Specificity of the biological effects of adrenal steroid hormones

The different classes of steroid hormones are all generated by the enzymatic modification of the cholesterol nucleus and the structures of steroid hormones in two-dimensional drawings appear very similar (Box 4.2). This raises the question of how different steroids exert specific actions in target cells. Specificity first requires structural differences between steroid hormones.

A number of empirical studies over many years has established that the basic structural requirement for a steroid to possess glucocorticoid or mineralocorticoid activity is that it should be a carbon 21 (C-21) compound with a -CO-CH₂OH side-chain attached at C-17. In addition, there must be an unsaturated bond between C-4 and C-5 (sometimes referred to as Δ4) and a keto group (-C=O) at C-3 of ring A, together termed 4-ene-3-one (or Δ4, 3-keto). Specific glucocorticoid activity requires a hydroxyl group at C-11 and this activity is enhanced by a similar group at C-17. Mineralocorticoids, on the other hand, require a hydroxyl group on C-21 whilst the presence of hydroxyl groups at C-11 and C-17 decrease mineralocorticoid activity.

Androgenic effects are generated by C-19 compounds containing a 17β-hydroxyl group. The latter is very important since oxidation to 17-keto results in marked loss of activity and it is also stereo-specific since steroids containing a 17α-hydroxyl group have little or no androgenic activity. The presence of either a 4-ene-3-one configuration or a 3-keto group in ring A is also necessary. The naturally occurring progestagens are, like cortisol and aldosterone, also 21-carbon molecules and possess keto groups on C-3 and C-20 for biological activity.

The second way in which specificity of steroid hormone action may be generated is, in large part, via the evolution of receptors that have much higher affinity for the active hormones than for metabolites or structurally similar steroids. This appears to be the case for estradiol and 1,25-dihydroxyvitamin D, the structures of which differ most from the other steroids. However, glucocorticoids, mineralocorticoids, progestagens and androgens have closer structural similarities and their specificities are markedly reduced. For example, the affinity of the mineralocorticoid receptor for cortisol is the same as that of the glucocorticoid receptor.

Cholesterol and steroid synthesis in the adrenal cortex

Cholesterol is either obtained from the diet or synthesized from acetate by a CoA reductase enzyme. Approximately 300 mg cholesterol is absorbed from the diet each day and about 600 mg synthesized from acetate. Cholesterol is insoluble in aqueous solutions and its transport from the main site of synthesis, the liver, requires apoproteins to form a lipoprotein complex. Circulating lipoproteins were first characterized by centrifugation and as a result are grouped by density.

In the adrenal cortex, about 80% of cholesterol required for steroid synthesis is captured by receptors which bind low-density lipoproteins (LDL) although recent evidence has shown that high-density lipoprotein (HDL) cholesterol may also be taken up by adrenal cells. The remaining 20% is synthesized from acetate within the adrenal cells by the normal biochemical route. The cholesterol can be stored as esters in lipid droplets or utilized directly (Box 4.4).

Box 4.4

Diagrammatic outline of the synthesis of cortisol from cholesterol in the adrenal cortex (see text and Box 4.5 for details). * Activities known to be regulated by ACTH.

The first stage in the synthesis of adrenal steroids is the hydrolysis of cholesterol esters and the active transfer of free cholesterol to the outer membrane of the mitochondria by a sterol transfer protein (Box 4.4). The transfer of hydrophobic cholesterol to the inner mitochondrial membrane is chaperoned by a steroidogenic acute regulatory (StAR) protein where the first enzymatic process in steroid hormone synthesis occurs. The enzyme, known as side chain cleavage enzyme, P450_scc, (which also has 20,22 desmolase activity), converts cholesterol to pregnenolone. Indeed, most of the subsequent steps in steroid hormone synthesis also involve cytochrome P450 heme-containing enzymes, so-called because light is maximally absorbed at 450 nm when the proteins are complexed with CO. The genes coding for the cytochrome P450 enzymes are abbreviated to CYP (Box 4.5) and they catalyze hydroxylations of the steroid molecule.

Box 4.5

Synthesis of the major steroid hormones secreted by the adrenal cortex. The genes coding for the P450 enzymes are abbreviated to CYP and the first step in the synthesis of hormones is the 6 carbon unit side chain cleavage of cholesterol. Many of the (more...)

Pregnenolone is then shuttled from the mitochondria to the smooth endoplasmic reticulum where it is converted to progesterone or to 17α-hydroxypregnenolone. Through subsequent hydroxylations, progesterone can be converted to corticosterone (another glucocorticoid that is only released in small amounts in the human) and then aldosterone, whilst 17α-hydroxypregnenolone can be converted to androgens and cortisol (Box 4.5). There is, however, considerable interconversion between these two pathways and it should be noted that the final stage in cortisol synthesis takes place back in the mitochondria.

In functional terms, the adrenal cortex is, therefore, not a single endocrine gland since it secretes different steroids with widely different activities and functions. This is achieved by differential expression of enzymes resulting in functional zonation that has anatomical correlates.

Anatomical and functional zonation in the adrenal cortex

Each adrenal gland weighs approximately 4 g and sits in close proximity to a kidney (in the UK, adrenal whilst, in the US and France, reference is made to a position above the kidney, viz suprarenal and sûrrénale (Box 4.6)). The cortex forms about 90% of its mass, the remaining core being the adrenal medulla. In the adult, it can be divided morphologically and functionally into three layers (the glomerulosa, fasciculata and reticularis). Each layer has a distinct histological appearance and secretes different steroid hormones (aldosterone, cortisol and androgens, respectively). A fourth or fetal zone is present during development. The inner 10–20% of the gland is the adrenal medulla secreting catecholamines. In the UK, these hormones are called adrenaline and noradrenaline; but the terms epinephrine and norepinephrine are also used for the same hormones.

Box 4.6

Histology and blood supply of the adrenal gland. Diagrammatic representation of the different zones of the adrenal gland and their hormone secretions Low power photomicrograph of a cross-sectional view of the adrenal gland

Embryologically, the adrenal gland develops from two cell types (Box 4.7). The innermost layers of the gland contain most of the apoptotic and senescent cells indicating that this is where the cells die, supporting the concept that cortical cells originate from the outer layers of the cortex and move inwards. In addition, the arrangement of blood flow within the gland appears to be crucial in developing and maintaining the morphological and functional zonation of the gland (Box 4.6). The arrangement is such that blood vessels supplied from branches of the aorta, phrenic and renal arteries flow from the outer cortex to drain inwardly into venules of the adrenal medulla. Thus, glomerulosa cells differentiate on the arterial side and reticularis cells on the venous side.

Box 4.7

Embryology of the adrenal gland. Two-dimensional diagram of a transverse section of the caudal region of a 6-week embryo. The fetal suprarenal cortex is derived from mesodermal cells. The medulla is formed from an adjacent sympathetic ganglion that is (more...)

The enzyme 17α-hydroxylase (CYP 17) is not present in the outer layer of the cortex and, thus, cortisol and androgens cannot be formed in this layer. Steroids and their metabolic by-products (notably lipid hydroperoxides) are released into the adrenal circulation and inhibit critical enzymes in subsequent layers through which the blood flows. As a result, no aldosterone can be synthesized by cells below the outer glomerulosa layer. In the inner layer, 17α-hydroxyprogesterone cannot be converted to cortisol but is shunted into the formation of androgens. Interestingly high cortisol concentrations reaching the adrenal medulla stimulate the synthesis of phenylethanolamine-N-methyltransferase which catalyzes the conversion of norepinephrine to epinephrine (see Box 4.39). Thus, the structural relationship between the cortex and medulla and its blood supply has additional functional implications within the medulla.

Box 4.39

Biosynthesis and control of catecholamines secreted by the adrenal medulla. Stimuli from the hypothalamus, medulla and pons activate preganglionic cholinergic nerves that stimulate the release of epinephrine and smaller amounts of norepinephrine from (more...)

Glucocorticoid receptors

Glucocorticoids are essential to life and after removal of both adrenals humans will not survive for long without glucocorticoid replacement. Cortisol has a wide range of actions, many of which are considered ‘permissive’. This is because it does not always initiate processes but allows them to occur by increasing the activity of enzymes, inducing enzymes or augmenting/inhibiting the action of other hormones.

Receptors for glucocorticoids (GRs) are usually intracellular and unlike thyroid hormones they usually exist in the cytoplasm, not the nucleus, and are associated with heat shock proteins (Box 4.8). These are displaced when cortisol diffuses across the cell membrane, and binds to these receptors in target cells. Subsequent phosphorylation of the receptors facilitates translocation of the hormone-receptor complex into the nucleus where it forms a homo- or heterodimer with another hormone-receptor complex. The effects of heterodimeric forms may differ from those of the homodimers.

