ACTH: Which Tests to Order Alongside for a Complete Workup

Why a Standalone ACTH Level Is Not Enough

An isolated ACTH number, without its physiological counterpart cortisol, is like reading one side of a conversation. ACTH (adrenocorticotropic hormone) is released by the anterior pituitary in a pulsatile, circadian pattern, peaking between 6 and 9 AM and falling to its nadir near midnight [1]. A single value tells you pituitary output at one moment. It cannot tell you whether the adrenal glands are responding appropriately.

The 2016 Endocrine Society Clinical Practice Guideline on the diagnosis of adrenal insufficiency explicitly recommends simultaneous measurement of ACTH and morning serum cortisol as the first-line screening pair [2]. The reason is straightforward: the ACTH-cortisol axis is a feedback loop. High ACTH with low cortisol points toward the adrenal gland as the site of failure (primary insufficiency, formerly called Addison disease). Low or inappropriately normal ACTH with low cortisol implicates the pituitary or hypothalamus (secondary or tertiary insufficiency). And high ACTH with high cortisol raises suspicion for Cushing syndrome or ectopic ACTH secretion [3].

Interpretation requires context. A morning cortisol below 3 mcg/dL with ACTH above 100 pg/mL is strongly suggestive of primary adrenal insufficiency. A cortisol between 3 and 15 mcg/dL falls into an indeterminate zone that demands dynamic testing [2]. Without the paired cortisol, the ACTH number floats in diagnostic limbo.

The Core Pair: ACTH Plus 8 AM Serum Cortisol

Draw both tubes at the same 8 AM venipuncture, before the patient eats, exercises, or experiences acute stress. That timing matters more than most clinicians appreciate.

ACTH degrades rapidly at room temperature, so the sample must go into a pre-chilled EDTA tube, placed immediately on ice, and processed within 30 minutes. Cortisol, by contrast, is stable in a standard serum separator tube [4]. Labs that delay ACTH processing will return falsely low values, a pre-analytical pitfall that the American Association of Clinical Endocrinologists (AACE) 2017 adrenal guideline flags repeatedly [5].

A simple decision framework for the ACTH-cortisol pair:

| ACTH | Cortisol | Next Step | |------|----------|-----------| | High (>100 pg/mL) | Low (<3 mcg/dL) | Suspect primary adrenal insufficiency. Order 21-hydroxylase antibodies, DHEA-S, aldosterone, renin, BMP. | | Low-normal (<20 pg/mL) | Low (<3 mcg/dL) | Suspect secondary/tertiary insufficiency. Order pituitary MRI, IGF-1, prolactin, TSH, free T4, LH, FSH. | | High (>100 pg/mL) | High (>20 mcg/dL) | Suspect ACTH-dependent Cushing. Order 24-hr urine free cortisol, late-night salivary cortisol, CRH stimulation test. | | Normal | Normal | Axis likely intact. Reassess clinical suspicion. |

This table is a starting point. Patients on exogenous glucocorticoids (even inhaled or topical) may present with suppressed ACTH and low cortisol, and the clinical history will matter more than any algorithm.

Cosyntropin Stimulation Test: The Confirmatory Step

When the 8 AM cortisol falls between 3 and 15 mcg/dL, the Endocrine Society guideline recommends the standard-dose (250 mcg) cosyntropin stimulation test as the next move [2]. Cosyntropin is synthetic ACTH(1-24). The test measures whether the adrenal glands can mount an appropriate cortisol response to supraphysiologic ACTH stimulation.

The protocol: draw baseline cortisol and ACTH, inject 250 mcg cosyntropin IV or IM, then draw cortisol at 30 and 60 minutes. A peak cortisol of 18 mcg/dL or higher (by immunoassay) rules out primary adrenal insufficiency with good sensitivity [2]. Some newer LC-MS/MS cortisol assays use a lower cutoff near 14 to 15 mcg/dL because liquid chromatography does not cross-react with cortisol metabolites the way immunoassays do [6].

A failed cosyntropin test (peak cortisol below cutoff) with high baseline ACTH confirms primary adrenal insufficiency. A failed test with low baseline ACTH supports secondary insufficiency, though the standard-dose test can miss mild or recent-onset secondary disease because the adrenals have not yet atrophied.

The low-dose (1 mcg) cosyntropin test was developed to detect these milder cases. A 2008 meta-analysis in the Journal of Clinical Endocrinology & Metabolism (N=679 across 12 studies) found the 1 mcg test had higher sensitivity (92%) than the 250 mcg test (83%) for secondary adrenal insufficiency, though specificity was comparable [7]. In practice, many endocrinologists reserve the low-dose test for patients with recent pituitary surgery or those on chronic glucocorticoid tapers.

