34

Disorders of the pituitary-adrenocortical axis

34

Disorders of the pituitary-adrenocortical axis

34

Disorders of the pituitary-adrenocortical axis

34

Disorders of the pituitary-adrenocortical axis

  34 Disorders of the pituitary-adrenocortical axis

Lothar Thomas

34.1 Pituitary-adrenocortical axis

The hypothalamic-pituitary-adrenocortical axis is a classic neuroendocrine system sub serving control of the adrenocortical glucocorticoid and mineralocorticoid secretion by the brain /1/. Pulsatile glucocorticoid production arises due to a subhypothalamic pulse generator and is the intrinsic property of the free-forward feedback interplay between the pituitary and adrenal glands. The pulsatile hormone signal is decoded at the ellular level by the intracellular glucocorticoid receptor (GR), the mineralocorticoid receptor (MR), or both GR and MR in cell types where the two are coexpressed. The adrenal cortex secretes cortisol under the control of pituitary adrenocorticotropic hormone (ACTH) and aldosterone under the control of angiotensin II and the K+ concentration in plasma.

34.1.1 Corticotropic cells of the anterior pituitary

The corticotropic cells of the anterior pituitary produce ACTH by proteolytic processing of proopiomelanocortin (POMC). The corticotropic cells are controlled by stimulatory hypothalamic factors (Fig. 34.1-1 – Hypothalamic-pituitary-adrenocortical axis). The most potent of these is corticotropin releasing hormone (CRH). CRH stimulates the expression of the POMC gene and increases ACTH secretion through the G-protein coupled CRH receptor-1. Unlike the cells of the adrenal cortex, which require constant stimulation by ACTH, the corticotrophic cells of the anterior pituitary do not need to be stimulated continuously by CRH. Weak ACTH secretion is also stimulated by angiotensin/vasopressin and oxytocin through the V1b vasopressin receptor, also known as the V3 receptor /1/.

34.1.2 Adrenal cortex

Cells in the zona fasciculate of the adrenal cortex synthesize and secrete glucocorticoids in response to ACTH stimulation. Adrenal steroid production results from the binding of ACTH to the G-protein coupled melanocortin-2 receptor. ACTH plays an important role for the trophic support of the adrenal cortex. Chronic ACTH deficiency causes apoptosis and loss of secretory capacity of the adrenal cortex. Chronic ACTH stimulation, on the other hand, leads to hyperplasia of the zona fasciculate with an increase in size, cell number and secretory activity.

Glucocorticoid secretion is dependent on the ACTH concentration and stimulation by adrenal nerves. Cortisol is the main glucocorticoid secreted in response to ACTH stimulation /2/.

Cells in the zona glomerulosa of the adrenal cortex synthesize and secrete aldosterone. The enzyme aldosterone synthase converts corticosterone to aldosterone with intermediate production of 18-hydroxycorticosterone. These conversions are controlled by the renin angiotensin-II-system and the concentration of K+ in serum.

The synthetic pathways of the adrenocortical steroids are shown in Fig. 34.1-2 – Biosynthesis of adrenocortical steroids.

34.1.3 Adrenal corticosteroid biosynthesis

Adrenal corticosteroid steroids are essential for life and the maintenance and adaption of their biosynthesis is regulated by the hypothalamic-pituitary-adrenocortical axis.

The 27 carbon-containing cholesterol molecule is the precursor for the 5 steroid hormone classes /3/ (Fig. 34.1-2 – Biosynthesis of adrenocortical steroids):

  • Glucocorticoids (e.g. cortisol, corticosterone), 21 carbons; synthesis site adrenal cortex
  • Mineralocorticoids (aldosterone, deoxycorticosterone), 21 carbons; synthesis site adrenal cortex
  • Androgens, 19 carbons; the androgen precursors dehydroepiandrosterone (DHEA) and androstenedione are synthesized in the adrenal cortex, in the testis testosterone is produced
  • Estrogens, 18 carbons (Fig. 37.1-1 – Biosynthesis of sex steroids); synthesis site ovary
  • Prostagens (e.g., progesterone), 21 carbon steroids; synthesis site corpus luteum (Fig. 37.1-1 – Biosynthesis of sex steroids).

Steroid hormones are produced from cholesterol precursors (Fig. 34.1-2 – Biosynthesis of adrenocortical steroids):

  • Through modification of bonds within the four fused rings; three cyclohexane (C6) rings and one cyclopentane (C5) ring
  • By altering the locations of single and double bonds between the carbon atoms
  • By oxidations and reductions at locations along the steroid backbone and at side chains.

Steroidogenesis for the synthesis of cholesterol precursors to cortisol, to sex hormones and to aldosterone occurs in four steps /3/ (Fig. 34.1-2 – Biosynthesis of adrenocortical steroids):

Step 1: Cholesterol to precursors

  • Cholesterol conversion to pregnenolone by side chain cleavage: key enzyme desmolase (P450scc), stimulated by ACTH
  • Pregnenolone conversion to progesterone: key enzyme 3β-hydroxy steroid dehydrogenase; pregnenolone is the precursor of all steroids
  • Progesterone is hydroxylated to 17-hydroxyprogeste- rone: key enzyme 17-hydroxylase (P450c17) encoded by the CYP17A1 gene; 17-hydroxyprogesterone is the precursor for the synthesis of cortisol and aldosterone
  • 17-hydroxyprogesterone conversion to androstenedione and dehydroepiandrosterone; key enzyme 3β-hydroxy steroid dehydrogenase (P450c17) which also possesses 17,20 lyase activity; androstenedione is the precursor for the sex hormones estrone, testosterone and estradiol

Step 2: Cortisol synthesis from 17-hydroxyprogesterone

  • 17-hydroxyprogesterone conversion to 11-deoxycortisol: key enzyme 21-hydroxylase (P450c21) a product of the CYP21A2 gene
  • 11-deoxycortisol conversion to cortisol: key enzyme 11-hydroxylase encoded by the CYP11B1 gene

Step 3: Sex hormones from androstenedione

  • Androstenedione conversion to estrone or to testosterone

Step 4: Aldosterone from progesterone

  • Progesterone conversion to 11-deoxycorticosterone: key enzyme 21-hydroxylase (P450c21) encoded by the CYP21A2 gene
  • 11-deoxycorticosterone conversion to corticosterone: key enzyme 11-hydroxylase, encoded by the gene CYP11B2
  • Corticosterone converted to aldosterone; key enzyme aldosterone synthase encoded by CYP11B2.

34.1.4 Glucocorticoid receptors

The glucocorticoid receptors have a dual mode of action:

  • As a transcription factor that binds to glucocorticoid response elements, both for nuclear and mitochondrial DNA
  • As a modulator of other transcription factors.

In the tissues, two receptors mediate the effects of adrenocortical glucocorticoid hormones /1/:

  • The low-capacity but higher affinity mineralocorticoid receptor (MR, or type I)
  • The low-affinity, but higher capacity glucocorticoid receptor (GR, or type II).

The MR is present only in aldosterone target tissues (kidney, colon, certain brain regions) whereas the GR is distributed widely in peripheral tissues and brain regions. Cortisol binding to the MR is inhibited physiologically by action of the receptor associated 11β-hydroxy steroid dehydrogenase type 2, which converts cortisol and corticosterone to their inactive 11-dehydro forms /2/ (Fig. 34.1-2 – Biosynthesis of adrenocortical steroids).

34.1.5 Glucocorticoid negative feedback

Glucocorticoid negative feedback occurs at several time domains referred to as fast, delayed, and slow feedback in brain, hypothalamus, and pituitary /2/.

Slow feedback

This mode reflects chronic exposure to glucocorticoids over days to weeks and affects both basal and stimulated hypothalamic-pituitary activity. High glucocorticoid plasma levels (Cushing’s syndrome, immunosuppressant glucocorticoid therapy) suppress ACTH and reduce its apoptotic effect on the adrenal cortex. Depending on the duration and the increased level of glucocorticoid exposure the renal insufficiency can take up a year to reverse /2/.

Fast feedback

This mode reflects stress induced activation of the corticotrophin releasing hormone (CRH) neurons within seconds. Fast feedback depends on the rate of glucocorticoid synthesis and does not require protein synthesis. ACTH is secreted by the pituitary in response to neuronal activity at the receptor signaling level.

Intermediate feedback

This mode, also called delayed feedback, occurs within 30 min. to hours and can effect either adrenal or hypothalamic responses to stimulation. The response requires the synthesis of new proteins. At the hypothalamic level, CRH and vasopressin neurons are sensitive to glucocorticoid level responses.

34.1.6 Circadian hypothalamic-pituitary-adrenocortical activity

In the absence of stress, the plasma glucocorticoid level varies according to a circadian rhythm. Concentrations reach their peak within 2–4 hours of waking and their nadir within 2–4 hours of going to sleep. With normal patterns of daily activity, peak levels are reached in the early morning and the nadir occurs at around 11:00 p.m. Peak levels result from increased hypothalamic and pituitary activity and increased adrenocortical sensitivity to ACTH. No hypothalamic-pituitary-adrenocortical stimulation takes place at the nadir. Maintaining low glucocorticoid levels for 4–6 hours of the circadian nadir is important for avoiding the effects of glucocorticoid excess on peripheral tissues /1/. Intake of food increases the hypothalamic-pituitary-adrenocortical activity, leading to an increase in glucocorticoid level.

In healthy individuals circadian hypothalamic-pituitary-adrenocortical activity is disrupted by shifts in the activity cycle, sleep deprivation, and aging. Pathologic states alter the circadian rhythm in glucocorticoids (e.g., autonomic glucocorticoid secretion in Cushing’s syndrome). Hypopituitrism or pituitary insufficiency includes all these clinical conditions /4/.

34.1.7 Physiological effects of glucocorticoids

Glucocorticoids have a significant effect on glucose metabolism. The action of insulin is counter regulated by glucocorticoids. This can lead to hyperglycemia and insulin resistance. Glucocorticoids activate lipolysis in adipose tissue and exert a catabolic effect on muscle by inhibiting protein synthesis and activating proteolysis. Glucocorticoids also have an effect on the cardiovascular system by affecting myocardial contraction, vascular tone, and blood pressure.

References

1. Lightman SL, Birnie MT, Conway-campbell BL .Dynamics of ACTH and cortisol secretion and implications for disease. Endocr Rev 2020; 41 (3): 470–90.

2. Jacobsen L. Hypothalamic-pituitary-adrenocortical axis regulation. Endocrinol Metab Clin N Am 2005; 34: 271–92.

3. Arlt W, Stewart PN. Adrenal corticosteroid biosynthesis, metabolism and action. Endocrinol Metab Clin N Am 2005; 34: 293–313.

4. Alexandraki KI, Grossman AB. Management of hypopituitarism. J Clin Med 2019; 8: 2153; http://europepmc.org/article/MED/31817511.

34.2 Diagnosis of adrenal insufficiency

Adrenal insufficiency is a condition characterized by inadequate glucocorticoid production owing to the destruction of the adrenal cortex or lack of ACTH stimulation. The diagnosis of adrenal insufficiency is made by demonstrating low basal or and/or stimulated serum cortisol. Investigations to establish the underlying etiology should follow.

Adrenocortical insufficiency is differentiated in /1/:

  • Primary adrenal insufficiency; lack of glucocorticoids and mineralocorticoids are the features. Patients can present with an insidious onset of symptoms or acutely in adrenal crisis.
  • Secondary insufficiency (ACTH deficiency) is a result of pituitary tumors, infiltrative diseases, head injury or congenital hypopituitarism; there is impaired stimulation of the adrenal cortex as a result of reduced ACTH stimulation
  • Tertiary insufficiency; the hypothalamic CRH secretion is disturbed.

34.2.1 Basal cortisol level

In the early morning sample at the time of maximal secretion (7.00–9.00), intact adrenocortical reserve can usually be confirmed with serum cortisol levels above 18 μg/dL (500 nmol/L) /1/. In cases with adrenocortical insufficiency the serum cortisol concentration is less than 3–4 μg/dL (83–110 nmol/L). The exclusion of adrenocortical insufficiency for patients with intermediate cortisol levels (4–14 μg/dL; 110–390 nmol/L) requires further functional testing. According to a study /2/ using the an upper cutoff of 7.9 μg/dL (218 nmol/L) and a lower cutoff of 2.7 μg/dL (74 nmol/L) can reduce the number of individuals who need functional testing.

34.2.2 ACTH test

The ACTH test provides additional information if basal cortisol levels fall within the borderline range (Fig. 34.2-1 – Results of cortisol, ACTH, and functional tests of the pituitary-adrenocortical axis). The test assess the functioning of the adrenal glands stress response and the adrenocortical glucocorticoid reserve (Tab. 33.3-3 – Functional tests for evaluation of the hypothalamic-pituitary axes and target organs). The ACTH test leads to cortisol peak concentrations over the physiological peak /1/. A low or no increase in cortisol in the ACTH test suggests the presence of primary adrenocortical insufficiency. The ACTH test may be normal in the case of recent onset secondary adrenocortical insufficiency. Due to its unsatisfactory sensitivity the ACTH test is not used in the first two weeks following pituitary surgery to assess the function of the hypothalamic-pituitary-adrenocortical axis.

The CRH test and the metyrapone test are more useful in such cases /3/. Refer to Tab. 33.3-3 – Functional tests for evaluation of the hypothalamic-pituitary axes and target organs.

Threshold values have been proposed for the diagnosis of adrenocortical insufficiency in critically ill patients /3/. Refer to Fig. 34.2-2 – Diagnostic approach in suspected hypocortisolism in critically ill patients.

34.2.3 Basal ACTH level

Serum ACTH is determined if adequate stimulation was not achieved in the ACTH test (cortisol ≤ 18 μg/dL; 500 nmol/L) and a distinction needs to be made between primary and secondary adrenocortical insufficiency (Fig. 34.2-3 – Diagnostic approach to differentiate primary, secondary, and tertiary adrenocortical insufficiency).

34.2.4 CRH test, insulin hypoglycemia test

If the ACTH level is decreased, secondary or tertiary adrenocortical insufficiency must be considered. The CRH test and the insulin hypoglycemia test can be used to clarify the diagnosis (Fig. 34.2-3 – Diagnostic approach to differentiate primary, secondary and tertiary adrenocortical insufficiency). A lack of increase in ACTH in the CRH test suggests secondary adrenocortical insufficiency. An adequate rise in ACTH in the CRH test indicates tertiary adrenocortical insufficiency. If the patient has been receiving glucocorticoid therapy, this must be discontinued at least 24 h before the functional tests are performed /34/.

