Historical Review

In 1564, the Roman anatomist, Bartholomeus Eustachius (1520-1570), discovered the adrenal gland as “glandulae quae renibus incumbent”—glands with an unknown function. Multiple hypotheses on its possible role were posited over centuries, such as that by the anatomist Adrianus Spigelius (1570-1625), who described the adrenal gland as the “upholstering space holder” between kidney and diaphragm. In 1855, Thomas Addison (1793-1860) first described a phenomenon wherein the only pathologic finding in some deceased patients was a morphologic destruction of the adrenal gland. He concluded that this organ must have a crucial function, and he called the syndrome Morbus Addison (Addison’s disease). One year later, Brown-Séquard confirmed his hypothesis after performing a series of bilateral adrenalectomies in cats, demonstrating that these endocrine glands were necessary for life. Addison’s conclusions, however, were not accepted, and even 2 years after his death, the famous pathologist, Rudolf Virchow, declared that he had never heard such an illogical statement.

In the 19th and early 20th century, several key findings were made. In 1856, von Koelliker described the anatomic division of the adrenal gland into cortex and medulla; and in 1903, Biedl confirmed that the adrenal cortex is the essential part. In 1894, epinephrine (adrenaline) was isolated from the adrenal medulla as the first hormone. Its chemical structure was described 3 years later, and in 1901, epinephrine was synthesized. In patients with Addison’s disease, however, the administration of epinephrine had no success, whereas the use of an animal extract of the adrenal cortex was life saving. Purification techniques were rapidly improved, and the resulting “Cortin” was the first-choice drug for treatment of Addison’s disease until the middle of the 20th century. Three independent groups of biochemists (Kendall, Winterstein, and Reichstein) successfully isolated 17-hydroxy-11-dehydrocorticosterone (later called cortisone) from the adrenal cortex; the physiologic compound, cortisol, was first described by Reichstein in 1937. The extraction of cortisone, however, remained arduous and uneconomical. Bovine adrenal glands of more than 20,000 animals were necessary to produce 1 kg of cortisone. The first synthesis and pharmaceutical preparation of cortisone was described in 1947 by an industrial company. Until that time, cortisone was only used in patients with Addison’s disease.

In the same decade, Hench and Kendall, two rheumatologists at the Mayo Clinic, found that in patients with different forms of rheumatism, the symptoms showed temporary remissions during pregnancy and inflammatory diseases like hepatitis. They speculated that this might be due to a general stimulation of the endocrine system and concluded that the use of cortisone might be beneficial in patients with acute rheumatoid arthritis. In September 1948, a female bedridden patient with severe and painful rheumatism that was resistant to all standard therapies at the time was the first documented case of cortisone treatment for inflammatory disease. After 3 days, the patient was able to stand up; 1 week later, she left the clinic without pain and on her own feet. Retrospectively, the speculations regarding pregnancy and hepatitis were obviously wrong, but the antiinflammatory character of cortisone was a key finding in pharmaceutical research. In contrast to Selye, who described cortisone as a crucial promoter of the physiologic stress response, the aforementioned finding that the adrenal gland cortex is the location for endogenous production of cortisone, an important inhibitor of stress and inflammation, has been confirmed. In 1950, Kendall, Hench, and Reichstein received the Nobel Prize in Medicine for their historical findings on the physiologic role of the adrenal gland.^1–4

Anatomy of the Adrenal Gland

The two paired adrenal glands are located in the retroperitoneal soft tissue near the top of each kidney. In neonates, the adrenal glands are relatively large (approximately one-third of the kidney’s size) compared with other organs. In the postnatal period, the cortex portion shrinks, leading not only to a relatively but also an absolutely smaller size of the organ. In adults, each adrenal gland weighs 4 to 5 g, has a flat form with a sagittal diameter of less than 1 cm, a transverse diameter of 3 cm, and a crani-caudal diameter of 4 to 5 cm. The right gland has a triangle/pyramid-like shape, whereas the left organ is shaped like a half-moon. The adrenal gland is composed of two embryologically distinct tissues. The adrenal cortex develops during the 5th week of gestation from a clump of mesodermal cells within the urogenital ridge known as the adrenal primordium. Later, during the 12th week of gestation, the adrenal medulla develops from neuroectodermal cells of the embryonic neural tube. In the fetal period, the cortex surrounds the medullar cells, resulting in the typical “sandwich” structure, consisting of a flat grey medulla with a yellow cortex.

