ADRENAL FUNCTION, TESTING AND DISORDERS I. Physiology and Anatomy

CLS 415 Clinical Chemistry II Endocrinology/Toxicology Lecture Series Adrenal Lecture Info Handout

1

ADRENAL FUNCTION, TESTING AND DISORDERS

I. Physiology and Anatomy of the Adrenal Gland A. Location

1. On upper pole of each kidney 2. Approximately 4 grams each 3. High vascular flow

B. Gland structure and appearance

1. ‘Pyramidal’ in shape 2. Consists of two distinct components

a. Outer cortex component (~90%) 1) Bright yellow color 2) Produce steroid hormones 3) Consists of 3 distinct layers of cells:

Zona glomerulosa: aldosterone Zona fasciculata: cortisol Zona reticularis: adrenal androgens

b. Inner medulla component (~10%) 1) Pearly gray color 2) Produce catecholamines 3) Consists of chromaffin cells

II. Characteristics of Hormones Produced by the Adrenal Gland

A. Cortical Hormones: Steroids 1. Corticosteroids

a. Mineralcorticoids: Aldosterone b. Glucocorticoids: Cortisol

2. Sex steroids a. Androgens: DHEAs (largest amount, 90% of plasma), testosterone b. Progesterone, estrogens (very small amounts, almost insignificant)

3. Common precursor: cholesterol 4. All share common carbon structure: basic steroid nucleus

(cyclopentanoperhydrophenanthrene nucleus, 17 carbons)


2

5. Steroid hormone classification based on number of carbons a. C18: estrogens (very minute amount produced) b. C19: androgens c. C21: corticosteroids

6. Not water soluble: requires carrier protein or conjugation a. Approximately 90-98% of all steroids circulate bound to carrier

protein 1) Corticosteroid-binding globulin 2) Sex-hormone binding globulin 3) Albumin

b. Conjugated to glucuronide or sulfate making hormone water soluble to circulate not bound to protein and excreted in urine

7. Metabolism/Excretion a. Liver: main function of the liver is to

1) Inactivate the steroid (to a metabolite) and/or 2) Make the steroid and/or metabolite water soluble (via

conjugation) for excretion by the kidney 3) Cytochrome P-450 metabolizing enzyme system 4) Considerable metabolism of steroids takes place outside of

their original site of synthesis b Kidney: main excretory route c. Biosynthesis pathway: cholesterol precursor and specific enzymes


3

B. Medullary Hormones: Catecholamines 1. Dopamine, norepinephrine, epinephrine 2. Common precursor: tyrosine (from diet or synthesized from phenylalanine

in liver) 3. Basic structure: catechol group + mono-amine

catechol group = benzene ring with two hydroxyls

4. Water soluble, transported in blood unbound to proteins, half life ~ 2 min 5. Metabolism/Excretion

a. Liver, most tissues, kidney, RBC: Catecholamines inactivated by catechol-O-methyl transferase (COMT) enzyme and oxidative deamination

b. Liver: conjugated with sulfuric and glucuronic acid c. Kidney: main excretory route d. Biosynthesis: tyrosine precursor and specific enzymes

Phenylalanine

This enzyme found only in adrenal gland


4

III. Hormones of the Adrenal Cortex: Aldosterone and Cortisol A. Mineralcorticoids: aldosterone

1. Function/physiologic effects a. Regulate salt homeostasis (Na+ and K+) b. Regulate extracellular fluid volume c. t1/2 = 15-30 minutes

2. Biosynthesis and regulation a. Synthesized only in the adrenal gland b. Major control mechanism: renin-angiotensin system

1) Stimulated by decreased plasma sodium, increased plasma potassium, decreased blood volume, decreased blood pressure

2) Renin: proteolytic enzyme synthesized and stored in juxtaglomerular cells of kidney