34.2.5 Clinical assessment

The clinical presentation of adrenal insufficiency depends on the tempo and extent of the loss of adrenal function. Common features are weight loss, anorexia, nausea, vomiting, lethargy and fatigue. Moderate plasma TSH elevation with normal FT4 is common at presentation and reflects lack of glucocorticoid inhibition of TSH release. Other laboratory abnormalities include moderate renal impairment, hypercalcemia mild normochromic anemia, eosinophilia and lymphocytosis /1/.

In primary adrenal failure, features of mineralocorticoid insufficiency (postural hypotension, muscle cramps, abdominal discomfort and salt craving are more pronounced) /1/. In acutely ill patients symptoms suggestive of primary adrenal insufficiency are volume depletion, hypotension, hyponatremia in 90% of cases, hyperkalemia in approximately 50% of cases, fever, abdominal pain, hyperpigmentation, or especially in children, hypoglycemia /4/.

The following tests are recommended in primary adrenal insufficiency /4/:

  • Corticotropin stimulation test (Synacthen; ACTH1-24 stimulation: 250 μg for adults and children > 2 years of age, 15 μg/kg for infants and 125 μg for children < 2 years of age) iv (30 or 60 min. test)
  • Peak levels < 500 nmol/L (18 mg/dL) at 30 or 60 minutes indicate adrenal insufficiency
  • If the corticotropin stimulation test is not feasable a morning cortisol < 140 nmol/l (5 μg/dL) in combination with ACTH as preliminary test is suggestive of adrenal insufficiency
  • Measurement of plasma ACTH. In patients with confirmed cortisol deficiency, a plasma ACTH > 2 fold the upper reference value is consistent with primary adrenal insufficiency.
  • The simultaneous measurement of plasma renin and aldosterone in primary adrenal insufficiency to determine the presence of mineralocorticoid deficiency is recommended.

Refer to

References

1. Pazderska, Pearce SHS. Adrenal insufficiency– recognition and management. Clin Medicine 2017; 17 (3): 258–62.

2. Schmidt IL, Lahner H, Mann K, Petersenn S. Diagnosis of adrenal insufficiency: evaluation of the corticotropin-releasing hormone test and basal serum cortisol in comparison to the insulin tolerance test in patients with hypothalamic-pituitary-adrenal disease. J Clin Endocrinol Metab 2003; 88: 4193–8.

3. Cooper MS, Stewart PM. Adrenal insufficiency in critical illness. J Intensive Care Med 2007; 22: 348–62.

4. Bornstein SR, Allolio B, Arlt W, Barthel A, Don-Wauchope A, Hammer GD, et al. .Diagnosis and treatment of primary adrenal insufficiency: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab 2016; 101: 364–89.

5. Michels A, Michels N. Addison disease: early detection and treatment principles. Am Fam Physician 2014; 89: 563–8.

6. Bornstein SR. Predisposing factors for adrenal insufficiency. N Engl J Med 2009; 360: 2328–39.

7. Shaikh MG, Lewis P, Kirk JMW. Thyroxine unmasks Addisons’s disease. Acta Paediatr 2004; 93: 1663–5.

8. Mody S, Brown MR, Parks JS. The spectrum of hypopituitarism caused by PROP1 mutations. Best Practice & Research Clinical Endocrinology and Metabolism 2002; 16: 421–31.

9. Rushworth RL, Torpy DJ, Falhammar H. Adrenal crisis. N. Engl J Med 2019; 381: 852–61.

10. Quinkler M, Beuschlein F, Hahner S, Meyer G, Schöfl C, Stalla GK. Adrenal cortical insufficiency – a life-threatening illness with multiple etiologies. Dtsch Arztebl Int 2013; 110: 882–8.

34.3 Diagnosis of hypercortisolism

Glucocorticoids are secreted in relative high amounts from the zona fasciculate of the adrenal cortex under the control of ACTH. In primary hypercortisolism, the adrenal gland has increased secretory function with intact hypothalamic and pituitary function. Secondary hypercortisolism results from a corticotropin cell tumor of the pituitary gland and causes Cushing’s syndrome.

34.3.1 Initial testing

Initial testing should follow clinical suspicion and include at least one of the following tests /1/:

  • Cortisol level in serum
  • Urinary or salivary free cortisol
  • Dexamethasone suppression test.

34.3.1.1 Serum cortisol level

In the early morning sample at the time of maximal secretion (7.00–9.00), intact adrenocortical reserve can usually be confirmed with cortisol levels above 18 μg/dL (500 nmol/L). A characteristic feature of subclinical hypercortisolism and Cushing’s syndrome is the loss of the normal nocturnal cortisol nadir. A cortisol level of greater than 5 μg/dL (138 nmol/L) in the resting, supine patient should prompt further functional tests such as the 1 mg or 2 mg dexamethasone test or determination of free urinary cortisol in order to rule out the presence of hypercortisolism.

34.3.1.2 Urine free cortisol

Urine free cortisol determination in a 24-hour urine collection is the mainstay of the diagnosis of hypercortisolism. Mild Cushing’s syndrome often results from small, but significant increases in nighttime cortisol secretion. Because most of the cortisol excretion during any 24-hour period is usually between 4 a.m. and 4 p.m., subtle cortisol increases require an adequate urine collection that must be verified with a measurement of urinary creatinine /2/.

34.3.1.3 Dexamethasone tests

The test uses the sensitivity to glucocorticoid negative feedback to diagnose Cushing’s syndrome and pseudo-Cushing’s states in individuals with hypercortisolism.

1 mg dexamethasone test

A consensus statement recommended that patients who have plasma cortisol greater than 1.8 μg/dL (50 nmol/L) after overnight 1 mg dexamethasone administration merit further evaluation /3/. Diagnostic sensitivity 95–98%, diagnostic specificity low.

Overnight 2 mg dexamethasone test

Cortisol suppression to less than 3 μg/dL (83 nmol/L) rules out hypercortisolism with a high degree of certainty.

Overnight 8 mg dexamethasone test

This test is performed on an inpatient basis. Blood is collected at midnight for the determination of cortisol and ACTH. Immediately afterward, 8 mg of dexamethasone is administered orally. When the serum cortisol concentration the following morning at 8:00 a.m. is less than 5 μg/dL (138 nmol/L), the presence of hypercortisolism is very unlikely. Higher concentrations together with a lack of suppression in the 2 mg dexamethasone test, indicate with high probability the presence of hypercortisolism.

The ACTH concentration prior to the dexamethasone administration at midnight and the cortisol level after the administration of 8 mg of dexamethasone the following morning will elucidate which type of hypercortisolism is present (Fig. 34.3-1 – Diagnostic approach in suspected Cushing’s syndrome):

  • An elevated ACTH concentration suggests Cushing’s syndrome whereas a low or normal concentration suggests the presence of an adrenocortical tumor
  • Cortisol suppression in the dexamethasone test suggests Cushing’s syndrome whereas a lack of cortisol suppression suggests ectopic ACTH syndrome or the presence of an adrenocortical tumor.

Steroid metabolome profiling

Some authors recommend metabolome profiling for diagnosis and sub typing patients with Cushing syndrome. Patients with different subtypes of Cushing’s syndrome show distinctive plasma steroid profiles that may offer a supplementary single-test alternative for screening purposes /4/.

34.3.1.4 Clinical assessment

Cushing’s syndome is characterized by excess concentrations of circulating glucocorticoid, most commonly due to exogenous treatment, more rarely by upregulated excessive production of ACTH by a pituitary tumor (Cushing’s disease), by ectopic secretion of ACTH, by cortisol secreting adrenal cortical tumors, or by multiple hypersecreting nodules in both adrenal cortices (primary macronodular adrenocortical hyperplasia) /5/.

Four challenges complicate the evaluation for Cushing’s syndome /6/:

  • Increasing global prevalence of obesity and diabetes
  • Increasing use of exogenous glucocorticoids, which cause a Cushing’s syndome phenotype
  • The confusion caused by nonpathologic hypercortisolism not associated with Cushing’s syndome
  • The difficulty identifying pathologic hypercortisolism when it is extrmely or cyclic or in renal failure
  • Incidental adrenal masses
  • Pregnany

For the clinical assessment of hypercortisolism refer to:

The diagnostics of adrenal subclinical Cushing’s syndome (incidentaloma) causes special problems for the endocrinologist /7/.

References

1. Nieman LK, Biller BM, Findling JW, Murad MH, Newell-Price J, Savage MO, et al. The diagnosis of Cushing’s syndrome: an endocrine society clinical practice guideline. J Clin Endocrinol Metab 2008; 93: 1526–40.

2. Findling JW, Raff H. Screening and diagnosis of Cushing’s syndrome. Endocrinol Metab Clin N Am 2005; 34: 385–402.

3. Findling JW, Raff H, Aron DC. The low-dose dexamethsone suppression test: a reevaluation in patients with Cushing’s syndrome. J Clin Endocrinol Metab 2004; 89: 1222–6.

4. Eisenhofer G, Masjkur J, Peitzsch M, Di Dalmazi G, Bidlingmaier M, Grüber M, et al. Pasma steroid metabolome profiling for diagnosis and subtyping patients with Cushing syndrome. Clin Chem 2018; 586–596.

5. Lightman SL, Birnie MT, Conway-Campbell BL. Dynamics of ACTH and cortisol secretion and implications for disease. Endocr Rev 2020; 41 (3): 470-90.

6. Nieman KL. Diagnosis of Cushing’s syndrome in the modern era. Endocrinol Metab Clin North Am 2018; 47 (2:) 259-73.

7. Yanase T, Oki Y, Katabami T, Otsuki M, Kageyama K, Tanaka T, et al. New diagnostic criteria of adrenal subclinical Cushing’s syndrome: opinion from the Japan Endocrine Society. Endocrine Journal 2018; 65 (4): 383–93.

8. Lindsay JR, Nieman LK. The hypothalamic-pituitary-adrenal axis in pregnancy: challenges in disease detection and treatment. Endocrine Reviews 2005; 26: 775–99.

9. Herman JP, Serology K. Hypothalamic-pituitary-adrenal axis, glucocorticoids, and neurologic disease. Neurol Clin 2006; 24: 461–81.

10. Mesotten D, Vanhorebeek I, van den Berghe G. The altered adrenal axis and treatment with glucocorticoids during critical illness. Nature Clinical Practice Endocrinology & Metabolism 2008; 4: 496–504.

11. Rodwell PM, Udwadia ZF, Lawler PG. Cortisol response to corticotropin and survival in septic shock. Lancet 1991; 337: 582–3.

12. Thevenot T, Borot S, Remy-Martin A, Sapin R, Cervoni JP, Richou C, et al. Assessment of adrenal function in cirrhotic patients using concentration of serum-free and salivary cortisol. Liver Int 2011; 31: 425–33.

13. Loriaux DL. Diagnosis and differential diagnosis of Cushing’s syndrome. N Engl J Med 2017; 376: 1451–9.

14. Grumbach MM; Biller BMK, Braunstein GD, Campbell KK, Carney A, Godley PA, et al. Management of the clinical inapparent adrenal mass( incidentaloma). Ann Intern Med 2003; 238: 424–9.

15. Allolio B, Fassnacht M. Clinical review: adrenocortical carcinoma: clinical update. J Clin Endocrinol Metab 2006; 91: 2027–37.

16. Fassnacht M, Arlt W, Bancos I, Dralle H, Newell-Price J, et al. Management of adrenal incidentalomas: European Society of Endocrinology Clinical Practice Guideline in collaboratorium with thr European Network for the study of Adrenal Tumors. Eur J Endocrinol 2016; 175: G1-G34.

17. Malchoff CD, Malchoff DM. Glucocorticoid resistance and hypersemsitivity. Endocrinol Metab North Am 2005; 34: 315–26.

18. Hopkins RL, Leinung MC. Exogenous Cushing’s syndrome and glucocorticoid withdrawal. Endocrinol Metab N Am 2005; 34: 371–84.

19. Jayasena CN, Gadhvi KA, Gohel B, Martin NM, Mendoza N, Meeran K, Dhillo WS. Day 5 morning serum cortisol predicts hypothalamic-pituitary-adrenal function after transsphenoidal surgery for pituitary tumors. Clin Chem 2009; 55: 972–7.

20. Kamenicky P, Droumaguet C, Salenave S, Blanchard A, Jublanc C, Gautier JF, et al. Mitotane, metyrapone, and ketoconazole combination therapy as an alternative to rescue adrenalectomy for severe ACTH-dependent Cushing’s syndrome. J Clin Endocrinol Metab 2011; https://doi.org/10.1210/jc.2011-0536.

34.4 Cortisol

Cortisol represents about 80% of the 17-hydroxycorticosteroids in the blood. Approximately 90% of cortisol is bound to cortisol binding globulin (CBG), 7% to albumin, and the rest exists as free cortisol. Conditions that lead to a change in the CBG concentration also alter the total cortisol level in plasma. Only free cortisol is biologically active and can be determined in serum/plasma, urine, and saliva. Pulsatile cortisol production arises due to a subhypothalamic pulse generator and is the intrinsic property of the feed-forward feedback interplay between the pituitary and adrenal glands. The pulsatile hormone signal is decoded at the cellular level by the intracellular glucocorticoid receptor (GR), the mineralocorticoid receptor (MR), or both GR and MR in cell types where the two are coexpressed /1/.

For diagnosing disorders of the hypothalamic-pituitary-adrenocortical axis, the determination of total cortisol, here referred to as cortisol, is the parameter of choice.

34.4.1 Indication

Cortisol

  • Diagnosis of hyper- and hypocortisolism
  • Differential diagnosis of hyper- and hypocortisolism as marker of numerous functional tests.

Free cortisol

Suspected Cushing’s syndrome, especially in patients with altered concentrations of steroid-binding globulin, as seen in conjunction with obesity, pregnancy, estrogen therapy, hormonal contraceptive use, hypothyroidism, anorexia nervosa, fasting, multiple myeloma, and nephrotic syndrome.

34.4.2 Method of determination

Cortisol (total cortisol)

High-pressure liquid chromatography (HPLC) in combination with spectrophotometry or fluorimetry /2/.

A candidate reference measurement procedure for the quantification of total serum cortisol with liquid chromatography tandem mass spectrometry (LC-MS/MS) is presented /3/.

Immunoassay: direct assays are employed, which do not require cortisol extraction from serum/plasma. Prior to the immunological determination, cortisol is released from protein binding by means of salicylic acid, 8-aniline-1-naphthol sulfonic acid, low pH, or heat. Monoclonal or polyclonal antibodies are used directed against protein conjugates of cortisol 21-hemisuccinate or cortisol 3-carboxy-methyloxime.