The circulatory supply, with a flow rate of about 5 mL per minute, is maintained by up to 50 arterial branches from the aorta, renal arteries, and inferior phrenic arteries for each adrenal gland. Blood flow is directed from the capsule into the subcapsular arteriolar plexus through the cortex towards the medulla, where a single vein drains the blood entering the vena cava or the renal vein, respectively. A direct blood supply to the medulla is maintained by medullary arteries.

The adrenal cortex receives afferent and efferent innervation. Direct contact of nerve terminals with adrenocortical cells has been suggested, and chemoreceptors and baroreceptors present in the adrenal cortex infer efferent innervation. Diurnal variation in cortisol secretion and compensatory adrenal hypertrophy are influenced by adrenal innervation. Splanchnic nerve innervation has an effect in regulating adrenal steroid release. The adrenal medulla secretes the catecholamines, epinephrine and norepinephrine, that affect blood pressure, heart rate, sweating, and other activities also regulated by the sympathetic nervous system. The adrenal cortex is divided into three layers: (1) the zona glomerulosa, just under the capsule, (2) the zona fasciculata, the middle layer, and (3) the zona reticularis, the innermost net-like patterned area with reticular veins draining into medullary capillaries. The zona glomerulosa exclusively produces the mineralocorticoid, aldosterone; the zonae fasciculate and reticularis produce glucocorticoids and androgens.⁵

Physiology of the Hypothalamic-Pituitary-Adrenal Axis

The adrenal glands are part of a complex system that produces interacting hormones to maintain physiologic integrity, especially during the stress response.^6,7 This system, the hypothalamic-pituitary-adrenal (HPA) axis, includes the hypothalamic region which produces corticotropin-releasing hormone (CRH), triggering the pituitary gland. The pituitary gland is composed of two major structures: the adenohypophysis (anterior pituitary) and neurohypophysis (posterior pituitary). The anterior pituitary is responsible for the secretion of corticotropin (adrenocorticotropic hormone [ACTH]), thyroid-stimulating hormone (TSH), growth hormone (GH), β-lipotropin, endorphins, prolactin, luteinizing hormone (LH), and follicle-stimulating hormone (FSH). The posterior pituitary secretes vasopressin (antidiuretic hormone [ADH]) and oxytocin. Corticotropin regulates the production of corticosteroids by the adrenal glands. Hypothalamic neurons receive input from many areas within the CNS; they integrate these inputs and initiate an output to the anterior pituitary via the median eminence. The median eminence secretes releasing hormones into a hypophyseal portal network of capillaries that connect the median eminence with the pituitary hormones.

The anterior pituitary gland secretes corticotropin (ACTH) under stimulation from hypothalamic CRH. ACTH in turn stimulates the synthesis and release of glucocorticoids, mineralocorticoids, and androgenic steroids from the adrenal gland. In terms of a feedback loop, ACTH release is inhibited by glucocorticoids, which act on both the pituitary corticotropic cells and hypothalamic neurons. ACTH is also released during stress, independent of the circulating serum cortisol level. CRH, vasopressin, and norepinephrine act synergistically to increase ACTH release during stress. Endorphinergic pathways also play a role in ACTH regulation. Acute administration of morphine stimulates release of ACTH, while chronic administration blocks ACTH secretion. ACTH and cortisol are secreted normally in a diurnal pattern, with lowest concentrations between 10 PM and 2 AM and highest levels around 8 AM. From a practical point of view, it is important to know about rhythms, because inadequate assessment of endocrine function must take into account the variability of hormone levels in the blood. Samples obtained at different times can provide useful dynamic information regarding hypothalamic-pituitary-adrenal function. Loss of diurnal rhythm may indicate hypothalamic dysfunction.