Box 4.8

The glucocorticoid receptor and activation by cortisol. Unbound, lipophilic cortisol readily crosses cell membranes and in target tissues will combine with the glucorticoid receptor (GR). Like the androgen and progesterone receptors, but unlike thyroid (more...)

The zinc fingers in the DNA-binding domain of the dimerized receptors interact with specific grooves of the DNA helix containing a consensus sequence. The site of receptor binding on the DNA is known as the hormone response element (HRE) - in this case the glucocorticoid response element (GRE). In association with other transcription factors, the GRs stimulate or suppress gene transcription that is usually initiated down-stream of the GRE. The structural similarities of the DNA-binding domain of glucocortiocoid, estrogen, androgen and progesterone receptors are such that they can all bind to the same hormone response element, a consensus 15 nucleotide sequence. Additionally, cortisol has equal affinity for the aldosterone receptor in the kidney tubules but its rapid inactivation to cortisone in these cells normally prevents binding.

The expression of GR is ubiquitous and it occurs in two forms, GR-α and GR-β. The latter does not bind glucocorticoid and probably acts as a ligand-independent regulator of glucorticoid activity. Cortisol may also exert effects via membrane receptors as do other steroid hormones. The serum protein that transports cortisol, cortisol-binding globulin (CBG), can also bind to cell surface receptors. Cortisol may then bind to the CBG-receptor complex and activate adenylate cyclase, thereby providing a mechanism by which cortisol exerts non-genomic actions.

Actions of glucocorticoids and clinical features of Cushing's syndrome

Cortisol, like the thyroid hormone T₃, has potent metabolic effects on many tissues (Box 4.9). These are essentially anabolic in the liver and catabolic in muscle and fat; the overall effect is to increase blood glucose concentrations. Thus, like growth hormone, epinephrine and glucagon, cortisol is also considered diabetogenic. It does this by opposing the action of insulin in peripheral tissues (decreasing glucose uptake via GLUT4 receptors) and increasing glucose production and release from the liver. The latter is accomplished through gluconeogenesis using amino acids (from the catabolic actions on muscle) as the primary carbon source (Box 4.9). Thus, Clinical Cases 4.1 and 4.2 had thin arms and legs caused by the catabolic actions of excess glucocorticoids on peripheral muscle. Patients with Cushing's syndrome tend to have a particular weakness of the muscles around the hips and shoulders, termed a proximal myopathy.

Box 4.9

Diagram showing the major actions of cortisol on metabolism. Cortisol stimulates the release of amino acids from muscle. These are taken up by the liver and converted to glucose. The increased circulating concentration of glucose stimulates insulin release. (more...)

Although cortisol has some minor lipolytic activity, this effect is overshadowed in a patient with Cushing's syndrome by the increased insulin secretion in response to the diabetogenic actions of cortisol. Insulin has a strong lipogenic action (see Box 2.8) and, thus, the excess glucocorticoids seen in Clinical Cases 4.1 and 4.2 increased fat deposition. The reason for the centripetal distribution of fat is not fully explained but probably results from metabolic differences between adipocytes in the omentum and those situated in subcutaneous tissues.

Bruising, scarring and purple striae around the abdomen are other classical signs of Cushing's syndrome (Box 4.10). Cortisol inhibits fibroblast proliferation and also the formation of interstitial materials such as collagen. Excess glucocorticoids result in a thinning of the skin and the loss of connective tissue support of capillaries. This makes them more susceptible to injury and leads to bruising. Bones are also affected by excess glucocorticoids. Cortisol decreases osteoblast function and decreases new bone formation; osteoclast numbers increase and measures of their activity increase. Furthermore, glucocorticoids decrease gut calcium absorption and decrease renal calcium reabsorption, thus adversely affecting calcium balance. Overall excess glucocorticoids cause osteoporosis.

Box 4.10

Clinical features of Cushing's syndrome. Common Moon face (with plethora) (~100%)

Glucocorticoids have other diverse actions including those on the cardiovascular system, central nervous system, kidney and the fetus. In the cardiovascular system, it is required for sustaining normal blood pressure by maintaining normal myocardial function and the responsiveness of arterioles to catecholamines and angiotensin II. In the CNS, cortisol can alter the excitability of neurons, induce neuronal death (particularly in the hippocampus) and can affect the mood and behavior of individuals. Depression may be a feature of glucocorticoid therapy. Furthermore, depressed patients may show increased cortisol secretion with alteration in the circadian rhythm of cortisol secretion.

In the kidney, cortisol increases glomerular filtration rate by increasing glomerular blood flow and increases phosphate excretion by decreasing its reabsorption in the proximal tubules. In excess, cortisol has aldosterone-like effects in the kidney causing salt and water retention. This is because the capacity of 11β-hydroxysteroid dehydrogenase type 1 enzyme that converts active cortisol to inactive cortisone in the kidney tubule is overwhelmed. Cortisol is then available to interact with the aldosterone receptor for which it has equal affinity (Box 4.11). This may be a factor in the hypertension seen in patients with Cushing's syndrome.

Box 4.11

Cortisol and the aldosterone receptor in the kidney. Cortisol and aldosterone have equal affinity for the mineralocorticoid (AT1) receptor. Circulating concentrations of cortisol are 100 times higher than aldosterone but it does not normally interact (more...)

Cortisol also facilitates fetal maturation of the central nervous system, retina, skin, gastrointestinal tract and lungs. It is particularly important in the synthesis of alveolar surfactant which occurs during the last weeks of gestation. Babies born prematurely may suffer respiratory distress syndrome and mothers with pre-term labor may be treated with glucocorticoids to stimulate fetal synthesis of surfactant.

One of the most important actions of glucocorticoids is on inflammatory and immune responses (Box 4.12) and it is these actions which led to the development of a multi-million dollar pharmaceutical industry in synthetic glucocorticoid preparations. Inflammation (increased capillary permeability, attraction of leukocytes etc.) results from injury and these effects are mediated by several factors the production of which is inhibited by cortisol.

Box 4.12

Major effects of glucocorticoids on inflammation and immune responses. Glucocorticoids inhibit the conversion of phosphatidyl choline to arachidonic acid by inducing the production of lipocortin which inhibits phospholipase-A₂ (PL-A₂). They inhibit the (more...)

Some of these factors are synthesized from arachidonic acid and cortisol inhibits the synthesis and release of arachidonic acid by inducing lipocortin which inhibits phospholipase A₂. This enzyme releases arachidonic acid from phosphatidyl choline and, thus, the availability of arachidonic acid for the synthesis of inflammatory mediators is reduced. In addition glucocorticoids stabilize lysosomes, preventing the release of proteolytic enzymes. They inhibit the proliferation of mast cells, production of cytokines and also the recruitment of leukocytes to the site of infection or trauma. They also affect the numbers and functions of circulating neutrophils, eosinophils and fibroblasts. In addition, glucorticocoids reduce the number of circulating thymus derived lymphocytes (T- cells) and as a result the recruitment of B lymphocytes. The net result is to reduce both cellular and humoral immunity.

Adrenal cortical androgens

The two steroids produced in greatest quantities by the adrenal cortex, DHEA and its sulfate have an ill-defined role in normal physiology. Together with androstenedione, they are generally termed ‘weak androgens’ and have a much lower affinity for the androgen receptor than testosterone. These adrenal androgens are, however, converted peripherally to the more active testosterone (Box 4.13). In males, the amount released from the adrenal glands and converted to testosterone is physiologically insignificant compared to the amount secreted by the testes but, in females, adrenal-derived testosterone is important in maintaining normal pubic and axillary hair.

Box 4.13

Peripheral metabolism of adrenal androgens. Androstenedione, DHEA and its sulfate DHEAS are metabolized to the more potent testosterone by 17β-hydroxysteroid dehydrogenase via androstenedione. In some target tissues, testosterone is 5α-hydroxylated (more...)

After the menopause, adrenal androgens may also be an important source of estradiol, again due to peripheral conversion. Adrenal androgen hypersecretion does not cause any clinical signs in adult males but is detectable in females by signs of hirsutism and masculinization. These effects are examined in more detail in Clinical Case 4.3 and the role of adrenal androgens in the adrenarche of puberty is discussed on page 238.

To understand the biochemical investigations of Clinical Cases 4.1 and 4.2 it is not only important to know the actions of adrenal cortical hormones, but also the control of their secretions.

Hypothalamic control of adrenocortical steroid synthesis - CRH and vasopressin

Corticotrophin-releasing hormone (CRH) is a 41-amino-acid peptide secreted by neurosecretory cells predominantly located in the paraventricular nucleus of the hypothalamus (Box 4.14). Released from nerve terminals in the median eminence, this peptide is transported to the anterior pituitary corticotrophs in the hypophyseal portal capillaries where it acts on a G-protein linked receptor to stimulate an increase in cAMP. The subsequent signal transduction pathways stimulate both the synthesis and release of adrenocorticotrophin (ACTH).

