Adrenal Function Tests DETAILS OF ADRENAL FUNCTION TESTS AND CLINICAL ASPECTS

FOR MBBS STUDENTS

1. Adrenal Function Tests Dr. Apeksha Niraula Assistant Professor Clinical Biochemistry Institute of Medicine TUTH

2. Objectives Stimulus Test (For Glucocorticoids) ACTH Stimulation (Cosyntropin) Test CRH Stimulation Test Insulin-Induced Hypoglycemia Stimulation Test Metyrapone Stimulation Test Suppression Test (For Glucocorticoids) Dexamethasone Suppression Test  Mineralocorticoid Stimulation Tests Mineralocorticoid Suppression Tests Adrenal Androgen Stimulation Tests Adrenal Androgen Suppression Tests

3. Several strategies are used to assess adrenal function: Tests are typically designed to differentiate between primary and secondary causes of disease or to detect abnormalities that may not be apparent in the results of static, baseline laboratory measurements They can be: Provocative stimulation tests: Useful in documenting reduced secretory capacity of adrenocortical hormones A specific stimulus is applied , and the release of a given hormone is measured during a specified time interval 2. Suppression tests: used to document hypersecretion of the adrenocortical hormones

4. Adrenal Function Test (Adrenal Cortex) Hormone/Test Range Inference Serum Cortisol (Diurnal rhythm of Cortisol) 08:00 AM: 8–20𝜇g/dl 04:00 pm: 3-16 𝜇g/dl Evaluation of Cortical function in Addison’s disease Loss of dirunal rhythm in Cushing’s Syndrome Urinary Free Cortisol 20-80 𝜇g/day Stress, Adrenal Adenoma ACTH 10-60 pg/dl Overnight Dexamethasone Suppression Test Measuring Cortisol at 8:00 am after 1 mg oral dexamethasone at midnight Exclusion of Cushing’s if Plasma cortisol is <5 𝜇g/dl CRH Test (Corticotropin releasing hormone) Plasma Cortisol determination at 0, 30, 45,60,90 minutes of iv 100 𝜇g CRH Impaired response in ACTH deficiency

5. Metyrapone Test Increased ACTH secretion after Metyrapone administration Increased ACTH secretion in Adrenal Hyperplasia No response in Adrenal tumo r (Benign/ Malignant) ACTH Stimulation Test Determination of Plasma Cortisol after i.m synthetic form of ACTH No increase in Cortisol after ACTH administration in Addison’s Disease Insulin Induced Hypoglycemia Plasma ACTH determination at 0,30,45,60,90,120 min after i.v. 0.5 U/kg body weight soluble insulin Exclusion of Cushing’s if peak plasma cortisol is more than basal

6. Referred to as the Cosyntropin test , assess es the functional capacity of the adrenal gland to synthesize cortisol Cosyntropin/ Synacthen/Cortrosyn: N-terminal 24 amino acid sequence of ACTH, which includes the biologically active domain Synacthen is a potent stimulator of cortisol secretion and has a very brief half-life and minimal antigenicity 1-hour (also known as the “Short Synacthen Test”) and Multiple-day ACTH stimulation test (or “Long or Prolonged Synacthen Test”) Addison Disease  ACTH Stimulation (Cosyntropin) Test

7. Rationale Administration of ACTH to normal subjects causes a rapid rise in the serum cortisol concentration Patients with adrenal destruction (eg, Addison disease ) show no change or an inadequate change in serum cortisol concentration after ACTH stimulation Patients with atrophy of the adrenal cortex caused by exogenous glucocorticoid treatment or dysfunction of the pituitary gland or hypothalamus may show a slight rise in serum cortisol concentration , but not one of normal magnitude

8. Procedure A morning (baseline) blood specimen is collected for determination of serum cortisol concentration; then 250 µg of cosyntropin is administered intramuscularly or intravenously Blood specimens for serum cortisol determination are collected 30 and 60 minutes after injection Interpretation A peak serum cortisol concentration of 18 to 20 µg/dL (500– 550 nmol/L) or greater is a normal response

9. Multiple-Day Adrenocorticotropic Hormone Stimulation Test Rationale To evaluate adrenal cortisol responsiveness A common situation is adrenal insufficiency treated with glucocorticosteroids before the cause has been established Prolonged ACTH stimulation is used to distinguish primary from secondary and/or tertiary (central) causes of adrenal insufficiency Procedure A total of 250 µg of cosyntropin is injected daily for 3 days This is followed by an 8-hour infusion of 250 µg of cosyntropin. Urinary-free cortisol and serum cortisol are measured daily