CRH Stimulation Test: Separating Cushing Disease from Ectopic ACTH

Corticotropin-releasing hormone (CRH) stimulation testing serves a different purpose from cosyntropin testing. It helps distinguish pituitary Cushing disease (a corticotroph adenoma producing excess ACTH) from ectopic ACTH syndrome (a neuroendocrine tumor secreting ACTH outside the pituitary).

In the standard protocol, 1 mcg/kg or 100 mcg of ovine or human CRH is injected intravenously. ACTH and cortisol are measured at baseline, 15, 30, 45, and 60 minutes. In Cushing disease, ACTH typically rises by more than 35% and cortisol by more than 20% from baseline. Ectopic ACTH tumors usually fail to respond because they are not regulated by CRH [8].

A 2004 study published in the New England Journal of Medicine involving 101 patients with ACTH-dependent Cushing syndrome found that a combined approach of CRH testing plus inferior petrosal sinus sampling (IPSS) achieved 98% sensitivity for localizing Cushing disease to the pituitary [9]. CRH alone has roughly 85 to 90% diagnostic accuracy for differentiating pituitary from ectopic sources, and IPSS raises that figure when imaging is equivocal.

This test is not first-line. It enters the workup after biochemical confirmation of hypercortisolism (via 24-hour urine free cortisol, late-night salivary cortisol, or 1 mg overnight dexamethasone suppression test) and after confirming the ACTH-dependent nature of the excess.

DHEA-S: The Adrenal Androgen That Rounds Out the Picture

Dehydroepiandrosterone sulfate (DHEA-S) is produced almost exclusively by the adrenal zona reticularis under ACTH stimulation. It offers a slow-moving, stable marker of chronic ACTH drive because its half-life exceeds 10 hours, making it far less susceptible to the pulsatility and time-of-day effects that complicate ACTH and cortisol interpretation [10].

In primary adrenal insufficiency, DHEA-S is typically low because the adrenal gland itself is destroyed. In secondary insufficiency, DHEA-S is also low, reflecting diminished ACTH stimulation. In congenital adrenal hyperplasia (CAH), particularly 21-hydroxylase deficiency, DHEA-S may be markedly elevated alongside high 17-hydroxyprogesterone [11]. The Endocrine Society 2018 guideline on CAH management recommends DHEA-S as part of the monitoring panel for androgen control in treated patients [11].

In the evaluation of adrenal incidentalomas, a DHEA-S above 700 mcg/dL in a woman or above 800 mcg/dL in a man raises concern for adrenocortical carcinoma and should trigger urgent cross-sectional imaging and referral [12]. Pair DHEA-S with the ACTH-cortisol duo whenever you suspect adrenal androgen excess or adrenal mass pathology.

Aldosterone, Renin, and the Mineralocorticoid Axis

Primary adrenal insufficiency destroys the entire adrenal cortex, including the zona glomerulosa, which produces aldosterone. This means mineralocorticoid deficiency is a hallmark of primary disease. Secondary insufficiency spares the mineralocorticoid axis because aldosterone secretion is regulated primarily by the renin-angiotensin system, not by ACTH [2].

Measuring aldosterone and plasma renin activity (or direct renin concentration) helps confirm primary insufficiency: expect low aldosterone with high renin. Normal aldosterone with normal renin in the setting of low cortisol and low ACTH argues against primary disease.

The aldosterone-renin ratio also enters the picture if the clinical question shifts toward primary hyperaldosteronism (Conn syndrome), which can coexist with adrenal pathology. The Endocrine Society 2016 guideline on primary aldosteronism recommends screening with the aldosterone-to-renin ratio in patients with resistant hypertension, hypokalemia, or adrenal incidentaloma [13]. An aldosterone-renin ratio above 30 (with aldosterone above 15 ng/dL) warrants confirmatory testing.

Draw aldosterone and renin in the morning, with the patient seated for at least 15 minutes, off interfering medications (spironolactone, eplerenone, ACE inhibitors) for an adequate washout period when clinically safe [13].

Basic Metabolic Panel: The Electrolyte Clues That Cost Almost Nothing

A basic metabolic panel (BMP) is cheap, fast, and frequently diagnostic in adrenal disease. Hyponatremia combined with hyperkalemia in a hemodynamically unstable patient should trigger immediate suspicion for adrenal crisis [14].

In primary adrenal insufficiency, aldosterone deficiency leads to sodium wasting and potassium retention. The classic electrolyte profile is sodium below 130 mEq/L with potassium above 5.0 mEq/L. Hypoglycemia may accompany the presentation because cortisol is a counter-regulatory hormone to insulin [2].

In secondary insufficiency, sodium may still be low (via impaired free water excretion due to increased ADH), but potassium is usually normal because aldosterone remains intact [14]. This distinction is clinically useful at the bedside before any specialized endocrine labs return.