Free cortisol

The determination of free cortisol in serum/plasma is technically demanding; therefore, free cortisol is determined preferentially in saliva or in urine.

Free cortisol in saliva: immunoassays for the determination of total cortisol can be used since cortisol occurs in saliva only in its free form.

Free cortisol in urine: can be determined using HPLC or immunoassay. Cortisol extraction is necessary since a greater quantity of cortisol metabolites and cortisol conjugates are present in urine. Cortisol is less water soluble than these substances and can therefore be extracted using dichloromethane or ethyl acetate. The extract is vaporized and solubilized in a buffer, followed by the determination of cortisol.

34.4.3 Specimen

  • Serum, heparin plasma: 1 mL
  • Saliva collected on a piece of gauze inside the mouth for about 5 minutes: 0.1–1 mL
  • Urine sample collected over a 24-hour period; cool temperatures are required during urine storage during the collection period to prevent the pH rising to greater than 7.5 as a result of bacterial growth: 5–10 mL

34.4.4 Reference interval

Refer to Tab. 34.4-1 – Reference intervals for cortisol.

34.4.5 Clinical significance

Basal cortisol levels are of limited clinical value because they are subject to individual variation and are strongly influenced by episodic cortisol secretion as well as by exogenous stimuli such as food intake, adiposity, physical and mental stress. Significant variation can also occur due to the pulsatile secretion patterns of ACTH /4/.

It is important to consider that the nocturnal cortisol nadir may appear excessively high because of stress due to serious systemic illness or pain. The nocturnal cortisol nadir increases linearly with advancing age and during the eighth decade of life is often near the upper reference interval value /4/. With advancing age, the increase in cortisol during the early morning occurs up to 2 h earlier, a fact that must be considered in the event of a delay in collecting blood samples.

Marked physically and mentally stressful situations during the day may influence cortisol secretion late into the night, thus causing falsely abnormal cortisol nadir levels /5/. The circadian cortisol rhythm may also be abolished in conjunction with acute psychosis or serious systemic disease /6/.

34.4.5.1 Hypercortisolism

In individuals with hypercortisolism differentiation between pseudo-Cushing’s state must be distinguished from Cushing’s syndrome. The world wide prevalence of the metabolic syndrome among obese persons is estimated at 10%. The clinical picture of this syndrome is almost the same as the Cushing’s syndrome. The prevalence of undiagnosed Cushing’s syndrome is about 75 cases per 1 million population. The chance that a person with obesity, hypertension, hirsutism, type 2 diabetes, and dyslipidemia has Cushing’s syndrome is about 1 in 500 /7/.

Refer to:

Free cortisol in saliva

The cortisol in saliva closely corresponds to that of free, biologically active cortisol in plasma and is independent of salivary flow. Unlike plasma cortisol, the concentration of salivary cortisol is not influenced by changes in the concentration of cortisol binding globulin. Salivary cortisol determinations are also suited for use in functional tests. An increased salivary cortisol concentration late in the evening (e.g., at 11:00 p.m.) increases the suspicion of hypercortisolism /3/.

Free cortisol in urine

The determination of free cortisol in urine is a reliable method for the detection of hypercortisolism if based on the correct collection of a 24-hour urine sample /89/. Free urinary cortisol is elevated in patients with any type of Cushing’s syndrome but not in adipose individuals or in patients with elevated estrogen concentrations. With a threshold value of 55 μg (153 nmol)/24 h, the diagnostic sensitivity and specificity for a diagnosis of Cushing’s syndrome are 100% and 73%, respectively. With a threshold value of 100 μg (276 nmol)/24 h, the diagnostic sensitivity and specificity are both 94% /10/.

Problems with the free cortisol test can be /11/:

  • Mild Cushing’s syndrome often results from low level but nevertheless significant cortisol secretion from 4:00 p.m. to 4:00 a.m. and may not be recognized based on a urine sample collected over 24 hours. Adequate urine collection that must be verified with a measurement of urinary creatinine is required.
  • Renal function and fluid intake are important factors associated with cortisol excretion. High fluid intake causes increased glomerular filtration of cortisol and reduced renal metabolism of the filtered cortisol, which results in increased urine cortisol excretion. For example, increased urinary excretion of free cortisol is observed in 76% of individuals who have a daily fluid intake of around 5 liters /12/.
  • If renal insufficiency is present, more cortisol is metabolized and cortisol excretion can be normal even in the presence of Cushing’s syndrome. Cortisol excretion is also increased in pseudo-Cushing’s states such as those that occur in association with endogenous depression, alcoholism, and eating disorders.

34.4.5.2 Hypocortisolism

The question of whether the cortisol concentration collected in a random blood sample during the day is of diagnostic value for the diagnosis of hypocortisolism when compared to the ACTH test is important because it is often not possible to conduct an ACTH test in outpatients. According to a study /13/ a cortisol level of greater than 15 μg/dL (420 nmol/L) has the same diagnostic sensitivity and specificity as the ACTH test for excluding adrenocortical insufficiency and a level of less than 5.1 μg/dL (142 nmol/L) has the same sensitivity and specificity for confirming the diagnosis.

Disorders associated with hypocortisolism are shown in Tab. 34.2-2 – Hypocortisolism.

34.4.6 Comments and problems

Interference factors

Immunoassays for the determination of cortisol exhibit cross reactivity with other corticosteroids. The cross reactivity rate is 1–5% for 11-deoxycortisol and corticosterone and above 20% for prednisolone /14/. The latter is converted into prednisone in the tissues. In patients on prednisolone therapy, the determination of cortisol cannot be performed by immunoassay. In patients who are being treated with metyrapone for a pituitary adenoma, cortisol determination should not be performed using immunoassay since the high concentrations of 11-deoxycortisol produce falsely high cortisol levels.

Biological influence factors

Blood should not be collected postprandially because food intake leads to a mean increase in cortisol after one hour of 90% if the food is eaten at lunch time and about 50% if it is eaten in the evening /15/.

Stability

Storage is possible at 22 °C or 4 °C for 4 days.

References

1. Lightman SL, Birnie MT, Conway-Campbell BL. Dynamics of ACTH and cortisol secretion and implications for disease. Endocr Rev 2020; 41 (3): 470–90.

2 Ching SYL, Lim EM, Beilby J, Bhagat C, Rossi E, Walsh JP, Pullan P. Urine free cortisol analysis by automated immunoassay and high-performance liquid chromatography for the investigation of Cushing’s syndrome. Ann Clin Biochem 2006; 43: 402–7.

3. Hawley JM, Own LJ, McKenzie F, Mussell C, Cowen S, Keevil BG. Candidate reference measurement procedure for the quantification of total serum cortisol with LC-MS/MS. Clin Chem 2016; 62: 262–9.

4 Schmidt IL, Lahner H, Mann K, Petersenn S. Diagnosis of adrenal insufficiency: evaluation of the corticotropin-releasing hormone test and basal serum cortisol in comparison to the insulin tolerance test in patients with hypothalamic-pituitary-adrenal disease. J Clin Endocrinol Metab 2003; 88: 4193–8.

5. Cooper MS, Stewart PM. Adrenal insufficiency in critical illness. J Intensive Care Med 2007; 22: 348–62.

6. Findling JW, Raff H, Aron DC. The low-dose dexamethsone suppression test: a reevaluation in patients with Cushing’s syndrome. J Clin Endocrinol Metab 2004; 89: 1222–6.

7. Findling JW, Raff H. Screening and diagnosis of Cushing’s syndrome. Endocrinol Metab Clin N Am 2005; 34: 385–402.

8. Lin CL, Wu TJ, Machcek DA, Jiang NS, Kao PC. Urinary free cortisol and cortisone determined by high performance liquid chromatography in the diagnosis of Cushing’s syndrome. J Clin Endocrinol Metab 1997; 82: 151–5.

9. Heckmann M, Wudy SA, Haack D, Pohlandt F. Reference range for cortisol in well preterm infants. Arch Dis Child Fetal Neonatal Ed 1999; 81: F171–4.

10. Jonetz-Mentzel L, Wiedemann G. Establishment of reference ranges for cortisol in neonates, infants, children and adolescents. J Clin Chem Clin Biochem 1993; 31: 525–9.

11. Murphy BEP. Some studies of the protein-binding of steroids and their application to the routine micro and ultramicro measurement of various steroids in body fluids by competitive protein binding-assay. J Clin Endocrinol Metab 1967; 27: 973–90.

12. Owen S, Haslam S, Adaway JE, Wood P, Glenn G, Keevil BG. A simplified liquid chromatography tandem mass spectrometry assay using on-line solid-phase extraction, for the quantitation of cortisol in saliva and comparison with a routine DELFIA method. Ann Clin Biochem 2010; 47: 131–6.

13. Bierwolf C, Kern W, Mölle M, Born J, Fehm HL. Rhythms of pituitary-adrenal activity during sleep in patients with Cushing’s disease. Exp Clin Endocrinol Diabetes 2000; 108: 490–9.

14. Kern W, Dodt C, Born J, Fehm HL. Changes of cortisol and growth hormone secretion during nocturnal sleep in the course of aging. J Gerontol 1996; 51A: M3–M9.

15. Kern W, Perras B, Wodick R, Fehm HL, Born J. Hormonal secretion during nighttime sleep indicating stress of daytime exercise. J Appl Physiol 1995; 79: 1461–8.

16. Titos MA, Biller BMK, Swearingen B. Management of Cushing disease. Nar Rev Endocrinol 2011; 7: 279–89.

17. Kadiyala R, Kamath C, Baglioni P, Green J, Okosieme OE. Can a random cortisol reduce the need for short synacthen tests in acute medical admissions? Ann Clin Biochem 2010; 47: 378–80.

18. Duplessis C, Rascona D, Cullum M, Yeung E. Salivary and free serum cortisol evaluation. Mil Med 2010; 175: 340–6.

19. Görges R, Knappe G, Gerl H, Ventz M, Stahl F. Diagnosis of Cushing’s syndrome: re-evaluation of midnight plasma cortisol vs urinary free cortisol and low dose dexamethasone suppression test in a large patient group. J Endocrinol Invest 1999; 22: 241–9.

34.5 Adrenocorticotropic hormone (ACTH)

Within the hypothalamic-pituitary-adrenal system the paraventricular nucleus are a group of densely packed neurons that are highly responsive to external physiological stimuli. These cells project to the capillaries of the median eminence, where they secrete corticotropin-releasing hormone (CRH) and arginine-vasopressin (AVP) into the portal system and thence pituitary corticotrophs to regulate adrenocorticotropin (ACTH) secretion /1/.

ACTH and related peptides are derived by proteolytic cleavage of the glycoprotein proopiomelanocortin (POMC) /2/. POMC has a molecular weight of 32 kDa and is cleaved by the enzyme pro hormone convertase 1 in the pituitary into an N-terminal glycopeptide (N-POC), joining peptide (JP), ACTH, and a C-terminal fragment called β-lipotrophin (βLPH). In addition to ACTH, the ACTH precursors POMC and pro-ACTH are present in the circulation at concentrations about 5 times greater than ACTH. The plasma concentrations are as follows /1/:

  • POMC: 5–33 pmol/L
  • Pro-ACTH: 5–33 pmol/L
  • N-POC: 5.6–16.8 pmol/L
  • β-LPH: 2.5–6.7 pmol/L
  • ACTH: 0.9–11.3 pmol/L
  • β-endorphin: ≤ 1.7 pmol/L.

Refer to Fig. 34.5-1 – Proopiomelanocortin processing by pro hormone convertases.

Amino acids 1–18 are responsible for the biological activity of ACTH, while amino acids 19–39 influence its half life. ACTH has a half life of 8–14 minutes, depending on whether it is determined by an immunoassay or a bioassay. The half life is shorter if it is determined by immunoassay.

In comparison to ACTH, POMC is relatively inactive while pro-ACTH exhibits comparable activity. It is not known, however, whether both bind to the ACTH receptor (MC-2R). ACTH precursors can be found in patients with ectopic ACTH syndrome due to small cell lung cancer. These ACTH precursors only stimulate cortisol synthesis if they are present in high concentrations or be cleaved to ACTH in the circulation.

34.5.1 Indication

  • Differential diagnosis of hypercortisolism: the diagnosis must first be established by cortisol determination and/or corresponding functional tests
  • Differential diagnosis of adrenocortical insufficiency
  • Suspected ectopic ACTH secretion (e.g., hypokalemia and metabolic alkalosis) in the case of a known underlying tumor; in any case of small cell lung cancer even without clinical signs of hypercortisolism
  • Follow-up of patients after surgical treatment for pituitary dependent Cushing’s syndrome.

34.5.2 Method of determination

Immunometric assay

The two site immunoassay relies on two antibodies binding to different epitopes of ACTH. One antibody is bound to a solid phase; the other is free in solution and labeled with an enzyme or a luminescent tracer. One antibody is directed against the amino terminal end of ACTH 1–39 (e.g., amino acids 1–17) while the other is directed against the carboxy terminal region (e.g., amino acids 34–39). The immunometric assay therefore detects only intact ACTH molecules. The detection limit is 0.6–9 ng/L (0.12–2 pmol/L) /2/.

Radioimmunoassay

Usually, polyclonal antibodies that are directed against an epitope against the N-terminal region of the molecule, are employed. The test detects intact ACTH and biologically active ACTH fragments as well as parts of POMC. The detection limit is 10–20 ng/L (2.2–4.4 pmol/L). The radioimmunoassay is not suitable for measuring low ACTH concentrations /3/.

34.5.3 Specimen

EDTA plasma, lithium heparin plasma: 1 mL

34.5.4 Reference interval

Adults /4/

  • In the morning

8:00–9:00 a.m.

5–60

(1.1–13.3)

  • At night

Midnight

< 10

(2.2)

Data expressed in ng/L (pmol/L). Conversion: ng/L × 0.2202 = pmol/L

34.5.5 Clinical significance

In patients with hypercortisolism, a low ACTH plasma level suggests the presence of an adrenocortical tumor while a normal or increased level suggests a pituitary cause or the presence of an ectopic ACTH syndrome. ACTH determination cannot be used to differentiate between secondary Cushing’s syndrome and ectopic ACTH syndrome since the ACTH concentrations in these two conditions overlap.

ACTH determination is not suitable for diagnosing Addison’s disease. However, when adrenocortical insufficiency is detected, an increased ACTH level suggests an underlying adrenal cause whereas a normal or reduced concentration suggests a pituitary cause.

