosyntropin is unavailable, though they require sampling at 1 and 2 hours due to slower absorption; as of 2025, compounded gels are validated but less preferred due to variability in efficacy. Performing the test in veterinary patients presents challenges, including from handling, which can artificially elevate and mimic hyperadrenocorticism, necessitating minimal restraint or mild in fractious animals. increases responses and should be avoided prior to testing, with effects resolving within 6 hours; and have no significant effects on and do not require washout. In equine practice, the test is adapted to investigate adrenal involvement in conditions like pituitary dysfunction (PPID), often linked to , using low doses of cosyntropin (0.1–1 μg/kg intravenously) and measuring at baseline and 30– post-administration to assess exaggerated responses. For exotic such as ferrets, where adrenal manifests as hyperadrenocorticism independent of pituitary control, the ACTH stimulation test is not effective for and is not recommended; relies on clinical signs, ultrasonography, and hormone panels for steroids and 17-hydroxyprogesterone. Recent 2024 veterinary guidelines and studies continue to validate compounded ACTH as a cost-effective option for small , with dosing adjustments to match cosyntropin efficacy in resource-limited settings.

Emerging Research and Alternatives

Recent validation studies have explored salivary cortisol measurements as a non-invasive alternative to serum sampling during the ACTH stimulation test, demonstrating strong correlations between salivary and serum levels post-stimulation in patients with suspected . These approaches aim to improve patient comfort and accessibility, particularly in outpatient settings, by leveraging late-night or stimulated salivary samples to assess adrenal reserve without . Emerging applications of artificial intelligence in endocrinology include machine learning models for analyzing dynamic hormone response curves from stimulation tests, enhancing diagnostic precision by identifying subtle patterns in cortisol trajectories that may indicate early or mild insufficiency. Such AI tools process serial data to predict outcomes with higher accuracy than traditional thresholds, potentially reducing interpretive variability across assays. As an alternative for differentiating pituitary from hypothalamic causes of secondary adrenal insufficiency, the corticotropin-releasing hormone (CRH) stimulation test evaluates ACTH responsiveness, offering complementary insights when the standard ACTH test shows blunted cortisol responses. For autoimmune Addison's disease, detection of 21-hydroxylase autoantibodies serves as a targeted serological alternative, confirming adrenal autoimmunity and guiding risk stratification without functional stimulation. Ongoing research highlights gaps in understanding long-term outcomes for patients with mild , where subtle deficiencies may contribute to increased morbidity, including cardiovascular risks and reduced , despite normal ACTH test results. Future directions emphasize integrating wearable biosensors for continuous axis monitoring, enabling real-time profiling through sweat or interstitial fluid analysis to detect responses and potentially obviate periodic invasive ACTH testing. These devices, combining with electrochemical detection, could facilitate proactive management of adrenal disorders by tracking dynamic hormone fluctuations over extended periods.

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