The HPA axis is stimulated not only by physical or psychic stress but also by peptides like ADH and cytokines. Thus, the HPA axis plays an important role during infections and immunologic disorders.^8,9 By interaction with the renin-angiotensin-aldosterone system (RAAS) regulating fluid and salt balance, synthesis of androgens (e.g., dehydroepiandrosterone) with possible impact on immunomodulation, and the sympathoadrenergic system, the HPA axis is probably the most important organ of stress response. Stimulation of the immune system by infections induces the release of proinflammatory cytokines like tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, or IL-6. Following a cascade, these cytokines stimulate both the hypothalamus and the anterior pituitary gland, which finally leads to the release of glucocorticoids. IL-6 is also able to induce steroid release directly from the adrenal gland. The adequate increase of glucocorticoid levels during inflammation is a crucial factor for appropriate stress response. In acute infections, this release maintains metabolic and energy integrity. If the process is chronic, the HPA axis develops an adaptation which induces typical clinical manifestations such as hypercatabolic states, hyperglycemia, and suppression of androgens and growth and thyroid hormones. These changes, however, may increase the risk for secondary infections. Increased cortisol levels suppress higher regulatory levels of the HPA axis in terms of a negative feedback loop. Hence, after major surgery or during sepsis and septic shock, high cortisol and low ACTH levels are detectable.^10,11 Even the infusion of dexamethasone or CRH is not able to suppress increased cortisol levels in these patients.^12,13 This phenomenon leads to the question of how cortisol release is induced. Several investigations demonstrated that adrenal cortisol synthesis in critically ill patients is not regulated by ACTH, but by paracrine pathways via endothelin, atrial natriuretic peptide (ANP), or cytokines like IL-6.^14–16 IL-6 directly induces the adrenal cortex to release cortisol, which in chronic courses, can worsen the prognosis.¹⁷

Cellular Response to Adrenocortical Hormones and Related Drugs

Cortisol, the major free circulating adrenocortical hormone, is a hydrophobic hormone; being a steroid, it circulates bound to protein. Complexed cortisol-binding globulin (CBG, or transcortin) accounts for about 95% of circulating cortisol, but only the free form is biologically active. Its plasma half-life is 60 to 120 minutes. Cortisol is metabolized by hydroxylation in the liver, and the metabolites are excreted in urine. Steroid hormones enter the cytoplasm of cells where they combine with a receptor protein. Metabolic, immunologic, and hemodynamic responses to adrenocortical steroid hormones are regulated in a very complex manner that includes transactivation, transrepression, posttranscriptional/translational regulation, and nongenomic effects. The immediate nongenomic effects of steroid hormones were primarily attributed to mineralocorticoids (aldosterone). Rapid activation of the sodium-proton exchanger, increase of intracellular Ca⁺⁺, and activation of second messenger pathways were described.^18,19 A randomized trial in patients during cardiac catheterization revealed that within minutes after aldosterone injection, cardiac index and arterial pressure increased significantly for 10 minutes and returned to baseline afterwards.²⁰ Interestingly, the genomic effects of aldosterone seemed to be mediated by binding to glucocorticoid (GC) receptors and not to mineralocorticoid receptors.²¹ There is evidence that GC, like cortisol, also modulates immune functions by rapid nongenomic effects via nonspecific interactions with cellular membranes and specific binding to membrane-bound GC receptors (GR).²² Nonspecific membrane effects have been demonstrated for inhibition of sodium and calcium cycling across plasma membranes by impairing Na⁺/K⁺-ATPase and Ca⁺⁺-ATPase. Moreover, the rapid activation of lipocortin-1 and inhibition of arachidonic acid release after GC was independent from GR translocation. Finally, high-sensitivity immunofluorescence staining revealed membrane-bound GR on circulating B lymphocytes and monocytes.²²

The multiple mechanisms by which GCs modulate cellular responses include mainly genomic pathways.^23–25 Nongenomic effects are thought to account for immediate immune effects of high doses of GC, whereas membrane-bound receptors probably mediate low-dose GC effects. The classic model is that GCs bind to the cytoplasmatic ligand-regulated GC receptor alpha (GRα), which is an inactive multiprotein complex consisting of two heat shock proteins (hsp90) acting as molecular chaperones and other proteins (Figure 165-1). Upon GC binding to GRα, conformational change causes dissociation of hsp90, with subsequent nuclear translocation of GRα homodimers, binding of GRα to GC response elements (GRE) of DNA, and transcription of responsive genes (transactivation) such as lipocortin-1 and β₂-adrenoreceptors. Alternatively, GRα may bind to negative GRE (nGRE) and repress transcription of genes (transrepression) such as pro-opiomelanocortin (POMC). More importantly, transrepression without direct binding of GRα to GRE by protein-protein interactions of GRα with transcription factors, nuclear factor kappa B (NF-κB) and AP-1, has been recognized as a key step by which GC suppress inflammation,²⁶ inhibiting synthesis of TNF-α, IL-1β, IL-2, IL-6, IL-8, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, cell adhesion molecules, and growth factors, and promoting apoptosis.²⁷ In addition, NF-κB repression may be mediated by GC-induced up-regulation of the cytoplasmatic NF-κB inhibitor, IκBα (see Figure 165-1) which prevents translocation of NF-κB.²⁸ Clinical investigations provide support for the presence of endogenous GC inadequacy in the control of inflammation and peripheral GC resistance.²⁹ With GC treatment, the intracellular relations between the NF-κB and GRα signaling pathways change from an initial NF-κB-driven and GRα-resistant state to a GRα-sensitive one. However, data are conflicting and probably do not explain early (<2 hours) suppressive effects of GC but may account for the longer-term dampening effect of GC on inflammatory processes.²³

Figure 165-1 Cellular mechanisms of glucocorticoid effects (right) and glucocorticoid resistance (left).