For assessment of ACTH in combination with different markers of the hypothalamic-adrenocortical axis refer to Tab. 34.2-1 – Results of cortisol, ACTH and functional tests in disorders of the hypothalamic- adrenocortical axis.

Diseases and syndromes with abnormal ACTH levels are listed in Tab. 34.5-1 – Diseases and syndromes associated with abnormal ACTH levels.

34.5.6 Comments and problems

Biological influence factors

ACTH secretion is pulsatile; the mean pulse frequency in a 24-hour period is 10 for women and 18 for men and the mean peak amplitude is 10.3 ng/L (2.3 pmol/L) for women and 16.8 ng/L (3.7 pmol/L) for men /4/. This pulsatile secretion is superimposed on a circadian rhythm, with a secretory maximum between 6:00 and 8:00 a.m.

Blood sampling

For blood sampling plastic tubes containing EDTA or heparin should be used, since ACTH is strongly adsorbed by glass surfaces. Whole blood should be centrifuged within 4 hours of sample collection.

Method of determination

Immunometric assays with monoclonal antibodies have higher analytical specificity for ACTH than competitive binding assays such as the radioimmunoassay. Immunometric assays, however, are not suitable for measuring the ACTH related peptides that occur in high concentration of ectopic ACTH syndrome. For ACTH related peptides, the radioimmunoassay appears to be the least specific of all assays because the assay is dependent on the detection of a single epitope present on the ACTH N-terminal region /1/.

Standardization

Results obtained from commercially available test kits are not completely comparable to each other. Usually, the test kits are standardized against one of the following reference preparations /1/:

  • National Institute for Biological Standards and Control (United Kingdom), MRC 74/555; 6.2 IU per 25 μg ACTH 1-39
  • National Hormone and Pituitary Program (Baltimore); 4.71 IU per 50 μg ACTH 1-39.

Stability

The plasma should be separated from the blood cells within 4 hours. After storage at 4 °C for 24 h or 22 °C for 19 h, the ACTH concentration decreases by more than 10% /4/.

References

1. Lightman SL, Birnie MT, Conway-Campbell BL. Dynamics of ACTH and cortisol secretion and implications for disease. Endocr Rev 2020; 41 (3): 470–90.

2. Stewart PM, Gibson S, Crosby SR, Penn R, Holder R, Ferry D, et al. ACTH precursors characterize the ectopic ACTH syndrome. Clin Endocrinol 1994; 33: 199–204.

3. Van Rijn JLML, van Landeghem BAJ, Haima P, Goldschmidt HMJ. Evaluation of ACTH immunoradiometric assays. Clin Biochem 1996; 29: 93–5.

4. Horrocks PM, Jones AF, Ratcliffe WA, Holder G, White A, Holder R, et al. Patterns of ACTH and cortisol pulsatility over twenty-four hours in normal males and females. Clin Endocrinol 1990; 32: 127–34.

5. Reisch N, Reincke M, Bidlingmaier M. Preanalytical stability of adrenocorticotropic hormone depends on time to centrifugation rather than temperature. Clin Chem 2007; 53: 358–9.

6. Janzen N, Peter M, Sander S, Steuerwald U, Terhardt M, Holtkamp U, Sander J. Newborn screening for congenital adrenal hyperplasia: additional steroid profile using liquid chromatography tandem mass spectrometry. J Clin Endocrinol Metab 2007; 92: 2581–9.

34.6 17-hydroxyprogesterone

The vast majority of hyperandogenism due to deregulation of androgen secretion are:

  • The congenital adrenal hyperplasia (CAH) due to steroid 21-hydroxylase deficiency
  • The polycystic ovarian syndrome (PCOS) that results from functional ovarian hyperandrogenism.

The first tier screening-test is the determination of 17-hydroxy progesterone (17-OHP) im Plasma.

Refer to Fig. 37.1-1 – Biosynthesis of sexual steroids.

34.6.1 Indication

  • Screening of congenital adrenal hyperplasia
  • Polycystic ovarian syndrome.

In many countries the screening for 21-hydroxylase deficiency is incorporated into newborn screening programs, using a two-tier protocol (initial immunoassay with further evaluation positive tests by liquid chromatography/tandem mass spectrometry).

34.6.2 Method of determination

  • Immunoassay with measurement of the time resolved immunofluorescence assay (TRIFA) /1/
  • Liquid chromatography tandem mass spectrometry LC-MS/MS (gold standard method) /2/.

34.6.3 Specimen

  • For CAH immunoassays to measure 17-OHP in dried blood spots are employed
  • Serum: 1 mL

34.6.4 Reference interval

Refer to Tab. 34.6-1 – 17-hydroxy progesterone reference intervals.

34.6.5 Clinical significance

The morbidities of congenital adrenal hyperplasia (CAH) and of polycystic ovary syndrome (PCOS) will be discussed.

34.6.5.1 Congenital adrenal hyperplasia (CAH)

CAH is a group of autosomal recessive disorders characterized by impaired cortisol synthesis. The most common form of CAH is caused by mutations in the gene CYP21A2 encoding the steroid 21-hydroxylase (P450c21) an adrenal enzyme that converts 17-OHP to 11-deoxycortisol and progesterone to deoxycorticosterone, respective precursors for cortisol and aldosterone /3/.

The CAH is differentiated into two forms /4/:

  • In the classic salt-wasting and simple virilizing forms of CAH the block of cortisol synthesis leads to corticotropin stimulation of the adrenal cortex with accumulation of cortisol precursors that are diverted to androgen biosynthesis leading to the virilizing form of CAH. About 75% of classic CAH patients suffer from aldosterone deficiency, neonatal salt loss and potentially fatal hypovolemia. Besides the salt-wasting form, the virilizing form of classic CAH has apparently normal aldosterone synthesis. Adrenal enzymatic deficiency causing hyperandrogenic symptoms presents later than the form with salt loss. The peri- and postpubertal onset of hyperandrogenism in females develops secondary to 21-hydroxylase deficiency.
  • The non classic mild form of CAH shows variable degrees of postnatal androgen excess but is sometimes asymptomatic.

The clinical symptoms of adrenal hyperplasia directly result from either the deficiencies in mineralocorticoid or glucocorticoid production or from overproduction of adrenal androgens. Mineralocorticoid deficiency leads to renal salt wasting, androgen excess causes virilization of females and glucocorticoid deficiency has many clinical ramifications /3/.

The gold standard for diagnosis of CAH is the measurement of 17OH progesterone (17-OHP) after ACTH (cosyntropin) stimulation /5/. The test employs a pharmacologic dose of 0.125–0.25 mg cosyntropin, which maximally stimulates the adrenal cortex (Tab. 33.3-3 – Functional tests for evaluation of the hypothalamic-pituitary axes and target organs).

If a diagnosis is highly probable on the basis of ambiguous genitalia in girls, and markedly elevated 17-OHP levels at neonatal screening, and/or electrolyte abnormalities in either sex, treatment should be instituted immediately without waiting for the results of the ACTH stimulation test /5/.

Enzyme deficiencies in CAH are shown in Tab. 34.6-2 – Enzyme deficiencies in adrenogenital syndrome.

34.6.5.1.1 Diagnosis of congenital andrenal hyperplasia (CAH)

The U. S. Endocrine Society Clinical Practice Guideline recommends /6/:

  • In infants with positive newborn screens for CAH referral to pediatric endocrinolologist and evaluation by cosyntropin (ACTH) stimulation testing
  • In symptomatic individuals past infancy an early-morning (before 8 a.m.) baseline 17-OHP measurement by LC-MS/MS.
  • In patients with borderline 17-OHP levels a complete adrenocortical profile after a cosyntropin stimulation to differentiate 21-hydroxylase deficiency from other enzyme defects (e.g., 17-OHP, cortisol, deoxycorticosterone, 11-deoxycortisol, 17OH pregnenolone, dehydroepiandrosterone).
  • In individuals with CAH genotyping is suggested only when results of the adrenocortical profile after a cosyntropin stimulation test are equivocal, or cosyntropin stimulation cannot be accurately performed (i.e., patient receiving glucocorticoid), or for purposes of genetic counseling.

An algorithm for the diagnosis of 21-hydroxylase deficiency is shown in Fig. 34.6-1 – Diagnosis of 21-hydroxylase deficiency.

34.6.5.1.2 Management of childhood CAH

Children with CAH are steroid dependent for life, and the goal of daily maintenance treatment is to replace deficient levels of cortisol and/or aldosterone while minimizing androgen excess, preventing virilization, optimizing growth, and protecting fertility. If children are prescribed excess hydrocortisone, side effects can include growth suppression and obesity. If the dose of hydrocortisone is insufficient, children are at a high risk for precocious puberty and adrenal crisis /78/.

Monitoring treatment by consistently timed hormone measurements is recommended /6/. Serum 17-OHP and androstendione are the traditional indicators of the adequacy of glucocorticoid treatment. Complete suppression of 17-OHP concentration is not a treatment goal but instead indicates over treatment. Androstendione levels should be referenced to age- and sex-specific norms. Acceptably treated patients with CAH generally have upper normal to mildly elevated 17-OHP and androstendione levels when measured in a consistent manner.

34.6.5.2 Polycystic ovary syndrome (PCOS)

PCOS is the result from functional ovarian hyperandrogenism due to deregulation of androgen secretion. Approximately two thirds of cases have functionally typical PCOS (PCOS-T) in which there is hypersensitivity to LH, characterized by hyper responsiveness of 17-OHP to a GnRH test or hCG test. The remaining one third of PCOS is functionally atypical (PCOS-A) lacking functional hyper responsiveness of 17-OHP. This is a heterogenous group, most of which have atypical functional ovarian hyperandogenism, in which ovarian androgen excess is indicated by a dexamethasone suppression test /9/.

The PCOS-A is demonstrated directly by the GnRH test or the hCG test.

In the GnRH test leuprolide acetate 10 ug/kg sc (or a comparable dose of any other short-acting GnRH) stimulates endogenous LH and FSH release that peaks after 3–4 hours and persists 24 hours; this in turn stimulates the increased secretion of sex steroids and their precursors, with serum levels peaking at 18–24 hours. In the absence of a steroidogenic block, an elevated 17-OHP response higher than 152 ng/dL (4.6 nmol/L) is typical for PCOS-A /9/.

In the hCG test exogenous administration of hCG 3,000 IU/m2 causes maximal stimulation of theca-interstitial cells and steroid secretion peaks at 24 hours. In the absence of a steroidogenic block, an elevated 17-OHP response higher than 152 ng/dL (4.6 nmol/L) is typical for PCOS /9/.

The short dexamethasone suppression test (short DAST) indicates functionally atypical PCOS (PCOS-A) lacking functional hyper responsiveness. Dexamethasone 0.25 mg/m2 is administered per os at 12 noon and a blood sample is collected at 4.00 h p.m. Dexamethasone rapidly suppresses adrenal testosterone and cortisol. Levels of total testosterone higher than 26 ng/mL (7.5 nmol/L) and cortisol below 5 ug/dL (138 nmol/L) suggests PCOS-A /9/.

34.6.6 Comments and problems

Blood sampling

17-OHP shows marked diurnal and menstrual cycle dependent variations. In adult females blood samples should be obtained between 8:00 and 9:00 a.m. and during the follicular phase.

Poor predictive value newborn screening

The high number of false positive screening tests for CAH is due to the fact that the 17-OHP concentration is high at birth and declines sharply in the first 2 days of life. In neonates with CAH, however, the concentration continues to increase. Newborn females have lower levels than newborn males. Pre term infants and healthy newborns under stress show elevated levels.

Stability

At 22 °C and 4 °C for up to 4 days.

34.6.7 Biochemistry and pathophysiology

17-OHP is a progestogen synthesized in the adrenal gland (molecular weight of 330 Da). The concentration increases together with LH during the menstrual cycle and reaches a peak at ovulation. 17-OHP is produced by the hydroxylation of progesterone, catalyzed by 17-hydroxylase. 17-OHP accumulates in 21-hydroxylase deficiency (Fig. 37.1-1 – Biosynthesis of sex steroids) is therefore the main biomarker used to diagnose this disorder.

Androgenization is associated with a spectrum of visible clinical manifestations that commonly include hirsutism, alopecia, and acne. The endocrine glands physiologically secrete the five androgens dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), androstenedione, testosterone, and androstenediol (Fig. 37.1-1 – Biosynthesis of sex steroids). Androstenediol binds to both androgen receptors and estrogen receptors.

The vast majority of enzyme deficiencies causing hyperandrogenic symptoms are:

  • 21-hydroxylase (CYP21A2) mutations in 90% of cases. 21-hydroxylase, a cytochrome P450 enzyme, converts 17-OHP to 11-deoxycortisol and progesterone to 11-deoxycorticosterone. As 11-deoxycortisol and 11-deoxycorticosterone are precursors for cortisol and aldosterone, respectively, loss of enzyme activity results in deficiencies in both of these corticoids and in the accumulation of 17-OHP, the most important endocrine biomarker for diagnosing this enzyme deficiency. More than 100 CYP21A2 mutations are known, but large deletions and a splicing mutation (intron 2,-13 from splice acceptor site, C-G substitution) that ablate enzyme activity comprise about 50% of classic CAH alleles. Because many patients are compound heterozygote for two or more different mutant CYP21A2 alleles, a wide spectrum of phenotypes are observed /4/.
  • 11β-hydroxylase (CYP11A1 mutations) in 5–8% of cases. This results in accumulation of 11-deoxycorticosterone and 11-deoxycortisol (steroid precursors with weak mineralocorticoid activity). Severe virilization occurs due to overproduction of androstenedione with enhanced conversion to testosterone.
  • 3β-hydroxysteroid dehydrogenase (HSD3B2 mutations) in less than 5% of cases. The synthesis pathways for cortisol and aldosterone are disrupted, with subsequent diversion of steroid precursors toward androgen synthesis.
  • 17-hydroxylase (CYP17A1 mutations) in only about 125 known cases. In this rare defect, the production of cortisol and adrenal androgens is reduced and steroid precursors are diverted toward aldosterone synthesis. 17-hydroxylase deficiency can manifest almost exclusively at puberty or thereafter.

References

1. Gonzalez RR, Mäentausta O, Solyom J, Vihko R. Direct solid phase time-resolved fluoroimmunoassay of 17-hydroxyprogesterone in serum and dried bood spots on filter paper. Clin Chem 1990; 36: 1667–72.