10. Inference No change in Cortisol: Primary Disorder (Adrenal) Increased Cortisol: Pituitary or Hypothalamic Disorder

11. CRH Stimulation Test Rationale:   The corticotropin-releasing hormone (CRH) stimulation test has been used as a diagnostic test in both adrenal insufficiency and Cushing syndrome In 95% of normal subjects, baseline ACTH increases two- to fourfold within 30-60 minutes of CRH administration Plasma cortisol typically peaks at >20 μ g/dL within the same period

12. Indications Adrenal Insufficiency: Patients with adrenal insufficiency exhibit one of three patterns of response to CRH stimulation depending on the cause: Individuals with primary adrenal insufficiency: High baseline ACTH (which increases in response to CRH) L ow cortisol levels before and after CRH 2. Individuals with secondary (pituitary) adrenal insufficiency: Low baseline ACTH levels that do not respond to CRH Cortisol levels in these patients are not affected by CRH 3. Patients with tertiary (hypothalamic) disease (ie, CRH deficiency) L ow baseline ACTH levels and an exaggerated and prolonged response to CRH Serum cortisol levels in these patients typically do not reach 20 μ g/dL

13. The CRH stimulation test has been used to identify the source of excess ACTH in ACTH-dependent Cushing syndrome Pituitary tumors tend to be sensitive to CRH stimulation while ectopic tumors do not usually respond Patients with hypercortisolism due to oversecretion of ACTH by the pituitary (Cushing disease) generally respond to CRH with a >20% rise in cortisol (average of 30- and 45-minute value and a >35% increase in ACTH (average of 15- and 30-minute value) In most cases, patients with primary adrenal hypercortisolism or ectopic ACTH syndrome do not respond to CRH Cushing Syndrome :

14. Insulin is administered to stimulate CRH release by hypoglycemia Plasma ACTH or cortisol concentrations are measured This test involves a risk of hypoglycemia (obtundation, seizure, coma, and death) and should be performed only in highly monitored settings The test should not be performed in patients with a seizure disorder or coronary artery disease Venous access must be maintained for immediate administration of intravenous glucose if hypoglycemic complications occur Used to diagnose Adrenal Insufficiency Insulin-Induced Hypoglycemia Stimulation Test

15. Metyrapone: an inhibitor of the 11-betahydroxylase enzyme that converts 11-deoxycortisol to cortisol Based upon the principle that decreasing serum cortisol concentrations normally produces an increase in corticotropin (ACTH) secretion due to a decrease in glucocorticoid negative feedback The test is performed primarily to detect partial defects in pituitary ACTH secretion 11-deoxycortisol does not inhibit ACTH secretion Metyrapone Stimulation Test

16. In healthy individuals; serum cortisol concentrations leads sequentially to decreased negative feedback at hypothalamic and anterior pituitary Corticotropin- releasing hormone (CRH) and ACTH secretion and adrenal steroidogenesis Resultant secretion of cortisol precursors, in particular, 11-deoxycortisol Can be measured by immunoassay, high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), or fast liquid chromatography-tandem mass spectrometry (LC-MS/MS) in blood or its metabolites in urine

17. In Cushing’s syndrome caused by a pituitary tumor, the ACTH response remains intact, and 11-deoxycortisol levels show a marked rise (200 nmol/L) Levels of 11-deoxycortisol that are less than this are consistent with adrenal tumor or ectopic ACTH

18. Elevated cortisol normally inhibits ACTH release from the pituitary gland, resulting in decreased production of cortisol and other ACTH-dependent steroids by the adrenal cortex The integrity of this feedback mechanism is tested by administering a potent glucocorticoid, such as dexamethasone and assessing ACTH secretion by measuring serum or urine cortisol concentrations Dexamethasone does not significantly cross-react with cortisol immunoassays, so the secreted endogenous glucocorticoid—cortisol—can be distinguished from the exogenous glucocorticoid, dexamethasone Dexamethasone Suppression Test

19.  A low dose of dexamethasone (1 mg in adults ; 0.3 mg/m 2 in children) administered at 22:00 hours is used to detect true hypersecretion of cortisol Patients with  endogenous Cushing syndrome normally fail to suppress their morning cortisol concentration to less than 5 µg/dL (138 nmol/L) with a low dose of dexamethasone This is a screening test Confirmation of Cushing syndrome requires measurement of urinary cortisol on at least two separate days or some other combination of tests (eg, midnight cortisol <5 µg/dL [<138 nmol/L], salivary cortisol <0.112 µg/ dL [<3.09 nmol/L]) A 2-mg dexamethasone dose has been used to exclude Cushing syndrome in obese patients

20. The renin-angiotensin-aldosterone system responds to electrolyte imbalance Sodium excretion and extracellular fluid volume are inversely correlated with plasma renin and aldosterone concentrations The fractional excretion of sodium (FENa) reflects the sodium status and is calculated as follows: Mineralocorticoid Stimulation Tests