After passive transport through the cell membrane, glucocorticoids (GC) bind to the intracellular GC receptor alpha (GRα), which is sequestered in the cytoplasm, bound to the heat-shock protein (HSP) complex that comprises chaperone molecules HSP70 and HSP90. Binding of GC to GRα allows formation of a homodimer which is transported into the nucleus. GR-mediated transcription induces inhibitor kappa B alpha (IκBα), which binds to and inhibits nuclear factor kappa B (NF-κB). Thus, GC inhibits the NF-κB-mediated synthesis of proinflammatory cytokines like tumor necrosis factor alpha (TNFα). Impaired GC sensitivity (GC resistance) includes three major pathways (dotted arrows): (1) decreased cytoplasmatic GC concentrations secondary to increased P-glycoprotein-mediated efflux of GC due to overexpression of the MDR-1 gene; (2) increased expression of a truncated splice variant of the GR which is unable to transactivate GC-sensitive genes (GRβ); and (3) activation of proinflammatory mediators via upstream kinases (JNK), which can directly inhibit GR transcription activity. GC, glucocorticoids; GR, glucocorticoid receptor; HSP, heat shock protein; JNK, c-Jun N-terminal kinase; MDR-1, multidrug resistance gene 1.

Besides transcriptional regulation, posttranscriptional, translational, or posttranslational processes have been described for GC-induced modulation of COX-2, TNF-α, GM-CSF, IL-1β, IL-6, IL-8, and interferon gamma (IFN-γ).²³ Furthermore, GCs act at multiple levels to regulate iNOS expression by decreased iNOS gene transcription and mRNA stability; reduced translation and increased degradation of the iNOS protein by the cysteine protease, calpain³⁰; limitation of the availability of the NOS cofactor, tetrahydrobiopterin; reduced transmembranous transport and de novo synthesis of the NOS substrate, L-arginine; and lipocortin-1-induced inhibition of iNOS.^31,32 Together, these complex mechanisms result in the considerable effect of GC to inhibit inflammation and to stabilize hemodynamics. Finally, GC receptors have been found in nearly every nucleated cell in the body, and since each cell type has its own expression of GC effect, it follows that GCs have many effects in the body, which is equally true of endogenously produced GC hormones or exogenously administered GC medications. Both increase hepatic production of glucose and glycogen and decrease peripheral use of glucose. Steroids also affect fat and protein metabolism. They increase lipolysis both directly and indirectly by elevating free fatty acid levels in the plasma and enhancing any tendency to ketosis. GCs further stimulate peripheral protein metabolism, using the amino acid products as gluconeogenic precursors.

Definitions of Adrenal Insufficiency

Adrenal glands may stop functioning when the HPA axis fails to produce sufficient amounts of the appropriate hormones. Primary adrenal insufficiency is defined by the inability of the adrenal gland to produce steroid hormones even when the stimulus by the pituitary gland via corticotropin is adequate or increased. Primary adrenal insufficiency affects 4 to 6 out of 100,000 people. The disease can strike at any age, with a peak between 30 and 50 years, and affects males and females about equally. In 70%, the cause is a primary destruction of the adrenal glands by an autoimmune reaction (“classical” Addison’s disease or autoimmune adrenalitis), with about 40% of patients having a history of associated endocrinopathies. Most adult patients have antibodies against the steroidogenic enzyme, 21-hydroxylase,³³ but their role in the pathogenesis of autoimmune adrenalitis is uncertain. In the other 30%, the adrenal glands are destroyed by cancer, amyloidosis, antiphospholipid syndrome, adrenomyeloneuropathy, acquired immunodeficiency syndrome (AIDS), infections (e.g., tuberculosis, cytomegaly, fungi), or other identifiable diseases (Box 165-1). In these cases, the typical morphologic changes of the adrenal cortex are atrophy, inflammation, and/or necrosis. In primary adrenal insufficiency, the whole adrenal cortex is involved, resulting in a deficiency of GCs, mineralocorticoids, and adrenal androgenes.^34,35