Box 4.14

Control of cortisol and androgens from the adrenal cortex. Diagram depicting the factors which control the secretion of cortisol. 24 h secretory pattern of cortisol. 24 h secretory patterns of androstenedione (Andro) and dehydroepiandrostenedione (DHEA). (more...)

The action of CRH on pituitary corticotrophs is potentiated by arginine vasopressin (AVP), also known as antidiuretic hormone (ADH). AVP, secreted by parvocellular (small) neurosecretory cells in the supraoptic and paraventricular nuclei of the hypothalamus, is also released into the hypophyseal portal capillaries. This contrasts with the magnocellular (large) neurosecretory cells in the same nuclei whose axons terminate in the posterior pituitary and release AVP into the general circulation (see Box 7.43).

Recent evidence has shown that there are at least two types of CRH receptors that differ in their anatomical location and in their pharmacology. It may well be that these two receptors mediate different functions of CRH. For example hyperactivity of CRH neurons both in the hypothalamus and other brain regions may not only activate the increased ACTH/adrenal activity associated with stress but also certain associated behavioral symptoms such as depression, sleep and appetite disturbances and psychomotor changes. CRH is also produced in the placenta, as is a specific binding protein, CRH-BP. This binding protein may modulate the paracrine effects of CRH within the placenta and its reduced production at term suggests that CRH/CRH-BP may play a role in parturition. CRH synthesis and CRH receptors have also been identified in immune cells and there is evidence that CRH may not only be anti-inflammatory through its central action on glucorticoid secretion but also pro-inflammatory through direct effects of peripherally released CRH. Thus, CRH is not simply a neuro-hormone that controls the secretion of ACTH.

Pituitary control of adrenocortical steroids - ACTH

ACTH is derived from a large precursor molecule pro-opiomelanocortin (POMC) that is cleaved by the action of specific peptidase enzymes (Box 4.15). Whilst this prohormone can give rise to numerous hormones, including opioid peptides and melanocyte stimulating hormone (MSH), the main product of POMC cleavage in the corticotroph cells is ACTH. In the brain, other products predominate.

Box 4.15

Synthesis of adrenocorticotrophic hormone (ACTH). The pro-opiomelanocortin (POMC) gene codes for a large pro-hormone plus signal sequence that is subsequently cleaved at the numbers indicated into smaller active molecules under the action of peptidases. (more...)

The main action of ACTH on the adrenal cortex is to stimulate the synthesis and release of glucorticoids and androgens via cAMP-dependent mechanisms via a G-protein coupled receptor. The immediate actions of ACTH on steroid synthesis are to increase cholesterol esterase, the transport of cholesterol to and across the mitochondrial membrane, cholesterol binding to P450_SCC and, hence, an increase in pregnenolone production (Box 4.4). Subsequent actions include the induction of steroidogenic enzymes and conspicuous structural changes characterized by hypervascularization, cellular hypertrophy and hyperplasia. This is particularly notable when excess ACTH is secreted over prolonged periods of time (e.g. pituitary-dependent Cushing's). Whether androgen synthesis and secretion is under some other control remains uncertain. In contrast, the primary stimulus for aldosterone secretion is through the renin-angiotensin system (see Box 4.33).

Box 4.33

Control of aldosterone secretion. Juxtaglomerula cells are modified smooth muscle cells which secrete renin The macula densa are tubular cells of the thick ascending limb of the loop of Henle which can detect circulating concentrations of sodium

Feedback control of glucocorticoids

The production of glucocorticoids is controlled by a classical negative feedback loop in which neurons in the hypothalamus detect circulating concentrations of glucocorticoids and consequently stimulate or inhibit the release of CRH and AVP from the parvicellular neurons (Box 4.14). AVP secreted by the magnocellular neurons is controlled by different stimuli, namely serum osmolarity and blood volume.

This feedback loop can, however, be over-ridden by both internal and external factors. Human biological clocks (normally entrained to the light-dark cycle) produce a circadian rhythm in the release of ACTH and, consequently cortisol, with peak concentrations of these hormones in the early morning and a nadir in the evening (Box 4.14). Thus, for patients requiring cortisol replacement therapy a larger dose of the steroid is given in the morning with a lower dose in the evening to simulate the normal endogenous rhythm. Stress, whether generated by physical or emotional trauma, is also a potent stimulus to cortisol secretion and can over-ride negative-feedback effects.

In addition to ACTH drive of the adrenal cortex, there is also evidence for non-ACTH-mediated regulation that could partly explain why, in some clinical situations, there is a dissociation between ACTH and cortisol secretions. The nerve supply of the adrenal cortex may modulate adrenocortical function and activation of the adrenomedullary system, that releases both catecholamines and peptides, is also implicated as a local control mechanism. In addition, immunomodulatory peptides such as cytokines, which can be released within the gland or by circulating leukocytes, also stimulate cortisol secretion. This could, in part, account for the rise in cortisol seen during chronic infection and sepsis.

The control of glucocorticoid production is, indeed, complex, but patients with suspected Cushing's syndrome are investigated using the physiological principles inherent in the control system.

Excess glucocorticoids: biochemical investigation of Cushing's syndrome

Once suspected, the diagnosis of Cushing's syndrome requires biochemical confirmation and elucidation of its cause. Endogenous causes may be primary (due to adrenal dysfunction) or secondary due to excess secretion of ACTH either from the pituitary gland or another (termed ectopic) source (Box 4.16). Alternatively therapeutic glucocorticoids may be the cause.

Box 4.16

Causes of Cushing's syndrome. Exogenous therapeutic glucocorticoids Anterior pituitary adenoma

Clinical Case 4.1 not only showed signs of excess glucocorticoid but also of excess androgen secretion. Thus, it is highly unlikely that her cushingoid symptoms were the outcome of her steroid treatment. These were inhaled beclomethasone and topical betamethasone, both of which are ‘pure’ glucocorticoids, without androgenic effects (Boxes 4.17 and 18). Thus, the cause of her cushingoid symptoms is likely to be endogenous rather than exogenous. There are a variety of tests to investigate and confirm the different causes of Cushing's secretion; these include measurements of cortisol and ACTH secretion and dynamic functional tests.

Box 4.17

Structures of various immunosuppressive steroids compared with those of progesterone and cortisol. All these steroids are C-21 or C-22 compounds with the 4-ene-3 keto core typical of all adrenal corticosteroids. Different side chains determine the potency (more...)

Box 4.18

Approximate relative activities of adrenal steroids and synthetic steroids.

Measurements of cortisol in blood, urine and saliva

Random measurements of peripheral blood cortisol concentrations are generally unhelpful in the diagnosis of Cushing's because the diurnal rhythm in cortisol secretion together with inter-individual differences makes interpretation of the results difficult. However, since the diurnal rhythm in cortisol secretion is lost in any endogenous cause of Cushing's syndrome, measurements of serum cortisol at 09.00 to give a ‘peak’ value and those at 24.00 to give ‘trough’ values can be useful in the diagnosis (Box 4.19). It is important that the midnight sample is taken with the patient unstressed. Since, however, this test requires hospital admission, a more frequently used alternative is to measure salivary cortisol in which concentration is independent of saliva flow rates due to its lipid solubility. This allows patients to collect their own samples in the comfort of their own homes.

Box 4.19

The investigation of the cause of Cushing's syndrome. In iatrogenic Cushing's syndrome due to the ingestion of synthetic or natural glucocorticoids, the concentration of cortisol in blood and urine is low and often unmeasurable (unless the offending steroid (more...)

Clinical Case 4.1 showed no diurnal variation in her cortisol secretion, mean concentrations being 1031 nmol/l and 915 nmol/l at 09.00 and midnight respectively and the corresponding mean plasma ACTH concentrations measured with a two site IRMA (see Box 3.25) were 30 and 12 pmol/l. These measurements indicate that the cause of the Cushing's is ACTH-dependent and not due to primary adrenal over-activity that would have suppressed ACTH due to negative feedback effects.

Another way of estimating cortisol secretion is to measure the small fraction of unmetabolized, unconjugated cortisol that is excreted in the urine over a 24 h period. Termed urinary free cortisol, this measurement allows assessment of total cortisol secretion throughout the day. The 24 h urinary free cortisol excretions measured by radioimmunoassay (see Box 6.14) for two consecutive days were 1245 and 1456 nmol/l day (NR 100–220 nmol/d) for Clinical Case 4.1 whilst those for Clinical Case 4.2 were low, at 42 and 34 nmol/day.

In conjunction with cortisol and ACTH measurements, dexamethasone, metyrapone and CRH tests are used to confirm the cause of Cushing's syndrome (Box 4.19).