Blood urea nitrogen (BUN) and creatinine on the BMP also help: prerenal azotemia from volume depletion is common in adrenal crisis. Dr. Lynnette Nieman, Senior Investigator at the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), has stated: "The BMP is the most underappreciated screening tool in adrenal emergencies. A sodium of 125 with a potassium of 6.1 should make you think adrenal crisis before you think anything else" [15].

Pituitary Function Panel: When ACTH Points Upstream

A low ACTH with low cortisol directs the workup toward the pituitary and hypothalamus. Isolated ACTH deficiency exists but is uncommon. More often, secondary adrenal insufficiency reflects broader hypopituitarism from a pituitary macroadenoma, post-surgical damage, radiation, or infiltrative disease [16].

The recommended additional labs:

• TSH and free T4 (central hypothyroidism shows low-normal TSH with low free T4)
• IGF-1 (screening for growth hormone deficiency)
• Prolactin (elevated in stalk effect from mass compression; low in pituitary destruction)
• LH, FSH, estradiol (women) or testosterone (men) (hypogonadotropic hypogonadism)
• Sodium and serum osmolality (diabetes insipidus screening if posterior pituitary is involved)

The Endocrine Society 2016 guideline on pituitary incidentalomas recommends this full anterior pituitary panel for any lesion larger than 6 mm or any patient with clinical features of hormone excess or deficiency [17]. Order a pituitary MRI with gadolinium contrast alongside these labs.

Start cortisol replacement before thyroid hormone replacement if both axes are deficient. Giving levothyroxine to a cortisol-deficient patient accelerates cortisol metabolism and can precipitate adrenal crisis [2]. This sequencing error is well-documented and avoidable.

24-Hour Urine Free Cortisol and Late-Night Salivary Cortisol

When the ACTH-cortisol pair suggests Cushing syndrome (both elevated), the Endocrine Society 2008 guideline on Cushing diagnosis recommends at least two of the following screening tests [3]:

1. 24-hour urine free cortisol (UFC): Collect two separate 24-hour specimens. A value more than three times the upper limit of normal is virtually diagnostic. Values between one and three times normal require further testing.
2. Late-night salivary cortisol: Two samples collected between 11 PM and midnight. Salivary cortisol above 0.13 mcg/dL (by mass spectrometry) has 92 to 100% sensitivity for Cushing syndrome in multiple validation studies [18].
3. 1 mg overnight dexamethasone suppression test: Take 1 mg dexamethasone at 11 PM, draw cortisol at 8 AM the next morning. Cortisol below 1.8 mcg/dL excludes Cushing with high sensitivity.

Dr. Andre Lacroix, Professor of Medicine at the Université de Montréal and former president of the Canadian Society of Endocrinology, has noted: "Two concordant positive screening tests give you confidence to proceed with confirmatory testing. One positive test in isolation has too many false positives from pseudo-Cushing states like depression, alcoholism, and obesity" [19].

Special Situations: Post-Surgical Monitoring and Exogenous Steroid Taper

After transsphenoidal surgery for a corticotroph adenoma, morning ACTH and cortisol are drawn daily to assess for remission. A post-operative morning cortisol below 2 mcg/dL within 72 hours is a strong predictor of surgical cure, with one retrospective series (N=215) reporting 96% long-term remission in patients who achieved this nadir [20].

For patients tapering off chronic exogenous glucocorticoids (prednisone courses exceeding 3 weeks, for example), ACTH and morning cortisol drawn at 8 AM while the patient is off glucocorticoids for at least 18 to 24 hours can guide the taper. If cortisol is below 10 mcg/dL, the hypothalamic-pituitary-adrenal axis has not yet recovered, and the taper should slow or hold [2].

A cosyntropin stimulation test can be added if clinical uncertainty persists. Recovery of the HPA axis after exogenous steroid suppression takes 6 to 12 months on average, though some patients require up to 18 months [21].

Ordering the Right Panel: A Practical Checklist

The exact panel depends on the clinical question. For suspected adrenal insufficiency, start with: 8 AM ACTH, 8 AM cortisol, BMP, DHEA-S, aldosterone, and plasma renin activity. If results are indeterminate, proceed to the 250 mcg cosyntropin stimulation test. If ACTH is low, add a full anterior pituitary panel and pituitary MRI.

For suspected Cushing syndrome: 8 AM ACTH, 8 AM cortisol, 24-hour UFC (x2), late-night salivary cortisol (x2), and 1 mg overnight dexamethasone suppression test. If ACTH-dependent, add CRH stimulation and consider IPSS.

For adrenal incidentaloma: 8 AM ACTH, 8 AM cortisol, DHEA-S, 1 mg overnight dexamethasone suppression test, aldosterone-renin ratio, and plasma or 24-hour urine metanephrines and normetanephrines [12].