Secondary adrenal insufficiency is characterized by adrenal hypofunction due to the lack of pituitary ACTH or hypothalamic CRH. Diseases of the anterior pituitary that can cause secondary adrenal insufficiency include neoplasms (e.g., craniopharyngiomas, adenomas), infarction (e.g., Sheehan’s syndrome, trauma), granulomatous disease (e.g., tuberculosis, sarcoidosis), hypophysectomy, and infection.³⁶ Causes also include hypothalamic dysfunction, such as after irradiation or surgical interventions (see Box 165-1). Because aldosterone secretion is more dependent on angiotensin II than on ACTH, aldosterone deficiency is not a problem in secondary adrenal insufficiency. Selective aldosterone deficiency can occur as a result of depressed renin secretion and angiotensin II formation.³⁴ Rare patients have an isolated deficiency of CRH,³⁷ and lymphocytic hypophysitis with subsequent adrenal insufficiency was described in women.³⁸ These disorders may lead to an isolated ACTH deficiency.³⁴

The so-called tertiary adrenal insufficiency, which is often summarized together with secondary forms, commonly occurs after withdrawal of exogenous GCs. Many of these patients do well during normal activities but are unable to mount an appropriate GC response to stress. This effect depends on the dose and duration of treatment and varies greatly from person to person. It should be anticipated in any patient who has been receiving more than 30 mg of hydrocortisone per day (or 7.5 mg of prednisolone or 0.75 mg of dexamethasone per day) for more than 3 weeks.³⁵ If supraphysiologic doses of GCs have been administered to a patient for more than 1 to 2 weeks, the drug should be tapered to allow for adrenal gland recovery. It may take 6 to 12 months for the adrenal glands to recover fully after prolonged use of exogenous GCs.³⁹ Since ACTH is not a major determinant of mineralocorticoid production, the basic deficit in adrenal insufficiency is that of deficient GC production. It is important that neither the dose of applied glucocorticoids, nor the time of treatment, nor the basal plasma level of cortisol allow sufficient assessment of the function of the HPA axis. Some drugs have also been described to induce adrenal insufficiency, either by directly affecting adrenocortical steroid release (e.g., fluconazole, etomidate)^40,41 or by enhanced hepatic metabolism of cortisol (e.g., rifampicin, phenytoin).³⁵

Isolated hypoaldosteronism is very rare and should be suspected in cases of hyperkalemia in the absence of renal insufficiency. The main causes for isolated deficiency of aldosterone secretion are congenital deficiency of aldosterone synthetase, hyporeninemia due to defects in the juxtaglomerular apparatus, or treatment with angiotensin-converting enzyme inhibitors that lead to loss of angiotensin stimulation. Other forms of hypoaldosteronism usually occur in patients with chronic renal disease and/or diabetes mellitus.

Relative Adrenal Insufficiency

The aforementioned forms of adrenal insufficiency which lead to an absolute deficiency of steroids are rare in critically ill patients (0%-3%).⁴² They are mostly characterized by morphologic changes of the HPA axis. To reflect the notion that subnormal adrenal corticosteroid production during acute severe illness can also occur without obvious structural defects in the HPA axis, deficiency syndromes due to a dysregulation have been termed functional adrenal insufficiency.⁴³ Functional adrenal insufficiency can develop during the course of an illness and is usually transient.³⁵ Decreased levels of GCs are registered much more often; these levels might be sufficient in normal subjects but are too low for stress situations, owing to higher need, and are associated with a worse outcome.⁴⁴ This led to the concept of relative adrenal insufficiency (RAI). The major cause for RAI is inadequate synthesis of cortisol due to cellular dysfunction. Hence, in contrast to absolute adrenal insufficiency, the morphologic changes in RAI may be minor, sometimes characterized by cellular hyperplasia within the adrenal cortex. This is often combined with peripheral GC resistance of the target cells, which is caused by inflammatory events and aggravates the clinical course, although the absolute cortisol serum levels might be normal.⁴⁵ In septic shock, RAI may be due to impaired pituitary corticotropin release, attenuated adrenal response to corticotropin, and reduced cortisol synthesis (Figure 165-2).^35,46,47 In addition, cortisol transport capacity to effect sites may be reduced, and response to cortisol may be impaired at the tissue level by cytokines modulating GC receptor affinity to cortisol and/or GC response elements.^48,49 In clinical trials, it was demonstrated that prolonged treatment of systemic inflammation in patients with severe acute respiratory distress syndrome (ARDS) with methylprednisolone can improve the decreased GC response by increasing the GC receptor affinity and reducing the NF-κB-mediated DNA binding and transcription of proinflammatory cytokines.²⁹ Thus, if RAI can be identified, treatment with supplemental corticosteroids may be of benefit.³⁵ Prevalence of RAI in the critically ill varied from 0% to 77% with different definitions, cutoff values, study populations, and adrenal function tests^{34,35,46,50,51} and may be as high as 50% to 75 % in severe septic shock.⁵²