2. Janzen N, Peter M, Sander S, Steuerwald U, Terhardt M, Holtkamp U, Sander J. Newborn screening for congenital adrenal hyperplasia: additional steroid profile using liquid chromatography tandem mass spectrometry. J Clin Endocrinol Metab 2007; 92: 2581–9.

3. Dauber A, Kellogg M, Majzoub JA. Monitoring of therapy in congenital adrenal hyperplasia. Clin Chem 2010; 56: 1245–51.

4. Merke DP, Auchus RJ. Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. N Engl J Med 2020; 383: 1248-61.

5. White PC. Neonatal screening for congenital adrenal hyperplasia. Nature Endocrinology 2009; 5: 490–8.

6. Speiser PW, Arlt W, Auchus RJ, Baskin LS, Conway GS, Merkr DP, et al. Congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab 2018; 103: 4043–88.

7. Merke DP, Bornstein SR. Congenital adrenal hyperplasia. Lancet 2005; 365 (94779): 2125–36.

8. Fleming L, van Riper M, Knafl K. Management of childhood congenital adrenal hyperplasia: an adrenal integrative review of the literature. J Pediatr Health Care 2017; 31: 560–77.

9. Rosenfield RL, Ehrmann DA. The pathogenesis of polycystic ovary syndrome (PCOS): the hypothesis of PCOS as functional ovarian hyperandrogenism revisited. Endocr Rev 2016; 37: 467–520.

10. Janzen N, Peter M, Sander S, Steuerwald U, Terhardt M, Holtkamp U, Sander J. Newborn screening for congenital adrenal hyperplasia: additional steroid profile using liquid chromatography tandem mass spectrometry. J Clin Endocrinol Metab 2007; 92: 2581–9.

11. Kratz A, Ferraro M, Sluss PM, Lewandrowski KB. Laboratory reference values. N Engl J Med 2004; 351: 1548–64.

12. Levy-Shraga Y, Pinhas-Hamiel O. High 17-hydroxyprogesterone level in newborn screening test for congenital adrenal hyperplasia. BMJ Case Report 2016; https://doi.org/10.1136/bcr-2015-213939.

13. Speiser PW, Arlt W, Auchus RJ, Baskin LS, Conway GS, Merke DP, et al. Congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency; an Endocrine Society Clinical practice Guideline. J Clin Endocrinol Metab 2018; 103 (11): 4043–88.

14. Turcu AF, Rege J, Chomic R, Liu J, Nishimoto HK, Else T, et al. Profiles of 21-carbon steroids in 21-hydroxylase deficiency. J Clin Endocrinol Metab 2015; 100 (6): 2283–90.

34.7 17-hydroxypregnenolone

17-hydroxypregnenolone is an intermediate in the biosynthesis of steroid hormones secreted by the gonads and adrenal cortex. 17-hydroxypregnenolone is produced by the hydroxylation of pregnenolone at the C17 position, catalyzed by the enzyme 17-hydroxylase. It has a molecular weight of 332 Da and is a pro hormone of DHEA. Refer to Fig. 37.1-1 – Biosynthesis of sexual steroids.

34.7.1 Indication

In the presence of hirsutism and suspicion of CAH:

  • Diagnosis of 3β-hydroxy steroid dehydrogenase (3β-HSD) deficiency
  • Diagnosis of 17-hydroxylase deficiency.

34.7.2 Method of determination

Radioimmunoassay

34.7.3 Specimen

Plasma: 1 mL

34.7.4 Reference interval

Refer to Tab. 34.7-1 – Reference intervals for 17-hydroxy pregnenolone.

34.7.5 Clinical significance

17-hydroxypregnenolone in plasma is almost entirely of adrenal origin and is elevatedin newborn screening for 21-hydroxylase deficiency . The mean plasma concentration /1/ of 17-hydroxypregnenolone in men is 1.9 μg/L and in women 3.5 μg/L .The mechanism where by plasma 17-hydroxypregnenolone levels are elevated in patients with 21-hydroxylase deficiency is unknown /2/. Acute stimulation with ACTH causes negligible changes in the plasma levels of the hormone. The ratio of 17-hydroxypregnenolone to 17-OHP can be useful for the diagnosis of 3β-HSD deficiency mediated CAH (Tab. 34.7-2 – Determination of the ratio of 17-OH pregnenolone to 17-OH progesterone).

References

1. Stroo Ca, Bermudez JA, Lipsett MB. Blood levels and production rate of 17-hydroxypregnenolone.

2. McKenna TJ, Jennings AS, Little Gw; Burr IM. Pregnenolone, 17-OH pregnenolone, and testosrerone in plasma of patients with congenital adrenal hyperplasia. J Clin Endocrinol Metab 1976; 42: 918–25

3. Mayo Clinic Medical Laboratories.

4. Frank-Raue K, Junga G, Raue F, Korth-Schatz S, Vecsei P, Ziegler R. 3-β-Hydroxysteroiddehydrogenase-Mangel und 21-Hydroxylase-Mangel bei Hirsutismus. Dtsch Med Wschr 1989; 114: 1955–9.

34.8 11-deoxycorticosterone

11-deoxycorticosterone, also known as deoxycorticosterone (DOC) or 21-hydroxyprogesterone is a steroid hormone produced by the adrenal gland that possesses mineralocorticoid activity and acts as a precursor to aldosterone. 11-deoxycorticosterone is produced from progesterone by 21-hydroxylase in the adrenal gland. 11-deoxycorticosterone has weak mineralocorticoid and no glucocorticoid activity. Addition of a 11-hydroxyl group produces glucocorticoid activity, yet further hydroxylation at C18 leads to the mineralocorticoid aldosterone.

In the zona fasciculata, 11β-hydroxylase converts 11-deoxycortisol and 11-deoxycorticosterone to cortisol and corticosterone, respectively, and is regulated by ACTH.

Cells in the zona glomerulosa of the adrenal cortex synthesize and secrete aldosterone. The enzyme aldosterone synthase converts corticosterone to aldosterone with intermediate production of 18-hydrocycorticosterone. These conversions are controlled by the renin angiotensin-II-system and the concentration of K+ in serum.

The homologous enzymes, 11β-hydroxylase and aldosterone synthase are encoded by the CYP11B1 and CYP11B2 genes, respectively.

Refer to Fig. 34.1-2 – Biosynthesis of adrenocortical steroids.

34.8.1 Indication

  • Mineralocorticoid excess symptoms of unknown origin
  • Congenital adrenal hyperplasia (CAH) due to 11β-hydoxylase deficiency
  • Glucocorticoid-remediable aldosteronism.

34.8.2 Method of determination

Radioimmunoassay following extraction and chromatography.

34.8.3 Specimen

Serum: 1 mL

24-hour urine collection, using boric acid as a preservative (0.1 g/100 mL); send the entire urine collection to the laboratory or measure the volume and send 10 mL to the laboratory.

34.8.4 Reference interval

  • Serum: 20–190 ng/L (61–576 nmol/L) /1/
  • Urine: 0.1–0.4 μg/24 h /2/

34.8.5 Clinical significance

Congenital adrenal hyperplasia (CAH) caused by steroid 11β-hydroxylase deficiency is considerably rare /3/, with a prevalence of 5–8% in Arabian countries, from which an overall frequency of 1 in 100,000 life births is estimated /4/. Unlike CAH caused by 21-hydroxylase deficiency, the disease is more common in the middle East and North Africa, where consanguinity is common resulting in identical mutations. According to a study /4/ clinically affected female newborns were profoundly virilized (Prader score of 4/5), and both genders displayed significantly advanced bone ages and were often times hypertensive. 11-deoxycortisol and 11-deoxycorticosterone were robust biochemical makers for diagnosis of 11β-hydroxylase deficiency. Computationel modeling of 25 missense mutations of CYP11B1 revealed that specific modifications in the heme-binding (R374W and R448C) or substrate-binding (W116C) site of the 11β-hydroxylase, or alterations in its stability (L299P and G267S), may predict severe disease /4/.

References

1. Kratz A, Ferraro M, Schluss PM, Lewandrowski KB. Laboratory reference values. N Engl J Med 2004; 351: 1548–63.

2. Hornung J, Gless KH, Abdelhamid S, Vielhauer W, Vecsei P. Radioimmunoassay of free urinary 18-hydroxy-deoxycorticosterone (18-OH-DOC) in patients with essential hypertension. Clin Chim Acta 1978; 87: 181.

3. Vinson GP. The mislabelling of deoxycorticosterone: making sense of corticosteroid structure and function. J Endocrinol 2011: 211: 3–16.

4. Khattab A, Haider S, Kumar A, Dhawan S, Alam D, Romero R, et al. Clinical, genetic, and structural basis of congenital adrenal hyperplasia due to 11β-hydroxylase deficiency. PNAS 2017; E1933-E1940. www.pnas.org/cgi/doi/10.1073/pnas.1621082114.

5. Elmlinger MW, Kühnel W, Ranke MB. Reference ranges for serum concentrations of lutropin (LH), follitropin (FSH), estradiol (E2), prolactin, progesterone, sex hormone-binding globulin (SHBG), dehydroepiandrosterone sulfate (DHEAS), cortisol and ferritin in neonates, children and young adults. Clin Chem Lab Med 2002; 40: 1151–60.

6. Mayo Clinic Medical Laboratories.

34.9 Dehydroepiandrosterone sulfate (DHEAS)

The circulation of large amounts of dehydroepiandrosterone (DHEA) and its sulfate derivative (DHEAS) suggests a physiological role in human physiology.

DHEA and DHEAS are the main secretion products of the adrenal glands in terms of quantity and are precursors of the androgenic and estrogenic steroids /1/. P450c17 is the single enzyme mediating both 17-hydroxylase (17 mono oxygenase, EC1.14.99.9) activities and 17,20 lyase activities in the synthesis of steroid hormones. Steroid 17-hydroxylase converts pregnenolone to 17-hydroxypregnenolone and converts progesterone to 17-hydroxyprogesterone. These 17 hydroxylated steroids may then be converted by 17,20-lyase to dehydroepiandrosterone and androstendione, respectively. The latter two steroids are precursors of testosterone and estrogen synthesis while 17-hydroxy­progesterone is a key precusor of cortisol synthesis /2/.

Refer to Fig. 34.1-2 – Biosynthesis of adrenocortical steroids.

The sulfation of DHEA into its more stable sulfate ester DHEAS is catalyzed by the enzyme DHEA sulfotransferase (hydroxy steroid sulfotransferase). The blood concentration of DHEAS is approximately 1,000 times that of DHEA.

The enzyme P450c17 is encoded by the gene CXYP17 and mutations can cause either 17-hydroxylase or 17,20 lyase deficiency or both.

The plasma concentration of DHEA is comparable to that of cortisol but shows significantly less intraindividual variation. Since DHEA and DHEAS are in a steady state relative to each other, DHEAS should be measured due to the fact that it is easier to determine and subject to less diurnal variation because of its longer half life (7–9 hours).

In the gonads and skin, steroid sulfatases convert DHEAS back to DHEA, which then acts as a precursor of stronger androgens and estrogens.

34.9.1 Indication

  • Suspected androgen excess in young women (deep voice, alopecia, acne, masculinization, ambiguous sex characteristics)
  • Suspected androgen excess in young men (precocious puberty, early pubic hair growth, early enlargement of the penis, deep voice)
  • Differential diagnosis of hirsutism and virilism
  • Suspected adrenocortical tumor, in particular carcinoma
  • Functional assessment of the zona reticularis in primary adrenocortical insufficiency
  • Non classic congenital adrenal hyperplasia.

34.9.2 Method of determination

  • DHEA: radioimmunoassay and immunoassays with and without extraction
  • DHEAS: immunoassays without extraction
  • DHEA and DHEAS: LC-MS/MS

34.9.3 Specimen

Serum: 1 mL

34.9.4 Reference interval

Refer to Tab. 34.9-1 – Reference intervals for DHEAS.

34.9.5 Clinical significance

In pregnancy, DHEAS is produced in large quantities by the fetal adrenal glands and serves as a precursor for estrogen synthesis in the placenta.

After birth, DHEAS declines sharply (80%) and does not start to increase again until the age of 7–8 years. DHEAS reaches a peak in both genders at the age of 20–25, when the concentration is approximately the same as at birth. At the age of 40–60 years, the concentration declines more sharply to only about 20% of the peak concentration and results in a fall in the formation of androgens and estrogens in peripheral target tissues.

In women, increased concentrations of DHEA and DHEAS cause symptoms of androgen excess; this is not the case in men. Mild to moderate increases are often idiopathic. When DHEA is significantly increased in men, it is converted into estrogen, resulting in increased estrogen levels.

DHEA mediates its action via multiple signaling pathways involving specific membrane receptors and via transformation into androgen and estrogen derivatives acting through their specific receptors /3/. These pathways include: nitric oxide synthase activation, modulation of γ-amino butyric acid receptors, N-methyl D-aspartate, differential expression of inflammatory factors and reactive oxygen species. Clinical and epidemiological studies suggest that low DHEA levels might be associated with ischemic heart disease, endothelial dysfunction, and atherosclerosis. DHEA, formerly believed to be only an intermediary steroid in the biosynthetic pathway of sex steroid hormones has documented unsuspected activities of its own /3/.

The clinical significance of DHEAS in hyperandrogenic disorders is shown in Tab. 34.9-2 – DHEAS in hyperandrogenism.

34.9.6 Comments and problems

Reference interval

Reference intervals are age and gender dependent. The reference intervals are lower with increasing age. Men have higher concentrations of DHEAS compared to women of the same age /4/.

Stability

Storage of serum samples is possible at 22 °C or 4 °C for 4 days.

Biological influence factors

The release of androgens synthesized in the adrenal cortex is stimulated by ACTH and not by gonadotropins. The synthesis of adrenocortical androgens can therefore be suppressed by the administration of glucocorticoids.

References

1. Goodarzi MO, Carmina E, Azziz R. DHEA, DHEAS and PCOS. J Seroid Biochem Molecular Biol 2015; 145: 213–25.

2. Maninger N, Wolkowitz OM, Reuss VI, Epel ES, Mellon SH. Neurobiological and neuropsychiatric effects of dehydoepiandrosterone (DHEA) and DHEA sulfate (DHEAS). Front Neuroendocrinol 2009; 30 (1): 65-91.

3. Traish AM, Kang HP, Saad F, Guay AT. Dehydroepiandrosterone (DHEA): a precursor steroid or an active hormone in human physiology. J Sex Med 2011; Nov; 8 (11): 2960–82.

