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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 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).
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, P450scc, (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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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 P450SCC 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).
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.
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.
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.
‘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.
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.
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.
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.
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.
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).
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.
A 26-year-old woman was referred to the Endocrine clinic because of increasing facial hair. A nursery school teacher with Greek parents, she had her menarche at 11 years of age but had always noted irregular periods and a tendency to be overweight. When seen, she was 1.70 m tall with a weight of 87 kg. On examination, she was obese but had no clinical evidence of glucocorticoid or mineralocorticoid excess and her blood pressure was normal. She had, however, excess facial hair, areolar hairs on the breasts, male pattern pubic hair with an extension up the linea alba in the midline of the lower abdomen. There was hair on the inner thighs but none on her back. There was no clitoral hypertrophy, breast atrophy or other signs of masculinization such as deep voice and muscular development. It had previously been suggested to her that the excess hair was a consequence of her Mediterranean origins and she was very resentful that her sister (a child of the same parents) was not similarly affected.
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.
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.
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.
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.
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.
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).
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).
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 26-year-old man was admitted to hospital via the Emergency Room with extreme fatigue and malaise. Some 7 weeks earlier he had been seen in the same department following a road traffic accident in which he had been knocked off his bicycle by a car. He was normally employed in the computer industry and his fiancée reported a general decrease in his intellectual abilities. When examined, there were no focal neurological signs and an emergency CT scan of his head was normal. He was normotensive with no abnormal physical signs; his blood pressure was 120/80 mmHg both lying and standing. His serum sodium was reported to be 109 mmol/l (NR 135–145 mmol/l).
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.
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.
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).
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.
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.
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.
A 36-year-old woman presented to hospital having suffered her first tonic-clonic epileptic seizure. She had fallen off a horse some 18 months previously while on holiday in Israel and had undergone neurosurgery to remove an intracranial hematoma. She had been told that her pituitary gland had been damaged by the head injury and had been treated with daily doses of hydrocortisone (15 mg), thyroxine (125 μg) and the synthetic AVP analog, desmopressin (20 μg). She was admitted and a CT scan of the brain confirmed structural brain damage presumed secondary to the previous injuries. She was discharged with the anti-epileptic medication, phenytoin, and the same doses of replacement therapy. Some 4 weeks later she was admitted with general malaise, vomiting and a low blood pressure of 70/40 mmHg. Her admission was precipitated by the initiation of her anti-epileptic medication.
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).
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.
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.
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.
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 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.
A 45-year-old female university lecturer was admitted via the Emergency Room with a 2-year history of short-lasting episodes of right-sided upper abdominal pain and faintness. Her gastrointestinal and hepatobiliary systems had been repeatedly investigated to no avail. Numerous visits to the primary care physician had not provided a diagnosis and she vigorously refuted previous suggestions that the episodes were due to depression or associated with hyperventilation or panic attacks. On the day of admission, a particularly severe attack had been precipitated by the activities required to defrost her deep-freeze. When she was seen in the Emergency Room, examination of the abdomen was normal but her blood pressure was recorded as 120/80 mmHg supine, falling to 80 mmHg on standing.
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.
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.
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 t1/2 in the circulation is very short (approximately 1–2 min).
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 α1A, α1B, α2A, α2B, β1, β2, β3 is also made according to the activation or inhibition of different signal transduction pathways.
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.
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.
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).
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.
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.
A 55-year-old woman presented with classical clinical features of Cushing's syndrome including hypertension, diabetes mellitus, central obesity and easy bruising. She denied previous depression or heavy alcohol intake and was not receiving any steroid containing medication.
Question 1: List the causes of Cushing's syndrome and discuss what initial investigations you would perform?
Question 2: In the light of these results, what additional tests would you perform?
Question 3: What is the likely cause of this patient's recurrent Cushing's and what further investigations should be performed?
A 33-year-old Afro-Caribbean woman presented to the Emergency Room with a 3 day history of sore throat and anorexia and a 1 day history of strange behavior. In retrospect, she said she had needed to take six teaspoons of sugar in each tea and felt odd if she did not eat. She was admitted having been found in bed convulsing and incontinent of urine and feces. There was no history of previous illness nor was she taking any medication. On examination, there were no visible injection sites and she was pyrexial (temperature 39.8°C) with a blood pressure of 100/60 and pulse 88 min/l. Chest examination was normal and neurological examination made difficult by un-cooperativity. Investigations showed normal hemoglobin and white cell count, a serum sodium of 130 mmol/l (NR 135–145 mmol/l), potassium of 3.0 mmol/l (NR 3.5–4.7 mmol/l) and urea 5.6 mmol/l (NR 2.5–8.0 mmol/l) but the initial serum glucose was 0.6 mmol/l. The supine chest X-ray was reported normal as were CT head scan and lumbar puncture but blood cultures grew Streptococcus viridans.
Question 1: This patient has a Streptococcus viridans septicaemia and a tonic-clonic fit secondary to hypoglycemia. She was treated with high doses of intravenous penicillin and a glucose infusion. How would you investigate the cause of hypoglycemia in this patient?
Question 2: Given these findings, what further investigations would you perform?
Question 3: In the light of these results, what investigation would you perform?
Question 4: How do you account for the normal cortisol response to tetracosactrin?
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.
Question 1: What initial investigations would you perform?
Question 2: Following the receipt of these results, what further investigations would you perform?