Dynamic tests of endocrine function

Dexamethasone is a potent synthetic glucocorticoid with negligible mineralocorticoid action and it is administered to suppress the endogenous release of ACTH and cortisol. ‘Low’ doses (i.e. 2 mg/day) suppress ACTH and cortisol in normal subjects and the overnight low-dose dexamethasone test is used to screen patients initially before deciding on a more formal investigation. Given that hypertension is associated with obesity and that obesity is epidemic, there is a relatively large number of obese hypertensive patients in whom Cushing's syndrome is suspected. The vast majority of these obese, hypertensive people have 09.00 cortisol concentrations that suppress normally (e.g. to <50 nmol/l, Box 4.19).

‘High’-dose dexamethasone (i.e. 8 mg/l day) suppresses ACTH and cortisol secretion in patients with pituitary dependent Cushing's, an effect that will not be seen in patients who have Cushing's syndrome due to an ectopic source of ACTH. Patients with primary adrenal overactivity have unmeasurably low ACTH concentrations and cortisol concentrations that are unaffected by dexamethasone at high or low dose.

Metyrapone is a drug that inhibits the final C-11 hydroxylation in the synthesis of cortisol (Box 4.5) and is used in a test in which the peripheral blood concentration of 11-deoxycortisol, the immediate precursor of cortisol, is measured. In pituitary-dependent Cushing's disease, metyrapone reduces serum cortisol concentrations and consequently increases ACTH secretion due to reduced negative feedback. The increased ACTH drive leads to an increase in serum 11-deoxycortisol concentration. This is not seen in cases in which the Cushing's syndrome is due to ectopic ACTH (there is no increase in ACTH secretion) nor when it is due to an adrenal tumor (cortisol production is independent of ACTH).

The CRH test investigates the functional capacity of the pituitary gland using measurements of ACTH or cortisol response to an injection of CRH. Cortisol is often measured because the assays are less expensive than those for ACTH. Alternatively, ACTH measurements can be made from venous drainage of the anterior pituitary gland by simultaneous bilateral catheterization of the inferior petrosal sinuses; many regard this as the definitive investigation of Cushing's syndrome. As a less invasive test, an overnight low-dose dexamethasone test followed by a CRH test has received recent support.

Other biochemical information can also be used to help diagnosis. For example, when both ACTH and its precursors are measured, the ratio of precursors to ACTH is higher in ectopic ACTH secreting tumors (e.g. small cell tumors of the lung) than in pituitary tumors. Similarly, when urine is subjected to specialized (gas-liquid) chromatographic analysis, the ratio of adrenal steroid precursors to products may be higher in cases of adrenal tumors than in ACTH-driven disease. In addition, the clinical features may vary. For example, adrenal tumors are associated with a greater degree of androgenization than ACTH-driven disease.

These tests were not performed in Clinical Case 4.1 because the results of the high-dose dexamethasone test (ACTH suppression) and CRH tests (an exaggerated response) were conclusive and confirmed that she had Cushing's disease as a result of an ACTH-secreting pituitary tumor. This is the commonest cause of endogenous Cushing's even though it has an incidence of only about 1 per million per year. It is three times more common in women and has a peak incidence between 20 and 60 years. The cause is not certain. Some have argued for a hypothalamic cause, others that it is due to the spontaneous development of pituitary tumors. These are often very small and, indeed, may not always be visible on MR scan.

Clinical Case 4.2 had low urinary cortisol concentrations and, despite her cushingoid appearance, underactive adrenal glands had been suspected. She underwent a tetracosactrin test (Box 4.20) to investigate the functional capacity of her adrenal cortex. Thirty minutes after a 250 μg bolus injection of tetracosactrin (the biologically active 24-amino terminal amino acids of ACTH) her serum cortisol concentration rose from 15 nmol/l to 120 nmol/l (NR >500 nmol/l). In view of this poor cortisol response and the low urinary cortisol excretion, she was started on hydrocortisone treatment (20 mg/day) and referred to the endocrine team.

Box 4.20

Tetracosactrin tests and cortisol assay. Tetracosactrin is the first 24-amino terminal amino acids of the native ACTH that has 39 amino acids. It has full biological activity. Cortisol is measured in peripheral venous blood before (i.e. at baseline) and (more...)

The endocrine team reinterpreted the data in view of the clear discrepancy between the patient's appearance and the biochemical findings; no further tests were required. Clinical Case 4.2 had been taking medroxyprogesterone acetate, a C-21 compound that has the 4-ene-3-one core structure. Whilst it lacks 11β,17α,21-trihydroxyl groups, the methylation at C-6 (Box 4.17) increases the glucocorticoid activity of prednisolone some six-fold. Thus, medroxyprogesterone whilst termed a progestagen, has approximately half the biological activity (per unit weight) of cortisol at the glucocorticoid receptor.

Clinical Case 4.2 was taking 600 mg medroxy-progesterone daily. This is approximately equivalent to 300 mg of cortisol a day; some 30-fold the normal daily endogenous production of the hormone and 120 times the normal dose of medroxy-progesterone (5 mg) prescribed in many combined oral contraceptives. This suppressed ACTH secretion and reduced adrenal function, resulting in the poor response to tetracosactrin. In addition, medroxyprogesterone is thought to have some activity at the androgen receptor and this may account for the changes in hairline of this patient (note that they are much milder than those of Clinical Case 4.1, Box 4.3).

Imaging the adrenal gland

In addition to biochemical investigations, a variety of scanning techniques is used to aid the diagnosis of Cushing's syndrome (Box 4.21). Though ultrasound is a cheap and non-invasive test, the best resolution of the adrenal glands is obtained by CT or MR scanning whilst MR imaging is preferred for the pituitary gland. It is to be emphasized that these techniques not only provide an anatomical diagnosis but also functional information. Thus, a single large adrenal in the presence of a contralateral small gland would point towards an adrenal tumor producing excess cortisol, suppressing ACTH leading to atrophy of the other gland. Bilaterally enlarged glands would tend to indicate ACTH-dependent disease, regardless of the source of the ACTH.

Box 4.21

The use of radiological techniques in the investigation of Cushing's syndrome. MR pituitary The imaging technique of choice for the hypothalamus and pituitary gland, though relatively expensive and not as widely available as CT scanning

Information from imaging modalities must always be interpreted in the light of the results from endocrine investigations. For example, bilateral nodular hyperplasia of the adrenal glands can occur in the absence of excessive ACTH drive and both the pituitary and the adrenal glands are predisposed to the formation of ‘incidentalomas’. This term is given to the radiological appearance of a tumor when no functional activity is clinically apparent. Published frequencies are approximately 5% for the adrenal gland and about 25% for the pituitary gland.

Treatment of Cushing's syndrome

The most common cause (indeed, the only common cause) of Cushing's syndrome, is an exogenous, usually therapeutic, source of glucocorticoid steroid as seen in Clinical Case 4.2. The ideal form of therapy is simply to reduce the dose of the prescribed steroid and, if necessary, to use additional drugs to facilitate this reduction without flare-up of the underlying disease activity. It is essential to do this slowly and progressively to avoid precipitating acute glucocorticoid deficiency that may be fatal. This was done in Clinical Case 4.2, the dose of medroxyprogesterone being gradually tailed off over many months. Her clinical symptoms improved markedly.

Cushing's syndrome with an endogenous cause is one of the most difficult endocrine diseases to diagnose and treat accurately. On a statistical basis, the odds will be in favor of a pituitary adenoma accounting for some 80% of cases of endogenous Cushing's syndrome. Difficulties arise, however, with rare cases of alcoholic pseudo-Cushing's (as was suspected in Clinical Case 4.1), cyclical Cushing's, well-differentiated sources of ectopic ACTH, such as carcinoid, and vanishingly rare causes of Cushing's syndrome associated with food intake or ectopic CRH production (Box 4.22).

Box 4.22

Problematic Cushing's syndrome. Only a small proportion of alcoholics develop Cushingoid features: a genetic factor has been implicated. There is a poor correlation between clinical features and biochemical estimates of adrenal activity. The presence (more...)

The preferred treatment of endogenous Cushing's syndrome (whether it is caused by overactivity of the adrenal gland or increased secretion of ACTH from the pituitary gland or an ectopic source) is usually surgical removal of the cause (Boxes 4.23 and 4.24). It can be difficult, however, to locate pituitary or ectopic tumors even with the best MR imaging equipment. Thus, radiation may be used as an adjunct to surgery or drugs such as metyrapone can be used to reduce activity of the adrenal cortex.

Box 4.23

Surface and surgical anatomy of the adrenal glands. The adrenal glands are retroperitoneal structures Each gland has three arteries, a superior from the inferior phrenic, a middle from the aorta and an inferior from the renal artery

Box 4.24

Treatment of Cushing's syndrome.