4. Kratz A, Ferraro M, Schluss PM, Lewandrowski KB. Laboratory reference values. N Engl J Med 2004; 351: 1548–63.

5. Elmlinger MW, Kühnel W, Ranke MB. Reference ranges for serum concentrations of lutropin (LH), follitropin (FSH), estradiol (E2), prolactin, progesterone, sex hormone-binding globulin (SHBG), dehydroepiandrosterone sulfate (DHEAS), cortisol and ferritin in neonates, children and young adults. Clin Chem Lab Med 2002; 40: 1151–60.

6. Mayo Clinic Medical Laboratories.

Table 34.2-1 Results of cortisol, ACTH, and functional tests in disorders of the hypothalamic-pituitary-adrenocortical axis /4/

Disorder

Plasma
cortisol
(basal)

Plasma
ACTH
(basal)

Free
cortisol
urine

Dexamet-
hasone test

CRH
test

LVP
test

Metyra-
pone
test

ACTH
test

Insulin-
hypoglyc-
emia test

2 mg

8 mg

Cushing’s
syndrome

↑–↑↑

↑–↑↑

↑–↑↑

↑–↑↑

↑↑

Ø

Autonomous
adrenal tumor

–↑↑

Ø

Ø

Ø

Ø

Ø

Ø–

Ø

Ectopic ACTH
syndrome

↑–↑↑

Ø

Ø

Ø

Ø

Ø

Ø

Hypo-pituitarism

Ø

Ø

Ø

Ø–

Ø

Isolated ACTH
deficiency

Ø

Ø

Ø

Ø–

Ø

Secondary
adrenal
insufficiency

Ø

Ø–

Ø

Prim. adrenal
insufficiency

Ø

Description of symbols for plasma cortisol, urinary free cortisol, and plasma ACTH: , elevated; ↑↑, elevated to strongly elevated; , decreased;

Description of symbols for functional tests: Ø no reaction, reaction with increase in cortisol; ↑↑ excessive reaction with increase in cortisol, decrease in cortisol, in the 8 mg dexamethason test suppression of cortisol; – not indicated.

Table 34.2-2 Hypocortisolism /456/

Clinical and laboratory findings

Adrenocortical insufficiency – Generalized

The clinical symptoms of adrenocortical insufficiency are nonspecific and are usually only recognized at a relatively late stage. A distinction is made between primary, secondary, and tertiary adrenocortical insufficiency, and the adrenal crisis.

Primary adrenocortical insufficiency (Addison’s disease)

The estimated prevalence of primary adrenal insufficiency is around 93–140 per million population with an incidence of 4.7–6.2 per million in white populations /5/. In industrialized countries, up to > 80% of primary adrenocortical insufficiency is due to an autoimmune adrenalitis. In 40% of cases, this occurs as isolated disease while in 60% of cases, it occurs in association with an autoimmune polyglandular syndrome (APS). Patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency most commonly present with acute adrenal insufficiency in the neonatal period (salt-wasting or with virilization).

Adrenalitis and APS are more common in women than men. APS is a component of MEN types 1 and 2. Type 1 has an autosomal recessive mode of inheritance and is caused by a mutation in the gene AIRE. In addition to adrenocortical insufficiency, it also features hypoparathyroidism and mucocutaneous candidiasis in childhood. MEN type 2 is associated with adrenocortical insufficiency, autoimmune thyroid disease, type 1 diabetes, and other organ specific autoimmune diseases. Most patients with the adult form of polyglandular syndrome (type 2) have antibodies against 21-hydroxylase.

Acquired hypothyroidism due to thyroiditis is a common disease. However, patients with hypothyroidism may have other underlying autoimmune endocrinopathies, such as Addison’s disease which are important to exclude. This is especially if the hypothyroidism begins in childhood /10/.

Other causes of primary adrenocortical insufficiency include: adrenal hemorrhage, infections (M. tuberculosis, Cryptococcus, Histoplasma, Toxoplasma, Pneumocystis, Cytomegalovirus), bilateral metastatic disease, drugs (e.g., megestrol, ketoconazole, metyrapone, mifepristone, amino glutethimide, etomidate); these inhibit corticosteroid metabolism.

In primary adrenocortical insufficiency, there is reduced secretory function with an intact hypothalamic-pituitary reserve. The symptoms of:

  • Glucocorticoid deficiency include anorexia, impaired performance, myalgia, arthralgia, nausea, abdominal pain, and orthostatic hypotension
  • Mineralocorticoid deficiency include hypotension, hypovolemia, orthostatic dysregulation, and salt wasting
  • Androgen deficiency include loss of axillary and pubic hair in women, dry skin, depression, and loss of libido
  • ACTH (POMC) stimulation include hyper pigmentation of the skin and mucous membranes due to β-lipotrophin and the production of melanin by melanocytes /8/.

Laboratory findings: morning cortisol < 3.6 μg/dL (100 nmol/L), ACTH elevated. Increase in cortisol in the ACTH test remains < 18 μg/dL (500 nmol/L). Antibodies against 21-hydroxylase. Glucocorticoid deficiency causes: tendency to hypoglycemia, hypercalcemia, mild TSH elevation, anemia, eosinophilia, and lymphocytosis. Mineralocorticoid deficiency in primary adrenocortical insufficiency causes: hyponatremia and hyperkalemia (Tab. 33.3-3 – Functional tests for evaluation of the hypothalamic hypothalamic-pituitary axes and target organs).

Secondary adrenocortical insufficiency

Secondary adrenocortical insufficiency (deficient ACTH secretion) and tertiary adrenocortical insufficiency (deficient CRH secretion) result in decreased stimulation of the adrenal cortex due to low levels of ACTH. Because of adrenal cortex atrophy cortisol secretion is reduced. Secondary adrenocortical insufficiency has a prevalence of 400 per million population with an incidence of 20 per million and year. The clinical symptoms are the same as the symptoms of glucocorticoid insufficiency seen in primary adrenocortical insufficiency but the skin is pale. Iatrogenic cases can occur as a result of treatment with external glucocorticoids (0.5–2% of this population).

Laboratory findings: low cortisol and low or low normal ACTH concentration. Mineralocorticoid function is usually intact in patients with secondary adrenocortical insufficiency. Important functional and differential diagnostic investigations include the ACTH test, followed by the insulin induced hypoglycemia test and CRH test (Tab. 33.3-3 – Functional tests for evaluation of the hypothalamic-pituitary axes and target organs). In the ACTH test and insulin induced hypoglycemia test (gold standard test), the cortisol concentration increases to > 18 μg/dL (500 nmol/L).

Adrenal crisis

Adrenal crisis in an adult is an acute deterioration in health status associated with absolute hypotension (systolic blood pressure <100 mmHg) or relative hypotension (systolic blood pressure > 20 mm Hg lower than usual) with features that resolve within 1–2 hours after parenteral glucocorticoid administration. In infants and young children adrenal crisis is defined as an acute hemodynamic disturbance (hypotension or sinus tachycardia relative to age-related normative data) or a marked electrolyte abnormality. After parenteral glucocorticoid administration the crisis resolves.

Clinical findings: Symptoms and signs of adrenal crisis are gastrointestinal symptoms (nausea, anorexia, vomiting), pain (back, limb, abdominal) fatigue, weakness, confusion, postural dizziness, abdominal tenderness, hyper pigmentation and pyrexia /9/.

Each year 6–8% of patients with adrenal insufficiency have an incident adrenal crisis.

Tissues become deficient within several hours after cortisol deprivation because cortisol has a circulating half-life of 90 minutes. Adrenal crisis is slightly more frequent in patients with secondary adrenal insufficiency. Risk factors of adrenal crisis include type 1 diabetes mellitus, older age, cardiac disease, asthma and autoimmune polyglandular syndromes.

Laboratory findings: hyponatremia, hyperkalemia, hypercalcemia, hypoglycemia, neutropenia, eosinophilia, lymphocytosis.

Anterior pituitary insufficiency

Complete or partial anterior pituitary insufficiency can present with clinical symptoms of GH, FSH, LH, prolactin, and TSH deficiency. It is caused by pituitary adenomas, traumatic brain injury, subarachnoid hemorrhage, surgery, and radiotherapy. Refer to Chapter 33 – Pituitary function.

PROP1 mutation

Mutations in the PROP1 gene are responsible for a high proportion of cases of isolated or combined deficiencies of anterior pituitary hormones /8/. The gene is located on chromosome 5q and codes for a transcription factor with 226 amino acids. This transcription factor, PROP1 (prophet of PIT-1), is responsible for the expression of another transcription factor in the anterior pituitary. PIT-1 dependent cells in the anterior pituitary synthesize GH, TSH, prolactin, FSH, LH, and ACTH.

The physical and hormonal phenotypes are variable and the condition is rarely diagnosed during the first year of life. GH and TSH deficiencies are common whereas ACTH deficiency is more likely to be diagnosed at a later stage. Often, the deficiency is only partial. Mutations of the PROP1 gene are the most common cause of the recessively inherited multiple pituitary hormone deficiency in individuals of European origin. Most patients with PROP1 mutations present in late childhood with growth disorders.

Sheehan’s syndrome

This syndrome is a common cause of hypopituitarism in developing countries, where it results from postpartum hemorrhage following home births. In many cases, the diagnosis is not made until years later. An early symptom is failure of lactation due to a deficiency of prolactin.

Pharmacotherapy with glucocorticoids

Glucocorticoid therapy can lead to suppression of the hypothalamic-pituitary-adrenocortical axis with atrophy of the pituitary corticotroph cells (tertiary adrenocortical insufficiency) and adrenal cortex. This can occur in patients who have received a dose equivalent to 20–30 mg/day of prednisone for several days [normal daily dose 10–25 mg hydrocortisone (cortisol)]. Blood should be collected for determination of endogenous cortisol production 24 h after the last dose /10/.

Monitoring of therapy in adrenocortical insufficiency

Adequate dosage of /10/:

  • Glucocorticoids: cannot be checked using biochemical tests
  • Mineralocorticoids: sodium and potassium in reference interval and normal blood pressure
  • Androgens: for women, 25–50 mg DHEA in the morning; DHEAS concentration in the reference interval.

Table 34.3-1 Conditions associated with pseudo Cushing’s states

Clinical and laboratory findings

Obesity

Approximately 15% of adipose individuals have elevated basal cortisol levels and inadequate suppression in the dexamethasone test but normal free cortisol concentrations in saliva and urine.

Chronic alcohol abuse

Alcoholics have elevated basal cortisol levels and inadequate suppression in the dexamethasone test. Values return to normal following 4 weeks of abstinence.

Pregnancy

Increased placental production of estrogen stimulates the synthesis of cortisol binding globulin (CBG) in the liver and increases the total plasma cortisol concentration in serum. A cortisol level at start of pregnancy of 15 μg/dL (414 nmol/L) increases in the 15th week of gestation to a median concentration of 25 μg/dL (690 nmol/L), in the 25th week of gestation to 35 μg/dL (966 nmol/L), and at the end of pregnancy to 40 μg/dL (1104 nmol/L). Free cortisol in the plasma and urine also increases continuously and, like the total cortisol, reaches levels similar to those seen in Cushing’s syndrome. ACTH also increases in parallel with cortisol. Causes include increased synthesis of CRH by the placenta as well as a progressive weakening of cortisol feedback inhibition. Cushing’s syndrome is rare in pregnancy. If it is present, however, fetal mortality can be as high as 20% in untreated cases. In the mother, it can lead to hypertension, hyperglycemia, and preeclampsia /8/.

Estrogen therapy

Estrogens increase the concentration of cortisol binding globulin (CBG). Because the total cortisol determination includes both the free and bound forms, basal cortisol levels of 33–60 μg/dL (1,100–1,660 nmol/l) are not unusual. The free cortisol concentration in the urine and saliva is normal, however.

Endogenous depression

Usually, increased basal cortisol levels and inadequate suppression in the dexamethasone test. Normalization of values during remission /9/.

Anorexia nervosa

Patients frequently exhibit similar cortisol patterns to patients with central Cushing’s syndrome but do not have clinical manifestations of the disease /9/.

Critically ill patients /10/

All types of acute and severe disease as well as severe trauma lead to a loss of the diurnal variation in cortisol secretion. In early phases of disease, cortisol levels increase due to the release of CRH and ACTH or to disruption of cortisol feedback inhibition. Levels usually remain elevated in the chronic phase because the concentration of CBG is elevated. In some patients, however, cortisol levels are low in relation to the disease severity and degree of stress. Cutoff values are shown in Fig. 34.2-2 – Diagnostic approach in suspected hypocortisolism in critically ill patients. In one study of patients with sepsis, all patients whose cortisol level did not increase above 9 μg/dL (250 nmol/L) in the ACTH test died, compared with just 26% of the patients in whom higher cortisol levels were reached /11/.

Liver cirrhosis

The synthesis of plasma proteins declines in liver cirrhosis. Over 90% of cortisol is bound to CBG and albumin. Patients with Child A to C cirrhosis show a continuous decrease in CBG, albumin, and cortisol. In a study of cirrhotics, patients with low albumin ≤ 25 g/L or low CBG ≤ 35 mg/L had a mean cortisol concentration of just 10.5 μg/dL (289.9 nmol/L) while those with near normal protein concentrations had a cortisol level of 15.0 μg/dL (414 nmol/L). However, free cortisol levels in serum and saliva were similar in both groups of patients /12/.

Table 34.3-2 Hypercortisolism and Cushing’s syndrome /13/

Clinical and laboratory findings

Cushing’s syndrome – Generalized

Cushing’s syndrome is characterized by a series of systemic complications, including abdominal obesity, systemic arterial hypertension, impairment of glucose tolerance, dyslipidemia, and thrombotic diathesis with increased cardiovascular risk /11/.

Endogenous Cushing’s syndrome: endogenous hypercortisolism is usually the result of Cushing’s syndrome. This is caused either by a tumor of the corticotroph cells of the pituitary or, less frequently, hyperplasia of the corticotrophs due to ectopic ACTH or CRH production.

ACTH independent Cushing’s syndrome accounts for 15% of cases, of which 50% are caused by adrenal adenomas and 50% by adrenal carcinomas. Bilateral hyperplasia is very uncommon. The 5-year prevalence of adrenal Cushing’s syndrome in Japan is 0.7 per 100,000 and that of ACTH dependent Cushing’s syndrome in Spain is 3.9 per 100,000. However, the prevalence of subclinical Cushing’s syndrome is much higher than that of clinically evident disease and is estimated to be around 2% /12/.