21. The r enin-angiotensin-aldosterone system responds to: Blood volume Blood pressure, and Electrolyte balance (and imbalance) Sodium excretion and extracellular fluid volume are inversely correlated with plasma renin and aldosterone concentrations Investigations: Plasma Renin activity Helps to differentiate between primary and secondary hyperaldosteronism The enzyme activity is high in secondary but low in primary hyperaldosteronism Aldosterone:  6–22 ng/dL for males and 5–30 ng/dL for females Increase in hyperaldosteronism and fall in hypofunction Na + /K +

22. Mineralocorticoid suppression tests are based on salt loading, such as saline infusion or oral salt loading Mineralocorticoid administration has also been used to suppress aldosterone secretion by the adrenal gland In healthy individuals, acute expansion of plasma volume with salt increases renal perfusion, suppresses renin release, and decreases aldosterone secretion This test should not be performed in patients with severe hypertension or heart failure Mineralocorticoid Suppression Tests

23. Administration of fludrocortisone, a synthetic mineralocorticoid: normally produces comparable suppression of aldosterone secretion Fludrocortisone should be administered with caution in patients with hypokalemia and heart or renal failure An alternate suppression test uses the ACE inhibitor Captopril It is recommended when risks from volume overload (e.g., the development of pulmonary edema or hypertension) preclude the use of other procedures Captopril inhibits the conversion of angiotensin I to angiotensin II, removing the angiotensin II stimulus for aldosterone secretion

24. The response of adrenal androgen secretion to ACTH stimulation is variable Plasma DHEA and Androstenedione are increased three- to fourfold after 90 minutes of stimulation with ACTH (10 µg/m 2 ) DHEA-S, on the other hand, is increased by 30% to 50% with ACTH administration ACTH stimulation studies are not considered useful in evaluating hypoandrogenic disorders Adrenal Androgen Stimulation Tests

25. Overnight suppression using dexamethasone produces small changes in adrenal androgen concentration compared with those of cortisol Dexamethasone, 0.75 mg, administered at midnight for several days, reliably suppresses adrenal androgen concentrations measured in blood Tissue stores of these androgens may account in part for the delay in response Adrenal Androgen Suppression Tests

26. Cushing syndrome Symptom complex that results from prolonged exposure to supraphysiologic concentrations of glucocorticoids

27. Prolonged exposure of body tissues to cortisol or other glucocorticoids Cushing’s syndrome: Named after the American neurosurgeon Harvey Cushing Two questions needs to be looked for: ‘Does the patient actually have Cushing’s syndrome?’ Once the diagnosis of Cushing’s syndrome is established, then a second question may be asked: ‘What is the cause of the excess cortisol secretion?’ Tests used in the differential diagnosis are different from those used to confirm the presence of cortisol overproduction Cortisol Excess

28. Diagnosis Clinical signs and symptoms Exclude Exogenous cortisol use Laboratory tests: Screening test Confirmatory test

29. Screening tests Three screening test can be used: 24-hour urinary free cortisol Salivary midnight cortisol level Overnight dexamethasone suppression test

30. 24-hour urinary free cortisol Reflection of the unbound circulating cortisol Creatinine should be measured in all collections Values greater than four times the upper limit of normal: suggestive of Cushing’s syndrome

31. Adrenal Insufficiency Primary adrenal insufficiency : Acquired (autoimmune destruction) Genetic (congenital adrenal hyperplasia) Secondary : Pituitary origin Tertiary: Hypothalamic origin Suppression of the HPA axis as a consequence of exogenous glucocorticoid treatment - more common

32. Diagnosis Hyponatremia, Hyperkalemia, metabolic acidosis Cosyntropin test

33. Mineralocorticoid Excess Most common cause: Primary hyperaldosteronism d/t aldosterone producing adrenal adenoma ( Conn’s syndrome )

34. Manifestations Hallmark : Hypokalemic hypertension Serum sodium tends to be normal due to the concurrent fluid retention Severe hypokalemia : muscle weakness, overt proximal myopathy, or even hypokalemic paralysis

35. Diagnosis Hypertension associated with drug resistance, hypokalemia , an adrenal mass , or hypertension before the age of 40 years: Initially measure the serum potassium Plasma renin activity is measured: LOW 24-hour urine collection for aldosterone and potassium excretion: HIGH Plasma Aldosterone: Renin ratio- HIGH (>750 pmol/l per ng/ml/hr) Aldosterone >450pmol/L= Conn’s Syndrome

36. Confirmatory test Confirmed by performing one of the four following: Saline loading test: Aldosterone <140 pmol/l [ Renin= Aldosterone] 2) Fludrocortisone suppression test 3) Captopril challenge test

37. Thank You