In some cases, when it is impossible to remove the ectopic source of ACTH (either because it cannot be located or because it has metastasized) bilateral adrenalectomy is performed. The surgical anatomy of the adrenal glands is complex (Box 4.23) and approaches to the adrenal gland include posterior, flank and anterior. Laparoscopic techniques have been developed and tend to be used for smaller tumors of the adrenal and for those considered benign.

Replacement hormone therapy after treatment of Cushing's syndrome will, of course, depend on the underlying diagnosis and the therapy used. Thus, in pituitary Cushing's when a small tumor has been removed, no replacement may be required in the long term. However, the remaining normal corticotroph cells will be atrophied as a result of the feedback inhibition and it may be some time before they recover. Thus, replacement may be required in the short term. Larger pituitary tumors may be associated with hypopituitarism requiring additional hormone replacement. Unilateral adrenalectomy will require no replacement therapy (though, again, it may be some time before the contralateral adrenal cells recover) but bilateral adrenalectomy will always require life-long glucocorticoid and mineralocorticoid replacement therapy.

Nelson's syndrome

Clinical Case 4.1 underwent bilateral adrenalectomy rather than removal of the pituitary tumor because she had previously undergone neurosurgery after a subarachnoid hemorrhage. Bilateral adrenalectomy in patients with pituitary-dependent Cushing's disease may be followed by a marked enlargement of the ACTH-secreting tumor and the enlarged pituitary gland may cause pressure on the optic chiasm and result in visual loss. At the same time the increased ACTH secretion can cause marked skin pigmentation. This is termed Nelson's syndrome and there has been vigorous debate as to whether these tumors are naturally aggressive or develop these tendencies on removal of feedback suppression from the adrenal glands. To prevent the development of Nelson's syndrome Clinical Case 4.1 received prophylactic pituitary radiotherapy in addition to both glucocorticoid and mineralocorticoid replacement therapy.

Excess adrenal androgens - congenital adrenal hyperplasia (CAH)

Clinical Case 4.1 illustrates the effects of an excess of all adrenal steroid hormones under the control of ACTH and Clinical Case 4.2 the more selective effects of an excess of exogenous steroid with glucocorticoid (and some androgenic) actions. Clinical Case 4.3 illustrates the clinical circumstances in which there is a selective excess of adrenal androgens caused by a deficiency of an enzyme required for normal steroidogenesis.

This young patient showed signs of mild androgen excess although ‘mild’ is not a word to use to a young female patient whose anxieties have driven her to seek medical attention. Excess hair growth may be distressing for young women, particularly in a culture where models in magazines appear with every body hair air-brushed away.

The majority of androgens in women originate from steroid precursors synthesized in the adrenal cortex (Box 4.5), rather than the ovaries, but in clinical practice attention should be paid to both glands. A gross excess of androgens after puberty leads to loss of female characteristics and masculinization, but assessing mild hyperandrogenism and hirsutism can be problematic. It is important to distinguish the fine vellus hair that covers most of the body from the stiffer and thicker terminal hair whose growth and distribution is dependent on androgens (see Box 6.22). Low concentrations of androgens are required for terminal hair growth in the axillae, lower abdomen and upper thighs but higher concentrations cause growth at distances away from these areas. Pale skins and dark vellus hair may cause undue worry, although it is perfectly normal. There is also ethnic variation in the amount of body hair (particularly in ethnic groups around the equator) that should also be taken into consideration.

In this patient, baseline concentrations of androgens and the progesterone precursor were abnormally high; serum testosterone was 6.3 nmol/l (NR <2.5 nmol/l), androstenedione 39.2 nmol/l (NR 4–10.6 nmol/l), 17α-hydroxyprogesterone 150 nmol/l (NR <18 nmol/l). Thus, this patient has evidence of pathological production of androgenic steroids. Clinical Case 4.3 has a loss of function mutation in the cytochrome P450 enzyme 21-hydroxylase (Box 4.25), an enzyme essential for the synthesis of glucocorticoids and mineralocorticoids. As a consequence, the loss of negative feedback from glucocorticoids, an increased ACTH drive and an increased steroid synthesis shunted into the androgen pathway, leads to the increased production of adrenal androgens.

Box 4.25

Congenital adrenal hyperplasia (CAH) - CYP21A2 deficiency. 21-hydroxylase (CYP21A2) deficiency is the most common form of CAH accounting for 90% of all such cases. Common in Alaskan Eskimos (~1 in 700 live births) but less common in most Western countries (more...)

Several suppression and stimulation tests of the adrenal gland and the ovary are available to define the source of the excess androgens. In this case, the elevated 09.00 h 17α-hydroxyprogesterone concentration together with the observation that 3 days treatment with 2 mg dexamethasone daily suppressed serum testosterone to 1.1 nmol/l (NR <2.5 nmol/l) was evidence that the excess androgens were due to a disorder of adrenal steroid synthesis. The treatment is to remove the ACTH drive to androgen synthesis by giving exogenous glucocorticoid.

Whilst a loss of function mutation in the CYP 21A2 gene is the most common form of CAH, other enzyme deficiencies occur and the clinical features of CAH vary according to the enzyme affected, the severity of the defect and the sex of the patient (Box 4.26). The more proximal the deficiency in the steroidogenic pathway the more widespread the defect so both the adrenal glands and gonads will be affected (Box 4.26). Measuring the relative concentrations of precursor molecules will generally allow diagnosis of the specific enzyme defect. The ‘milder’ cases such as Clinical Case 4.3 are characterized by the retention of some enzyme activity and later presentation. These may require ACTH stimulation (a tetracosactrin test) or specialized chromatographic analysis of a 24 h collection of urine to make a biochemical diagnosis.

Whilst CAH is rare, a common cause of the associated features of obesity, menstrual irregularity and hirsutism is polycystic ovary syndrome (PCOS, see page 266). The etiology of this syndrome is poorly understood but is associated with hyperandrogenism. However, in almost all patients with CAH ultrasound scanning of the ovaries will reveal polycystic ovary (PCO) appearances. This was the case in Clinical Case 4.3 and this led to a marked delay in the true diagnosis. Her PCO had been considered the cause of her hirsutism rather than CAH inducing the symptoms of PCOS.

Deficiency of adrenocortical secretions - Addison's disease

Worldwide, the most common cause of Addison's may still be tuberculosis but in Western countries autoimmune disease is a more common cause of adrenal failure (Box 4.27). A relatively recent development has been the effect of the human immunodeficiency virus (HIV). This has increased the incidence of hypoadrenalism due to infectious agents including viruses such as cytomegalovirus, fungi such as histoplasmosis, coccidiomycosis or blastomycosis, bacteria such as tuberculosis or the drugs that are used to treat these agents. AIDS itself may be associated (by an as yet unknown process) with generalized resistance to glucocorticoid effects of cortisol. Clinical Case 4.4 illustrates some of the clinical features of Addison's disease.

Box 4.27

Causes of hypoadrenalism. Abrupt cessation of exogenous sources of glucocorticoids Primary (Addison's disease)

Clinical Case 4.4

A 43-year-old married woman was referred to the outpatient department with increasing skin pigmentation and weight loss (Box 4.28). There was no obtainable family history of any illness and, apart from lethargy, she denied any other problem. She had two healthy children. She was taking no medication. A forthright lady, she professed a hearty dislike for both medical and dental surgeries. She had a supine systolic blood pressure of 50 mmHg (that became unrecordable when standing) but adamantly refused hospital admission.

Box 4.28

Clinical features of Clinical Case 4.4. Left: Photograph of face at presentation Above: Photograph of mouth showing marked pigentation of gums, buccal mucosa and tongue

Taken together, the major features of this case indicate primary adrenal failure i.e. Addison's disease (Box 4.29). The low systolic blood pressure is indicative of a deficiency of both glucocorticoids and mineralocorticoids, weight loss, due to reduced appetite, is a consequence of cortisol deficiency and skin pigmentation is caused by excess ACTH (the result of loss of glucocorticoid negative feedback).

Box 4.29

Clinical features of Addison's disease. Weakness (~100%) Weight loss (~100%)

Skin pigmentation

The pigmentation of Addison's disease is so noteworthy that few cases of primary hypoadrenalism reach the severity of that seen in Clinical Case 4.4 although difficulties may arise in the case of Afro-Caribbeans or Asians where the increase in pigmentation may not be detected so easily. In conditions associated with high circulating concentrations of ACTH (e.g. primary adrenal failure or ectopic production), melanosome function within the melanocytes is stimulated. This is because ACTH is equipotent with melanocyte-stimulating hormone (MSH) at the G-protein linked melanocortin-1 receptor. Thus, ACTH stimulates melanin production.