– Pituitary adenoma

The most common form of Cushing’s syndrome is caused by ACTH producing micro adenomas (≤ 10 mm) or macro adenomas (> 10 mm) of the pituitary. Around 5% of hormonally active pituitary adenomas secrete ACTH. Around 85% of cases of Cushing’s syndrome are ACTH dependent.

It is typical of pituitary adenomas that their production of ACTH is inadequately suppressed by elevated endogenous cortisol concentrations and exogenous glucocorticoid administration and that they react to stimulation by releasing hormones such as CRH by secreting excessive amounts of ACTH. In hypothalamic-pituitary Cushing’s syndrome, two different types of cortisol secretion can be distinguished. In some of these patients, pronounced hyper pulsatility is observed, whereas in other patients, the amplitude of secretory pulses cannot be distinguished from that seen in healthy individuals /13/.

Ectopic ACTH production

In 75% of cases, ectopic ACTH production is the result of small cell lung cancer (SCLC). The signs of Cushing’s syndrome are often not clinically apparent for the following reasons /13/:

  • The cleavage of proopiomelanocortin (POMC) is often incomplete, resulting in inactive fragments or fragments with reduced biological activity that are often not detected by immunoassays.
  • Because of the rapidly progressive course of the SCLC, signs of Cushing’s syndrome are often not clinically apparent. A slower course with all the symptoms of Cushing’s syndrome is found in conjunction with carcinoid tumors of the lung, thymus, and pancreas as well as in medullary thyroid cancer and pheochromocytoma. Usually, increases in ACTH secretion are extremely high. With the exception of around 50% of patients with pulmonary carcinoid tumors, ACTH secretion cannot be influenced by the administration of dexamethasone or CRH.

Adrenocortical hyperplasia

In the rare form of micronodular adrenocortical hyperplasia, stimulation of steroid hormone synthesis by autoantibodies is thought to occur. Cortisol secretion cannot be influenced by CRH, ACTH, or dexamethasone /13/.

Adrenocortical tumors

Adrenocortical tumors have a prevalence of at least 3% in people aged over 50 years. Carcinomas, however, are very rare, with an incidence of 1–2 per 1 million and year. Approximately 60% of carcinomas produce adrenocortical steroids and most result in a rapidly developing Cushing’s syndrome with or without virilization. Cortisol secretion cannot be influenced by CRH, ACTH, or dexamethasone. Carcinomas also frequently produce precursors of cortisol synthesis (e.g., 11-deoxycortisol and 17-hydroxycorticosterone) particularly associated with the secretion of androgens (DHEA, androstenedione) /14/.

Adrenal subclinical Cushing’s Syndrome (Incidentaloma)

Incidentalomas are small adrenal masses with autonomous glucocorticoid production without specific signs and symptoms of Cushing’s syndrome /14/. They are found at 1–8.7% of autopsies. All patients with incidentaloma should be investigated for hypersecretion of hormones. The most frequent findings in these patients are disorders of the hypothalamic-pituitary-adrenocortical axis. Autonomous subclinical secretion of glucocorticoids is observed in an average of 9% of patients with incidentaloma. The current diagnostic has elicited two main criteria /7/:

  • The relatively low reliability of a low range serum cortisol essential for the diagnosis by an overnight 1 mg dexamethasone suppression test
  • Different cutoff values for serum cortisol after 1 mg dexamethasone suppression test compared with those of other countries.

Three hierarchical cortisol cutoff values, 5.0, 3.0 and 1.8 μg/dL after a 1 mg dexamethsone suppression test are presented. Clinical assessment /7/:

  • Serum cortisol equal or higher than 5 μg/dL alone is considered sufficient to diagnose autonomous cortisol secretion and the current criterion based on serum cortisol equal or higher than 3 μg/dL can continue to be used
  • Clinical evidence suggests that serum cortisol 1.8-2.9 μg/dL is not always normal, so cases who meet the cutoff value as well as basal ACTH concentration <10 ng/L or poor ACTH response to corticosteron releasing hormone (CRH) and nocturnal serum cortisol equal or higher than 5 μg/dL are proposed to have subclinical Cushing’s syndrome.

Diagnostic criteria for subclinical Cushing’s syndrome in Europe and the USA are /15/:

  • The 1 mg dexamethasone suppression test overnight as the first test. Recommended are the aforementioned cutoffs
  • 24-h urine free cortisol as the second screening test
  • Low-dose 2-day dexamethasone suppression test as the confirmatory test
  • Determinatin of ACTH, DHEAS or adrenal scintigraphy for evaluation of autonomous cortisol secretion from adrenal adenoma.

Generalized glucocorticoid resistance

This familial disease is characterized by a generalized resistance to glucocorticoids at all glucocorticoid receptors in the tissues as well as the pituitary and hypothalamus /16/. Glucocorticoid negative feedback does not take place, which results in uninhibited secretion of cortisol, androgens, and mineralocorticoids. Symptoms of Cushing’s syndrome are absent but signs of androgen excess are evident. ACTH levels are normal or slightly elevated, there is an up to 200-fold increase in the excretion of free cortisol, and there is a 2–5-fold elevation in deoxycorticosterone.

Exogenous Cushing’s syndrome

Cushing’s syndrome can develop following the intake of high doses of glucocorticoids (GC) over a prolonged period /17/. There is a high prevalence of infections among these patients. The morning cortisol level and ACTH concentration are low. GCs such as prednisone that are used therapeutically show high cross reactivity in immunoassays. An exception to this is dexamethasone.

Trans sphenoidal surgery (TSS)

TSS is an effective method for treating pituitary adenomas /18/. However, its complications include secondary adrenocortical insufficiency. Morning cortisol levels on postoperative day 5 of greater than 14 μg/dL (392 nmol/L) indicate that the corticotroph system is intact and that glucocorticoid treatment can be discontinued. Concentrations of below 4 μg/dL (111 nmol/L) suggest the presence of a secondary adrenocortical insufficiency. Intermediate concentrations are inconclusive and indicate the need to perform an insulin induced hypoglycemia test several times in the following weeks.

Metyrapone is a drug used to treat Cushing’s syndrome /19/. It inhibits the enzyme 11β-hydroxylase that catalyses the synthesis of cortisol and corticosteroids. Serum cortisol or free cortisol in a 24-hour urine collection decline to within the reference interval within a few days.

Table 34.4-1 Reference intervals for cortisol

Plasma/serum cortisol: reference intervals can only be specified for defined times during a 24-h period.

Preterm
infants /16/

24th week

4.0–27 (110–744)

25th week

3.6–24 (100–671)

26th week

3.3–22 (90–605)

27th week

2.9–20 (81–545)

28th week

2.6–19 (73–491)

29th week

2.4–16 (66–443)

Children /17/

8 a.m.

5th day

0.6–20 (17–550)

2–12 months

2.4–23 (66–630)

2–15 years

2.5–23 (69–630)

16–18 years

2.4–29 (66–800)

Adults /18/

8 a.m.

5–25 (138–690)

Midnight

Up to 5 (up to 138)

Free cortisol in saliva /19/

Adults

8 a.m.

0.20–1.7 (5.4–45.7)

11 p.m.

< 0.23 (< 6.4)

Values in μg/dL (nmol/L); conversion formula: μg/dL × 27.6 = nmol/L

Free cortisol in urine: adults

HPLC /14/

< 62 μg/24 h (170 nmol/24 h)

LC-MS/MS /16/

< 60 μg/24 h (165 nmol/24 h)

Immuno­assay

17–68 μg/24 h (47–188 nmol/24 h)

Immuno­assay
after extraction
of cortisol

20–71 μg/24 h (55–196 nmol/24 h)

Conversion: μg/dL × 2.76 = nmol/L; threshold values are the 97.5th percentiles,

Table 34.5-1 Diseases and syndromes associated with abnormal ACTH levels /6/

Clinical and laboratory findings

Hypothalamic-pituitary Cushing’s syndrome

Plasma ACTH levels are within the upper reference interval or slightly elevated. Nevertheless, such ACTH levels are abnormal in the face of raised cortisol levels, which would normally suppress ACTH secretion. Therefore, interpretation of ACTH levels is only possible in combination with the corresponding cortisol levels. ACTH levels do not contribute directly to the diagnosis.

Cushing’s syndrome due to adrenocortical tumor

Plasma ACTH is suppressed to an undetectable level. Hypercortisolism in the absence of ACTH proves the autonomy of steroid production in these adrenal tumors.

Ectopic ACTH syndrome

Ectopic secretion of ACTH by non pituitary tumors is responsible for 10–20% of cases of ACTH-dependent Cushing’s syndrome. ACTH levels may be markedly elevated but cannot be used to distinguish this syndrome from pituitary Cushing’s syndrome. Over 50% of cases are caused by small cell lung cancer. The next most frequent underlying tumors, at 10% each, are malignant islet cell tumors of the pancreas and thymomas.

Primary adrenocortical insufficiency

Significantly elevated ACTH levels are present. Although the levels decrease in response to adequate substitution, they do not necessarily decrease to within the reference interval. ACTH determinations are therefore not suited to monitoring therapy.

Secondary adrenocortical insufficiency

The pattern of a low cortisol and a low ACTH level proves the presence of secondary or tertiary adrenocortical insufficiency. These can be differentiated using the CRH test.

Tertiary adrenocortical insufficiency

If the stimulatio of ACTH secretion remains intact in the CRH test, given a low cortisol level and a low ACTH level, it can be presumed that the primary defect is not localized within the corticotroph cells of the anterior pituitary but instead within the CRH-producing neurons of the hypothalamus (tertiary; hypothalamically caused adrenocortical insufficiency).

Table 34.6-1 17-hyroxyprogesterone reference intervals

Newborns > 36th week of gestation (blood spot on filter paper) /10/

Time resolved fluorescence immunoassay (TRFIA)

17.6 (< 7.5–56.7)

LC-MS/MS

2.10 (< 1.1–9.7)

Data expressed in nmol/L. Values are 5th and 95th percentiles

Serum /11/

Children

0.03–0.9

Men

Up to 0.15

Women

Follicular phase

0.6–3.0

Mid-cycle

3.0–7.5

Luteal phase

3.0–15

Postmenopausal

≤ 2.1

Data expressed in nmol/L. Conversion: nmol/L × 330 = ng/L

Table 34.6-2 Enzyme deficiencies in congenital adrenal hyperplasia (CAH)

Clinical and laboratory findings

21-hydroxylase deficiency

Mutations in CYP21A2 (the gene encoding 21-hydroxylase, a cytochrome P-450 enzyme)result in lack of 21-hydroxylase which is required for the production of cortisol and aldosterone. Insufficient production of cortisol leads to compensatory over stimulation of the adrenal cortex. Cortisol and aldosterone precursors accumulate as a result of the enzyme deficiencies /4/. Steroid 21-hydroxylase converts 17-hydroxyprogesterone (17-OHP) to 11-deoxycortisol, and progesterone to 11-deoxycorticosterone (Fig. 37.1-1 – Biosynthesis of sexual steroids). As 11-deoxycortisol and 11-deoxycorticosterone are precusors to cortisol and aldosterone, respectively, moderate to complete loss of 21-hydroxylase deficiency results in deficiencies of both of these vital corticosteroids, as well of high levels of 17-OHP.

Depending of the severity of the genetic defect, the accumulating steroid precursor may be variably shunted to androgen pathways not impaired by the block, this can lead to prenatal virilization in affected girls /12/. CAH has an incidence of approximately 1 per 16–20 thousand live births. CAH is traditionally divided into classical and non classical forms. The former presents shortly after birth while the latter does not appear until childhood. Classical CAH can be classified into salt wasting and virilizing form resulting from either the deficiencies in mineralocorticoid or glucocorticoid production or from over production of adrenal androgens.

Congenital adrenal hyperplasia (CAH)

Around 95% of cases of CAH are caused by mutations in CYP21A2, the gene encoding adrenal 21-hydroxylase cytochrome (P450c21). The enzyme converts 17-OHP to 11-deoxycortisol and progesterone to deoxycorticosterone, with these products being precursors for cortisol and aldosterone. The blockade of cortisol leads to corticotropin (ACTH) stimulation of the adrenal cortex, with the accumulation of cortisol precursors that are diverted to sex hormone biosynthesis. Depending on the severity of the mutation, 21-hydroxylase deficiency can lead to a deficiency of glucocorticoids and mineralocorticoids and, consequently, of cortisol and aldosterone. The absence of feedback inhibition by cortisol results in increased ACTH release with hyperplasia of the adrenal cortex and increased steroid production proximal to the enzymatic block (Fig. 37.1-1 – Biosynthesis of sexual steroids). The elevated concentrations of progesterone and 17-hydroxyprogesterone (17-OHP) are diverted toward androgen synthesis, leading to increased production of dehydroepiandrosterone and androstenedione, which in turn are converted into testosterone /46/. Clinical symptoms are due either to a deficiency in glucocorticoids and mineralocorticoids or an excess of androgens. Patients with a mildly virilizing form of the disorder synthesize inadequate amounts of cortisol but adequate amounts of aldosterone, which means that the Na+ balance is maintained.

Clinical findings: A cardinal feature of classic or severe virilizing CAH in newborn females is abnormal development of the external genitalia with variable extent of virilization. Evaluation for CAH needs to be cosidered for infants found to have bilateral nonpalpable gonads /13/. In 75% of cases with severe enzyme deficiency, inadequate aldosterone production causes salt wasting, failure to thrive, and potentially hypovolemia and shock. Missed diagnosis of salt-losing CAH is associated with increased risk for early neonatal morbidity and mortality. If simple virilizing CAH is not recognized and treated, both girls and boys may undergo rapid postnatal growth and virilization. In addition to the “classic salt wasting” and “simple virilizing” forms of CAH diagnosed in infancy, there is also a mild or “nonclassic” form which features variable degrees of postnatal androgen excess but is sometimes asymptomatic. The mild subclinical impairment of cortisol synthesis in nonclassic CAH generally does not lead to Addisonian crisis.

in both males and females, androgen excess leads to accelerated growth and premature epiphyseal growth plate closure with reduced final height. In females, androgen excess causes hirsutism, acne, menstrual abnormalities, clitoromegaly, and, in severe cases, masculinization of the external genitalia. 21-hydroxylase deficiency can cause shock, hyponatremia, and hyperkalemia in early childhood. In order to reduce morbidity and mortality, therefore, it is important to diagnose salt wasting disorders in newborns before they develop a full blown adrenal crisis. In the absence of a severe salt wasting syndrome, newborn boys with CAH show no obvious symptoms at birth, whereas girls with the disease are identifiable by their ambiguous genitalia.