Figure 165-2 Concept of relative adrenal insufficiency (RAI).

Unlike in adequate stress response (left), RAI may occur when causal or additional factors impair the function of the hypothalamic-pituitary-adrenal (HPA) axis. This may be due to microcirculatory failure, additional drugs like antibiotics, anesthetic drugs, infections, long-term use of steroids, or hemorrhages. Impaired HPA axis function results in insufficient antiinflammatory response and increased inflammatory response. Plus (+) denotes activation; minus (−), inhibition. CRH, corticotropin-releasing hormone; CBG, cortisol-binding globulin; GC, glucocorticoids.

Evaluation of Adrenal Insufficiency

In clinical practice, assessment of adrenal function is difficult, especially in critically ill patients, since the diurnal rhythm is lost. Values indicating normal adrenocortical function are listed in Box 165-2. Normally, serum cortisol concentrations in the morning (8 AM) of less than 3 µg/dL (80 nmol/L) are strongly suggestive of absolute adrenal insufficiency,⁵³ while values below 10 µg/dL (275 nmol/L) make the diagnosis likely. Basal urinary cortisol and 17-hydroxycorticosteroid excretion is low in patients with severe adrenal insufficiency but may be low-normal in patients with partial adrenal insufficiency. Generally, baseline urinary measurements are not recommended for the diagnosis of adrenal insufficiency. To differentiate between primary, secondary, and tertiary adrenal insufficiency in cases of low cortisol, it is recommended to measure plasma ACTH concentrations simultaneously. Inappropriately low serum cortisol concentrations in association with increased ACTH concentrations are suggestive of primary adrenal insufficiency, whereas the combination of low cortisol and ACTH concentrations indicates secondary or tertiary disease. This, however, should be confirmed by stimulation of the adrenal gland with exogenous ACTH. In secondary or tertiary adrenal insufficiency, the adrenal glands release cortisol, whereas in primary adrenal insufficiency, the adrenal glands are partially or completely destroyed and do not respond to ACTH.

ACTH stimulation tests usually consist of administering 250 µg (40 International Units) of ACTH (so-called high-dose ACTH stimulation test). For long-term stimulation tests, which are preferred for differentiating between secondary and tertiary adrenal insufficiency, 250 µg of ACTH are infused either over 8 hours or over 2 days.⁵⁴ Serum cortisol and 24-hour urinary cortisol and 17-hydroxycorticosteroid (17-OHCS) concentrations are determined before and after the infusion. This test may be helpful in distinguishing primary from secondary/tertiary adrenal insufficiency. In primary adrenal insufficiency, there is no or a minimal response of plasma or urinary cortisol and urinary 17-OHCS. Increases of these values in the 2 to 3 days of the test are indicative of a secondary/tertiary cause of adrenal insufficiency. In normal subjects, the 24-hour urinary 17-OHCS excretion increases 3- to 5-fold above baseline. Serum cortisol concentrations reach 20 µg/dL (550 nmol/L) at 30 to 60 min and exceed 25 µg/dL (690 nmol/L) at 6 to 8 hours post initiation of the infusion. Today this is not very often used, because clinical manifestations of adrenal insufficiency combined with basal cortisol levels, short-term ACTH stimulation tests, and CRH tests (see later) usually provide sufficient information.

A short-term stimulation test with 250 µg ACTH, mostly used for patients who are not critically ill, determines basal serum cortisol levels and the induced-response concentration 30 and 60 minutes after intravenous (IV) administration of ACTH. The advantage of the high-dose test is that pharmacologic plasma ACTH concentrations can be achieved by either IV or intramuscular injection.⁵⁵ This way of application, however, may be too high to identify mild cases of secondary adrenal insufficiency or chronic deficiencies.⁵⁶