Skin color, along with religion, politics and wealth, is one of the most divisive factors in the human condition yet it is simply the interplay between the pigments melanin and hemoglobin. Constitutive skin color is determined by: the different ratios of eumelanin (brown/black) and pheomelanin (yellow/red) in the skin; the number of melanosomes; the rate of melanogenesis and the rate of transport of melanin from the melanocytes to the keratinocytes. Facultative skin color depends on the response of melanocytes to UV light and hormones (Box 4.30).

Box 4.30

Skin pigmentation. Melanins are made from tyrosine. The initial reaction involves the enzyme tyrosinase; subsequent enzymic reactions form complex polymers. In the absence of cysteine, eumelanin (black) is formed, in the presence of this amino acid pheomelanin (more...)

Aldosterone and the control of salt and water balance

The next clinical case is one in whom the biochemical changes in the concentration of plasma sodium were the most noteworthy feature, not skin pigmentation. It serves to introduce the functions and control of the mineralocorticoid, aldosterone.

A low serum sodium concentration or hypo-natremia is one of the commonest medical problems, affecting approximately 5% of all hospital inpatients. This man caused great diagnostic confusion and, as a result, his case is very educative. In brief, he had euvolemic hyponatremia (Box 4.31). That is, there was no evidence of depletion in circulating blood volume (such as postural hypotension, decreased skin turgor and sense of thirst) nor was there any sign of excess extracellular fluid (ECF) such as edema or ascites (accumulation of fluid in the peritoneal cavity). To interpret this case, a more detailed understanding of the control of salt and water is essential.

Box 4.31

Classification of hyponatremia. This classification is based on simple clinical signs. Patients are thirsty, have a low blood pressure (that falls still further on standing), a tachycardia, and decreased skin turgor

Aldosterone, secreted by the glomerulosa cells of the adrenal cortex, stimulates the active uptake of sodium (Na⁺), and consequently water, from the glomerular filtrate in the distal tubules of the kidney. Aldosterone synthesis and release is controlled by the renin-angiotensin system (Box 4.33). Smooth muscle cells in the afferent and efferent arterioles of the kidney synthesize, store and release renin. The release of this enzyme is stimulated by a reduced perfusion pressure in the kidney, increased activity of sympathetic nerves innervating the smooth muscle cells or a reduction in Na⁺ delivery to the macula densa.

Once released, renin cleaves angiotensinogen to angiotensin I and this peptide is further converted by angiotensin-converting enzyme (ACE), found in the endothelial cells of the lung and kidney, to the octapeptide, angiotensin II. Angiotensin II then acts on the glomerulosa cells of the adrenal cortex to stimulate the production of aldosterone.

The action of aldosterone on the distal convoluted tubule cells of the kidney is mediated by cytoplasmic receptors that, like the glucocorticoid receptors, translocate to the nucleus of target cells after hormone binding (Box 4.32). The hormone-receptor complexes initiate the synthesis of proteins involved in active Na⁺ uptake in the kidney through Na⁺ selective ion channels. As a result of the sodium reabsorption, the transepithelial voltage is increased (tubular lumen negative) and there is a passive movement of Cl^- from the lumen to the blood. Thus, both Na⁺ and Cl^- are retained, water follows down the osmotic gradient and ECF volume increases.

Box 4.32

Aldosterone actions in the kidney. In the circulation aldosterone (A) is mainly bound to cortisol-binding globulin (CBG) or albumin. Free aldosterone enters the tubule cells of the kidney and binds to the mineralocorticoid receptor (MR). This induces (more...)

Integrated endocrine control of salt and water balance

Aldosterone stimulates Na⁺ and water retention, helping to maintain salt and water balance and, thus, blood pressure. The two other major hormones involved in this control are atrial natriuretic peptide (ANP) and arginine vasopressin (AVP), otherwise known as antidiuretic hormone (ADH) (Box 4.34).

Box 4.34

Simplified diagram illustrating the integrated actions of aldosterone, arginine vasopressin (AVP) and atrial natriuretic peptide (ANP) in the control of salt and water balance. The renin/angiotensin system stimulates aldosterone secretion, and angiotensin (more...)

ANP antagonizes the overall effects of aldosterone i.e. it promotes the excretion of sodium and, thus, reduces ECF volume. The 28 amino acid peptide is synthesized and stored in atrial myocytes. An increase in atrial tension caused by an increase in central venous pressure (CVP) stimulates ANP release. ANP inhibits Na⁺ reabsorption in the distal convoluted tubules and collecting ducts via a cGMP-dependent mechanism. It also inhibits AVP, aldosterone and renin secretion and increases the GFR (hence, the sodium load delivered to the kidneys). The overall effect is to reduce the ECF volume.

Normally, Na⁺ balance determines the ECF volume and thus blood pressure and the perfusion pressure within the vascular system. An increase in ECF stimulates Na⁺ and water excretion through ANP release. A decrease causes Na⁺ and water retention through aldosterone secretion. Sodium salts are the major determinants of osmolality in the ECF since they are the most abundant solutes. Changes in sodium balance affect serum osmolality.

Regulation of serum osmolality is achieved by the action of AVP decreasing solute-free water clearance by the kidney (i.e. retention of water without electrolytes). Increases in osmolality are detected by osmoreceptors in the hypothalamus and these stimulate AVP secretion from the magnocellular neurosecretory cells in the supraoptic and paraventricular nuclei of the hypothalamus. AVP increases the reabsorption of water in the kidney by inserting water channels (aquaporins) into the membranes of tubular cells in the distal convoluted tubules and collecting ducts. Its secretion is inhibited by a reduction in serum osmolality resulting in reduced water reabsorption and increased excretion.

AVP secretion is also stimulated by a reduced ECF volume. This is achieved through low-pressure volume receptors in the cardiac atria and pulmonary vessels. High-pressure sensors in the aortic arch, carotid sinus and the afferent arterioles of the kidney inhibit AVP secretion. Thus, whilst AVP controls solute-free water balance maintaining both osmolality and ECF volume, aldosterone and ANP regulate the ECF volume by controlling Na⁺ balance. The relationship between Na⁺ balance and ECF volume is complex, particularly under certain pathological conditions.

Hyponatremia

Hyponatremia can be classified into hypo- hyper- and euvolemic and the classification is based on simple clinical signs (Box 4.31). It is evident that hyponatremia, as seen in Clinical Case 4.5 can occur as a result of excess water intake, decreased water excretion, deficient Na⁺ intake or excess loss of the cation. The abundance of Na⁺ in the environment means that a deficient intake is virtually never seen. A deficiency can arise if doctors (unthinkingly or unwisely) replace the body's loss of salt-containing body fluids (e.g. diarrhea, sweat or plasma) with intravenous solutions containing only a sugar and water (e.g. 5% dextrose).

Since there was no evidence for a large oral intake of fluids or loss of any body fluid, the initial working diagnosis of Clinical Case 4.5 was decreased water excretion due to the inappropriate secretion of AVP. This was thought to have resulted as a result of a head injury in the road traffic accident. The syndrome of inappropriate antidiuresis (SIAD) is incompletely understood but results from a defect in the excretion of free-water. It is generally seen in conditions affecting the nervous system or the chest and is diagnosed when patients have euvolemic hyponatremia, normal renal function and when other causes of decreased free water clearance (i.e. hypothyroidism and hypoadrenalism) have been excluded. In normal circumstances AVP, secreted by the parvicellular neurons, potentiates the action of CRH but in hypoadrenalism (with the loss of negative feedback) AVP secretion into the hypophyseal portal capillaries leads to an increase in AVP concentration in the general circulation with secondary effects on renal free-water clearance.

After several days of fluid restriction (1000 ml/ day) (Box 4.35) a short tetracosactrin test (Box 4.20) was performed on Clinical Case 4.5. The baseline serum cortisol concentration was low (45 nmol/l) and the response to tetracosactrin subnormal (30 min value 210 nmol/l, NR >500 nmol/l). A diagnosis of Addison's disease was made and he was treated with 10 mg hydrocortisone (cortisol) twice daily and referred to the Endocrine team.

Box 4.35

Treatment of hyponatremia. Two important principles should always be applied: An accurate diagnosis should be made and the underlying cause e.g. hypoadrenalism or hypothyroidism in the case of euvolemic or, say, cardiac failure in edematous states, treated (more...)

The Endocrine team was concerned about the diagnosis because of the lack of family history of autoimmune disease and the absence of skin pigmentation. An alternative diagnosis of post-traumatic hypopituitarism was considered and pituitary function tests were performed. These showed that he was also hypogonadal (serum testosterone 4.5 nmol/l, NR 9–25 nmol/l) with normal gonadotrophins (serum luteinizing hormone concentration 2.3 UI/l, follicle stimulating hormone 1.5 UI/l) and somato-trophin deficient (peak serum somatotrophin 5.6 mU/l after insulin induced hypoglycemia). A ‘long’ tetracosactrin test (Box 4.20) was, therefore, performed when the patient was taking 5 mg prednisolone daily rather than cortisol that would have been measured along with endogenous cortisol in the radioimmunoassay. This showed that the basal serum cortisol was unmeasurable and that over the succeeding 5 days it rose to 123 nmol/l (NR >1000 nmol/l, i.e. there was a poor cortisol response despite adequate stimulation with ACTH).