Laboratory findings /5/: In cases of suspected CAH, Na+ and K+ are first determined. Serum Na+ concentrations are in the range of 130–135 mmol/L and K+ levels are in the range of 5.5–6.5 mmol/L. Currently, the 17-OHP concentration is usually measured by automated time-resolved lanthanide fluorescence immunoassay (DELFIA). Immunoassays to determine 17-OHP are often performed using a blood spot on filter paper. The positive predictive value of the screening methods in newborns is low (about 1%). Marked elevation of 17-OHP are characteristic of classic 21-hydroxylase deficiency. However, endocrinologists are frequently confronted with false positive results that require further investigation. It is recommended that screening laboratories employ a second-tier screen by LC-MS/MS in preference to all other methods (e.g., genotyping) to improve the positive predictive value of CAH screening /13/. Measuring additional analytes or ratios of analytes (sum of 17-OHP and 21-deoxycortisol divided by cortisol) can also improve screening outcomes. In principle, calculating the ratio of 17-OHP to 21-deoxycortisol divided by cortisol should increase the positive predictive value to 100% /10/. In a study /14/ five C21 steroids were significantly higher in patients with 21-hydroxylase deficiency: 17 OHP 67-fold, 21-deoxycortiosol 35-fold, 16-hydroxyprogesterone 28-fold, progesterone 2-fold. On the other hand, performing molecular biological investigations on the blood spot increases the positive predictive value to only 18% /5/. In asymptomatic patients with definite 17-OHP elevation, an ACTH test is performed to differentiate between classical and non classical CAH (Tab. 34.6-1 – 17-OH progesterone reference intervals).

Patients with classical CAH are treated using hydrocortisone and fludrocortisone. Determination of 17-OHP and androstenedione are used to monitor progress.

-hydroxysteroid dehydrogenase (-HSD) deficiency

There are two 3βHSD isoenzymes, types I and II, which share 93.5% homology. Type II isoenzyme (HSD3B2) is expressed in the adrenals and gonads, whereas type I (HSD3B1) is expressed in the placenta and peripheral tissues. The adrenals of a patient with HSD3B2 deficiency secrete very large amounts of of three Delta 5 steroids (Fig. 37.1-1 – Biosynthesis of sexual steroids): pregnenolone, 17-hydroxypregnenolone, and dehydroepiandrosterone (DHEA). These may serve as precursor substrates for 3βHSD1. Some of the secreted 17-hydroxypregnenolone is converted by the extraadrenal action of 3βHSD1, resulting in elevated levels of 17-OHP, confusing with the diagnosis as 21-hydroxylse deficiency /12/. Patients who have CAH caused by 3β-HSD deficiency usually die at an early age. Mild cases can lead to the development of polycystic ovary syndrome in women.

Laboratory findings: determination of the ratio of 17-OH pregnenolone to 17-OHP. This is particularly useful following ACTH stimulation (Tab. 34.7-1 – Reference intervals for 17-OH pregnenolone).

11β-hydroxylase deficiency

Corticosterone synthesis is inhibited, leading to an accumulation of 11-deoxycorticosterone (DOC) (Fig. 34.1-2 – Biosynthesis of sexual steroids). DOC has mineralocorticoid effects and causes arterial hypertension. There is also over production of androgens with the development of adrenogenital syndrome. The elevation in DOC prevents the development of a salt-wasting syndrome in spite of the aldosterone deficiency.

17-hydroxylase deficiency

This is a rare defect. The synthesis of cortisol and androgens is reduced and the synthesis of aldosterone is increased, resulting in hypernatremia, hypokalemia, and hypertension (Fig. 37.1-1 – Biosynthesis of sexual steroids). Because testosterone and estrogen synthesis in the gonads is also disrupted, female patients present with sexual immaturity and genetic males (XY karyotype) have a female phenotype.

Table 34.7-1 Reference intervals in plasma for 17-hyroxypregnenolone /3/

Age

Male

Female

Premature 26–28

12–98

12–98

Premature 29–36

3.5–89

3.5–89

Month 1–5

2.3–31

2.3–31

Month 6–12

2.2–19.8

2.2–19.8

1–2 yrs

0.35–7.1

0.35–7.2

3–6 yrs

< 2.77

< 2.77

7–9 yrs

< 1.88

< 2.13

10–12 yrs

< 3.93

< 3.99

13–15 yrs

0.35–4.65

< 4.08

16–17 yrs

0.32–4.78

< 2.24

Data expressed in μg/L. Conversion: μg/L × 3.026 = nmol/L

Table 34.7-2 Determination of the ratio of 17-hydroxy­pregnenolone to 17hydroxyprogesterone (17-OHP)

Indication: suspected 3β-HSD deficiency induced CAH.

Principle: ACTH stimulates the synthesis of 17 hydroxy- pregnenolone, but not of 17-OHP because the synthetic pathway is blocked as a result of the 3β-HSD deficiency.

Test protocol: on the evening before the ACTH test oral administration of 1 mg dexamethasone to patients who weigh less than 50 kg, 1.5 mg to patients who weigh 50–70 kg, and 2 mg to patients who weigh more than 70 kg. Blood collection on the following morning for determination of basal 17-hydroxypregnenolone and 17-OHP levels. I.v. administration of 0.25 mg ACTH followed by another blood collection after 60 min. for determination of the two steroids.

Clinical significance: Ratio of 17-OH pregnenolone to 17-OHP:

  • Is almost the same in healthy individuals before and after ACTH stimulation.
  • In 3β-HSD deficiency following ACTH stimulation the ratio is ≥ 2.0 /4/.

Table 34.9-1 Reference intervals for DHEAS

 

Female
(μmol/L)

Male
(μmol/L)

Children /5/

1–7 days

1.78–12.80

2.32–11.47

8–15 days

0.91–9.49

0.82–4.77

16 days–3 yrs

< 0.2–3.33

< 0.2–2.69

4–6 yrs

< 0.2–1.27

< 0.2–6.18

7–8 yrs

0.29–2.13

0.21–3.22

9–10 yrs

0.34–5.27

0.40–2.40

11 yrs

0.23–2.71

0.53–5.19

12 yrs

0.67–6.12

0.32–10.13

13 yrs

0.58–4.59

0.37–7.84

14 yrs

0.60–8.86

0.38–8.13

15 yrs

0.88–9.52

1.41–11.95

16 yrs

1.52–10.45

0.82–9.89

17 yrs

2.32–10.99

2.71–9.34

18–19 yrs

3.87–11.85

2.81–12.89

Men /6/

0.27–16.7 μmol/L

Women /6/

Reproductive age

≤ 14.3 μmol/L

Postmenopausal

≤ 3.2 μmol/L

Values are 2.5th and 97.5th percentiles. Conversion: μmol/L × 0.2714 = μg/L

Table 34.9-2 DHEAS in hyperandrogenism

Clinical and laboratory findings

Congenital adrenal hyperplasia (CAH)

In young children, the following enzyme deficiencies cause excessive elevation of DHEAS:

  • 3β-hydroxy steroid dehydrogenase (3β-HSD) deficiency with accumulation of 11-deoxycorticosterone that is not converted into corticosterone.
  • 11β-hydroxylase deficiency, typically with elevated 17-OH progesterone. Some patients also have reduced aldosterone production and a salt wasting syndrome.

Adrenocortical tumors

Significant elevations in DHEAS are measured in adrenocortical tumors because DHEA is the most frequently produced androgen in the adrenal cortex in terms of quantity. The higher the level of DHEAS in relation to other adrenocortical steroids, the more likely the presence of an adrenocortical carcinoma. Some adrenocortical carcinomas produce only slightly active steroids; Cushing’s syndrome does not develop in these cases. However, large quantities of DHEAS are usually produced. DHEAS can be used as a diagnostic marker and to assess disease progression.

Adrenocortical insufficiency

The serum concentration of DHEAS is reduced. However, DHEAS cannot be used as a diagnostic marker of adrenocortical insufficiency.

Polycystic ovary syndrome (PCOS)

The PCOS is a heterogeneous disorder in 10–20% of women in the reproductive age. A proportion of patients have increased secretion of adrenal precursor androgens measured by elevated serum levels of DHEAS and 11β-hydroxy androstenedione. For further information refer to Section 34.6.5.2 – Polycystic ovary syndrome.

Figure 34.1-1 The hypothalamic-pituitary-adrenocortical axis (HPA) is a neuroendocrine system subserving control of adrenocortical glucocorticoid secretion by the brain. Secretion via corticotropin-releasing hormone (CRH) released from the hypothalamus regulates ACTH synthesis from the anterior pituitary. The adrenal glucocorticoid production is regulated by a negative feedback loop in response to pituitary derived ACTH. Arginine-vasopressin (AVP) and oxytocin are weak stimulants of ACTH, but they potentiate the action of CRH. CRH binds to surface receptors on the anterior pituitary corticotroph cells leading to the release of ACTH and other proopiomelanocortin (POMC) related peptides. Glucocorticoid feedback to the hypothalamus down regulates CRH secretion. The brain reacts most sensitively to feedback inhibition through its glucocorticoid receptors and high affinity mineralocorticoid receptors. Feedback inhibition also takes place at the level of the anterior pituitary, which also contains glucocorticoid receptors.

Brain Circadian rhythm Stress Feedback Episodic secretion Neurotransmitter HypothalamusCRH/AVP synthesis Anterior lobe of the pituitary glandPOMC synthesis CRH AVP ACTH Adrenal cortexCortisol synthesis Cortisol (Plasma) Target organProtein synthesis

Figure 34.1-2 Biosynthesis of adrenocortical steroids. The synthetic pathways for aldosterone, cortisol, and testosterone are shown. 21-hydroxylase deficiency prevents the synthesis of aldosterone and cortisol and precursors such as 17-hydoxy pregnenolone are directed toward androgen synthesis /2/.

Cholesterol desmolase (CYP11A) 17α- Hydroxylase (CYP17) 17-OH Pregnenolone 17-OH- Progesterone 11-De- oxycortisol 17β-Hydroxy- steroid de- hydrogenase Cortisol 17,20-Lyase Cholesterol Aldosteron 3β-OH-steroid dehydrogenase Pregnenolone 21-Hydroxylase (CYP21) Progesterone 11β- Hydroxylase (CYP11B1) 5α- Reductase 11β-Hydroxylase (CYP11B2) 11-Deoxycorticosterone Dehydro- epiandrosterone Andro- stenedione Testosterone OH CH 3 CH 3 O Dihydro- testosterone 18-Hydroxylase (CYP11B2) Corticosterone 18-Hydroxylase (CYP11B2) 18-OH corticosterone HC 21 CH 2 OH O OH CH 2 HO 17 11 3 3 CH 3 O HC 21 CH 2 OH O O 18 CH HO 11 3 CH 3 O HC CH 2 CH 3 CH 3 HO CH 3 CH 2 CH 2 CH 3 CH 3 CH

Figure 34.2-1 Diagnostic approach in suspected hypocortisolism.

Cortisol 8 a.m. > 17 μg/dL (470 nmol/L) < 3 μg/dL (80 nmol/L) 3–17 μg/dL (80–470 nmol/L) ACTH test Peak value< 20 μg/dL (552 nmol/L) Corticosteroid insufficiencyprobably Corticosteroid insufficiencyunlikely Peak value≥ 20 μg/dL (552 nmol/L)

Figure 34.2-2 Diagnostic approach in suspected hypocortisolism in critically ill patients /3/.

Cortisol 8 a.m. > 25 μg/dL (690 nmol/L) < 15 μg/dL (414 nmol/L) 15–25 μg/dL (414–690 nmol/L) ACTH test Peak value< 25 μg/dL (690 nmol/L) Corticosteroid insufficiencyprobably Corticosteroid insufficiencyunlikely Peak value≥ 25 μg/dL (690 nmol/L)

Figure 34.2-3 Diagnostic approach to differentiate primary, secondary, and tertiary adrenocortical insufficiency.

No Yes Secondaryadrenal insufficiency Tertiaryadrenal insufficiency ACTH High Low Primaryadrenal insufficiency Secondaryadrenal insufficiency CRH test ACTH increase

Figure 34.3-1 Diagnostic approach in suspected Cushing’s syndrome.

Low High Cortisol 12 p.m. < 5 μg/dL (138 nmol/L) ≥ 5 μg/dL (138 nmol/L) Cortisol ≤ 3 μg/dL (83 nmol/L) Cortisol > 3 μg/dL (83 nmol/L) Exclusion M. Cushing 8 mg dexamethasone test M. Cushing unlikely 2 mg dexamethasone test Cortisol ≤ 3 μg/dL (83 nmol/L) Cortisol > 3 μg/dL (83 nmol/L) Ectopic ACTH syndrome Adrenal tumor Hypophysial/hypo-thalamic M. Cushing ACTH

Figure 34.4-1 Causes of endogenous Cushing’s syndrome, modified from Ref. /16/. Approximately 70 % of cases of Cushing’s syndrome are associated with macronodular hyperplasia of the adrenal cortex and McCune-Albright syndrome.

M. Cushing70% 10% ectopic ACTH formation1% ectopic CRH formation10% adrenal adenoma5% adrenal carcinoma4% Other adrenal causes

Figure 34.5-1 Proopiomelanocortin (POMC) processing by prohormone convertase 1 (PC1) and prohormone convertase 2 (PC2). Modified with kind permission from Ref. /1/. The numbers refer to the respective amino acid sequences. β-LP, β-lipotrophin; N-POC, N-terminal proopiomelanocortin; JP, joining peptide; γ3MSH, γ3-melanocyte stimulating hormone, αMSH, α-melanocyte stimulating hormone; CLIP, corticotropin-like intermediate lobe peptide; γLPG, γ-lipotrophin; βEP, β-endorphin.

γ 3 MSH β LPH POMC 1 241 Pro-ACTH 1 241 150 153 γ LPH β EP 208 153 211 241 1 76 79 108 N-POC JP 112 150 ACTH 112 α MSH 130 51 124 76 CLIP 150 PC1 PC2 PC2 PC2

Figure 34.6-1 Diagnosis of 21-hydroxylase (21 OHD) deficiency using steroid measurements of 17-hydroxyprogesterone (17-OHP) in serum. The diagnostic thresholds appear similar for LC-MS/MS assays. With kind allowance of Ref. /6/.

Morning 17-OHP (follicular phase) 200–1,000 ng/dL (6–30 nmol/L) < 200 ng/dL< 60 nmol/L > 1,000 ng/dL> 30 nmol/L 21 OHD excluded 21 OHD Cosyntropin stimulation test < 1,000 ng/dL (< 30 nmol/L)
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