A diagnosis of primary hypoadrenalism was made and subsequently autoantibodies against the adrenal cortex were detected by immunofluorescence supporting a diagnosis of autoimmune Addison's disease. The hypogonadism and somatotrophin deficiency were considered to be the result of pituitary damage resulting from the head injury (i.e. secondary hypogonadism) even though the apparent injury had been quite minor. An MR scan of the pituitary gland was normal.

This patient was treated with androgens and given advice about his replacement glucocorticoids. However, his subsequent course revealed the difficulties associated with adequate patient education and the potent effects of glucocorticoid steroids on the brain.

He was admitted to hospital as an emergency some weeks later in a psychotic state. It transpired that he had developed a chest infection and had taken additional quantities of his cortisol as well as the prednisolone that had been prescribed by his primary care physician together with antibiotics for his infection. He had taken to doubling the steroid dose whenever he felt in the least unwell and on admission was taking in excess of 100 mg of prednisolone daily (roughly equivalent to 20 times the daily cortisol production) plus his replacement cortisol. The dose was reduced to normal over several weeks and his mental state improved.

It has been estimated that 5% of all hospital admissions are due to the unwanted effects of prescribed drugs and the next clinical case is a further example. It is used to illustrate the clinical importance of the transport and metabolism of adrenal steroids.

Transport and metabolism of adrenocortical steroids

Whilst thyroid hormones are stored, there is virtually no storage of steroids within the adrenal gland and, thus, their secretion requires an activation of the biosynthetic pathway. However, adrenocortical steroids share a number of features with thyroid hormones. They are relatively insoluble in aqueous solution and are bound to circulating proteins, with relatively small quantities of each steroid (<10%) circulating in a biologically active- free state.

The main glucocorticoid, cortisol, binds to corticosteroid-binding globulin (CBG or transcortin) whilst the main androgens (and estrogens) are transported attached to sex-hormone-binding globulin (SHBG). Both these specific transport proteins have high affinities for their respective hormones (Box 3.36) and normally carry 75–80% circulating hormones. A smaller percentage is bound to albumin that has a low affinity but a high capacity for the hormones. Like thyroxine-binding globulin (TBG), these proteins are synthesized by the liver and their concentrations in blood are altered by a number of factors, particularly by pregnancy and estrogen administration when their synthesis increases. The uptake of steroids by cells from capillary blood occurs by diffusion from the free hormone pool although, as with thyroid hormones, there is experimental evidence for specific transport mechanisms.

In the circulation, cortisol is in equilbrium with its biologically inactive 11-keto analog, cortisone. 11β-hydroxydehydrogenase type 2 inactivates cortisol whilst the type 1 enzyme converts inactive cortisone to cortisol. The enzymes are present in many tissues but of particular note is the inactivation of cortisol in kidney cells to prevent cortisol interacting inappropriately with aldosterone receptors (Box 4.11).

Box 4.36

Transport of steroids in plasma.

Both cortisol and cortisone are mainly metabolized in the liver (Box 4.37) and the reduced metabolites are conjugated and excreted in the urine as glucuronides. Measurement of cortisol metabolites in the urine provide a useful clinical index of cortisol secretion. Particularly useful are the 17-hydroxycorticoids since these metabolites represent up to 50% of the total cortisol secretion. As discussed above, urinary free cortisol may be measured as a surrogate of daily secretion. The major androgens secreted from the adrenal cortex are androstenedione and dehydroepiandrostenedione (DHEA) and its sulfated form (DHEA-S). Androstenedione is reduced to androsterone in the liver prior to excretion whilst DHEA-S is excreted directly into the urine. Measurement of urinary or serum DHEA-S can indicate an adrenal abnormality.

Box 4.37

Metabolism of cortisol and aldosterone. Cortisol has a t_1/2 in the circulation of about 100 min. The small amount of cortisol excreted in the urine unchanged is representative of the daily secretion of cortisol. With high serum cortisol concentrations (more...)

In the case of Clinical Case 4.6, the patient was initiated on anti-epileptic medication with no change in the dosage of glucocorticoid replacement. The phenytoin led to the induction of liver enzymes involved in the metabolism of the steroids and subsequently to glucocorticoid deficiency. She responded well to an increase in hydrocortisone dose.

Selective mineralocorticoid excess and deficiency

The most common causes of increased mineralocorticoid secretion, accounting for approximately 99% of all cases of hyperaldosteronism, are not due to a primary increase in aldosterone synthesis but to a secondary cause (Box 4.38). As has been seen, the secretion of large amounts of adrenocorticoids with mineralocorticoid actions (though not necessarily aldosterone itself) occurs pari pasu in Cushing's syndrome (probably accounting at least in part for the hypertension in Clinical Case 4.1) and in some of the syndromes of congenital adrenal hyperplasia.

Box 4.38

Causes of mineralocorticoid excess. Secondary hyperaldosteronism Physiological

Excessive production of aldosterone itself (termed primary hyperaldosteronism) is a rare disease that is clinically nondescript. Patients present with systemic hypertension and the only distinguishing feature of this disease is that serum analysis will usually indicate hypokalemia with an alkalosis. This is due to the excess aldosterone-induced sodium retention by the kidney in exchange for K⁺ and H⁺ that are lost in urine. Primary hyperaldosteronism is usually due to an adrenal adenoma (Box 4.38). Typically, patients are between the ages of 30 and 50 years and are more often female than male. They form much less than 1% of all cases of systemic hypertension in this age group but are important in having a surgically curable form of hypertension.

Once suspected, hyperaldosteronism can be confirmed by the measurement of 24 h urine aldosterone and by investigation of the feedback loop between renin and aldosterone. Thus, serum renin concentrations are suppressed and serum aldosterone concentrations cannot be suppressed by normal measures. A variety of tests has been used to improve sensitivity in the detection of primary hyperaldosteronism in the at-risk hypertensive patient population. These include the use of aldosterone/renin ratios and the use of postural changes. It is important to emphasize that the sensitivity of many tests is reduced by a low-salt diet; investigation may require dietary supplementation with 6 g sodium chloride daily.

The same imaging techniques and analysis used for diagnosing Cushing's syndrome (Clinical Cases 4.1 and 4.2) also apply to hyperaldosteronism with CT or MR scans proving the most useful in detecting adrenal adenomas. It can either be treated surgically or medically with the aldosterone receptor antagonist spironolactone, the angiotensin II antagonists such as candesartan or losartan or the angiotensin-converting enzyme (ACE) inhibitors such as captopril or ramipril.

Mineralocorticoid deficiency occurs with glucocorticoid deficiency as a result of adrenal failure. Isolated hyporeninemic hypoaldosteronism occurs occasionally in diabetic patients with renal impairment. In severe cases, replacement therapy with fludrocortisone is required, though care is required to avoid inducing heart failure.

The adrenal medulla and pheochromocytoma

The adrenal medulla forms part of the sympatho-adrenal division of the autonomic nervous system. It has been known for over a hundred years that, when bilateral adrenalectomy is performed on experimental animals, replacement of adrenal cortical hormones is an absolute requirement for life. The same is not true of epinephrine and norepinephrine secreted by the adrenal medulla. One could conclude, therefore, that the adrenal medulla is not important clinically. Strictly speaking this may be true except for the rare tumors of the adrenal medulla that secrete excess catecholamines and often go undiagnosed.

The diagnosis of such pheochromocytomata is often made for the first time at post mortem and a study at the Mayo Clinic showed that nearly 80% of cases were unsuspected in life. The pathophysiology of the adrenal medulla is, thus, important in states associated with excess catecholamine secretion and this is illustrated in Clinical Case 4.7.

The clinical presentation in this case may seem bizarre and, indeed, it is probably the reason the diagnosis was not made for several years. In order to understand the presenting symptoms, it is important to detail the synthesis and actions of catecholamines.

Catecholamine synthesis and secretion

The adrenal medulla consititutes less than 20% of the adrenal gland. The cells are polygonal and arranged in cords. They receive blood either directly from medullary arterioles or from the venules of the cortex (rich in cortisol) that drain centripetally to medullary venules. Epinephrine and lesser amounts of norepinephrine are synthesized by and secreted from the chromaffin cells of the medulla in response to stimulation of pre-ganglionic (cholinergic) sympathetic nerves originating in the thoraco-lumbar lateral gray matter of the spinal cord. Chromaffin cells are so named because their affinity for chromium salts leads to characteristic staining. As modified post-ganglionic nerve cells, they are classical neurosecretory cells - neurons releasing hormones into the general circulation.

The catecholamines, like melatonin and thyroid hormones, are synthesized from tyrosine (Box 4.39) but unlike thyroid hormones, they are made from single tyrosine molecules. These are either synthesized from phenylalanine or imported from the circulation. The rate-limiting step in the synthesis of catecholamines is that catalyzed by tyrosine hydroxylase, converting tyrosine to dihydroxy- phenylalanine (DOPA). Subsequent decarboxylations and hydroxylations oulined in Box 4.39 convert DOPA to dopamine, norepinephrine and finally to epinephrine (catalyzed by the enzyme aromatic l-amino acid decarboxylase) results in the formation of dopamine. Further hydroxylation, catalyzed by dopamine β-hydroxylase takes place in secretory granules (unlike the other enzymatic processes) and results in norepinephrine. In some adrenal medullary cells, the synthetic process stops at norepinephrine but in most cells (and particularly those at the corticomedullary junction) it is converted to epinephrine by phenylethanolamine N-methyltransferase (PNMT). The activity of this enzyme is markedly increased by the high cortisol concentrations reaching the medulla.

Through an energy-requiring process, catecho-lamines are stored in secretory granules in association with ATP (four catecholamine molecules to one ATP) and a number of proteins, including adrenomedullin. Many functions of these proteins remain to be elucidated though some play a role in the storage mechanism since the intragranular concentration of catecholamine is such that they would cause osmotic damage if they existed free in solution. The output of the adrenal gland is controlled from nerve cells within the posterior hypothalamus which can ultimately stimulate acetyl- choline release from preganglionic nerve terminals. This induces depolarization of the chromaffin cells and exocytosis of the catecholamine containing granules following a transient rise in intracellular calcium concentration. Once secreted their t_1/2 in the circulation is very short (approximately 1–2 min).

Actions and metabolism of catecholamines

Catecholamines act on their target tissues through typical G-protein-linked membrane receptors. These receptors are classified as α or β on the basis of the physiological and pharmacological effects induced by hormone binding (Box 4.40). Further subclassification into α_1A, α_1B, α_2A, α_2B, β₁, β₂, β₃ is also made according to the activation or inhibition of different signal transduction pathways.

Box 4.40

Effects of catecholamines. Increase in heart rate and force via β₁-receptors Increased venous return via α-receptors

The physiological effects of the catecholamines are manifold and summarized in Box 4.40. They have been characterized as preparing us for ‘flight or fight’ with overall actions to increase heart rate and stroke volume, increase blood pressure, dilate bronchi, mobilize glucose and stimulate lipolysis. These actions are mediated by β-adrenergic receptors. Blood flow to the splanchnic bed is reduced by vasoconstriction of arterioles. This effect is mediated by α-adrenergic receptors and it helps to divert blood flow to skeletal muscles.

Whilst most catecholamines released from sympathetic nerves are taken back up into the pre-synaptic terminal (termed uptake₁), catecholamines released into the circulation are taken up by non-neuronal tissues (uptake₂) and rapidly converted to deaminated products by monoamine oxidase (MAO) or to O-methylated products by catechol O-methyltransferase. The latter enzyme also catalyzes the meta-O-methylation of the products of MAO action - metanephrine, normetanephrine, epinephrine and vanilyl mandelic acid (Box 4.41). These may then be conjugated with glucuronide or sulfate and excreted in the urine.

Box 4.41

Catecholamine metabolism - diagnosis of pheochromocytoma. Small quantities of free norepinephrine (~0.5%) or conjugated with sulfate (~2%) are excreted in the urine Catechol O-methyl transferase (COMT) converts the catecholamines to metanephrine and normetanephrine (more...)

Diagnosis and treatment of pheochromocytomas

The clinical features of pheochromocytomas, many of which could be predicted from the known actions of catecholamines, are given in Box 4.42. Surges of catecholamine secretion can induce paroxysmal symptoms and many precipitants of catecholamine secretion are known. These include tumor palpation and drugs, particularly anesthetic agents. Operations on people with undiagnosed pheochromocytomas can be fatal. In Clinical Case 4.7, it seems likely that her particularly severe attack of abdominal pain and faintness that led her to seek immediate medical attention was caused by leaning over the edge of a chest freezer to remove ice. The pressure on the abdomen could have released catecholamines from the tumor.

Box 4.42

Clinical features of pheochromocytoma. Headache (~60%) Palpitations (~60%)

The mechanism of her postural hypotension remains to be explained. Two mechanisms have been proposed, receptor down-regulation and the release of vasodilator peptides co-localized in the catecholamine secretory granules. Prolonged exposure of adrenergic receptors to high circulating concentrations of catecholamines reduces the number of receptors (down-regulation) and also causes desensitization. Desensitization occurs by phosphorylation of the receptors and eventually a complete functional uncoupling of the receptor from its G-protein. Receptors can then be internalized into the cell. Thus, down-regulation and desensitization may lead to a reduction in the normal sympathetic vasoconstrictive tone (β-receptor mediated) and postural hypotension. In addition, peptides released with the catecholamines, notably adrenomedullin, are potent vasodilators and may also play a role in the hypotension observed in Clinical Case 4.7.

Overall, these tumors are rare (~1 per million per annum) and usually benign. They are found in the sexes equally and have a maximum incidence between the ages of 20 and 50 years, though they can occur at any age. In general, it is said that 10% are bilateral, 10% are extra-adrenal, 10% occur in childhood and that 10% are malignant. The majority of pheochromocytomas are sporadic and without known cause. Some occur in MEN type 1 (Box 5.40).

The first step in the diagnosis of these tumors is awareness of their possibility. The second is confirmation of the diagnosis biochemically. By and large, physicochemical analysis of catecholamine concentrations is performed using high-performance liquid chromatography (HPLC) with electrochemical detection. The assays with the lowest false negative rates are those for urinary and plasma catecholamine metabolites, metanephrines and normetanephrines. Chromatographic analysis of several 24 h urine collections from Clinical Case 4.7 showed that her excretion of catecholamines was several times higher than normal.

Several stimulation and suppression tests are also available but the safest are the glucagon stimulation and the clonidine or pentolinium suppression tests. These are based on the principles that catecholamine secretion from a pheochromocytoma (but not normal adrenal medulla) is stimulated approximately 2–5-fold by glucagon whilst catecholamine secretion from a pheochromocytoma is not suppressed by clonidine or pentolinium. These drugs suppress catecholamine secretion by at least 50% from a normal adrenal medulla.

The third step in the diagnosis of pheochromocytomata is their localization, usually with CT or MR scanning. These have sensitivities of about 98% and specificities of about 70%. ‘Functional’ scans can be performed using meta-iodobenzylguanidine (or MIBG). MIBG is taken up by the tumor by the uptake₁ process and the technique has a sensitivity of about 80% but nearly 100% specificity. The tumor of Clinical Case 4.7 was localized to the right adrenal gland and lack of any other areas of uptake suggested that there were no functional metastases (Box 4.43). Since pheochromocytomata may occur in a number of positions outside the adrenal glands MIBG scanning is extremely helpful when it is positive.

Box 4.43

MIBG scanning. This image is available on the website.

The only form of curative therapy (Box 4.44) is complete surgical removal of the tumor after initial medical treatment. The latter is required to reduce the risks of acute release of catecholamines in response to anesthetic drugs and surgical handling. The usual pre-operative treatment is initially with α-adrenegic blockade followed by combination α- and β-adrenergic blockade (Box 4.44). This avoids the increase in blood pressure that can be seen if β-blockade is initiated and there is unopposed α-adrenergic vasoconstrictive activity. Other treatments such as radiotherapy or chemotherapy are rarely successful but may be used as palliative therapy. More recently, large doses of radiolabeled MIBG have also been given for the palliation of metastatic disease.

Box 4.44

Treatment of pheochromocytoma.

Clinical case questions

Clinical Case Study Q4.3

A 64-year-old woman was seen in the Endocrine clinic because she had noticed increasing hirsutism for 6 years but worse over the last year. She had also noted increasing fatigability, and some left-sided abdominal pain. Her voice had become deeper but she had a normal appetite and no weight loss. She had a past history of mild hypertension and had had one child. She smoked 10 cigarettes a day. Examination revealed marked hirsutism with temporal recession of the hairline and a beard (Box Q4.3a). The blood pressure was 200/110 mmHg.

Box Q4.3a

Clinical photograph of the face of Clinical Case Study Q4.3. Note the marked male pattern balding and the beard.

Question 1: What initial investigations would you perform?

Question 2: Following the receipt of these results, what further investigations would you perform?