3) Angiotensinogen: globulin synthesized in liver 4) ACE: circulating angiotensin-converting enzyme found in

abundance in lung (converts angiotensin-I to angiotensin-II) 5) Aldosterone: stimulates renal tubular cells (DCT) to reabsorb

sodium and water, excrete potassium 6) Minor control: ACTH


5

B. Glucocorticoids: cortisol 1. Function/physiologic effects:

a. Essential for life, especially when under major stress or severe trauma (plays role in protection under stress); t1/2 = 60-90 min

b. Influence on carbohydrate metabolism: promotion of gluconeogenesis, deposition of liver glycogen and reduction of glucose utilization (increase plasma glucose, antagonist to insulin)

c. Influence on protein metabolism: increased gluconeogenesis primarily due to stimulation of protein catabolism (breakdown); inhibits amino acid uptake and protein synthesis in peripheral tissues (especially muscle, skin, bone)

d. Affect fat metabolism: when in excess, causes a central distribution of fat in the neck, face, and trunk

e. Anti-inflammatory actions: used therapeutically to treat inflammatory conditions such as rheumatoid arthritis

f. Immunosuppressive actions; anti-allergic properties 2. Diurnal variation

a. under influence of ACTH b. high levels early morning, lowest levels late evening

3. Biosynthesis and regulation a. Synthesized primarily in the zona fasciculata in adrenal gland b. Negative feedback control mechanism: hypothalamus- pituitary-adrenal axis


6

IV. Laboratory Methodologies: Adrenal Cortex Hormones A. General information and considerations

1. Immunoassays (RIA, FPIA, MEIA, chemiluminescence) and chromatography methods such as high pressure liquid chromatography (HPLC) have been developed that can measure all clinically significant steroids

2. Assay sensitivity has increased so that urine is not exclusive sample

analyzed 3. Analysis of urine

a. 24 hour urine collection b. Problems: incompleteness of sample collection, altered renal function c. Usually represents the secretory activity of the endocrine gland

over a period of time d. Measures hormone and/or its metabolites and/or its conjugates e. Advantage: good for measuring amount of ‘free’ hormone since

this form will generally pass through glomerulus

4. Analysis of blood a. Heparinzied plasma is most common sample, some serum and

EDTA plasma for specific assays. b. Strict specimen collection and handling. Often sample must be

drawn in pre-chilled venipuncture tubes, transported to lab on ice, separated immediately in refrigerated centrifuge, frozen on dry ice, repeated freeze-thaw cycles avoided.

c. Largely replaced urine assays for routine purposes due to convenience for patient and laboratory

d. More appropriate than urine sample for stimulation and suppression testing because results represent the concentration of hormone at time of sampling

5. Analysis of saliva

a. Generally, most steroids of clinical interest can be measured, but is not used in routine clinical laboratories at this time

b. Generally, reliable indicator of ‘free’ hormone: cortisol, progesterone, estriol (E3)

c. Non-invasive, stress-free sampling technique

6. Free versus bound: measurement of physiologically active form may be of interest when alterations in binding protein occur a. Total assay: measures protein-bound and ‘free’ hormone b. Free assay: measures physiologically active form


7

7. Assay procedures used for steroid measurement have many similarities. One or more of the following steps may be required before final quantitation

a. Hydrolysis: usually done to measure both the steroid and its conjugates in urine or blood. Hydrolysis frees the conjugate from the steroid so you can measure the total amount of steroid present.

b. Extraction: after hydrolysis, steroids that were conjugated are now insoluble in aqueous (urine) solution. By mixing with an organic, the steroid will extract into the solvent (like with like). Note that addition of an organic solvent to the plasma denatures the binding proteins. Thus, the extracted steroid represents both protein-bound and unbound fractions.

c. Purification/separation: chromogenic substances and other nonspecific material will also be extracted with the steroid of interest. The degree of purification will depend on the method of quantitation

B. Plasma cortisol

1. Rationale/diagnostic value a. Useful in evaluating adrenal cortex function

1) adrenal hormone excess, adrenal hormone deficiency 2) increased or decreased pituitary function 3) congenital abnormalities

b. Levels will be increased with stress and low blood sugar c. Lack of diurnal variation a key indicator of dysfunction in the

hypothalamus-pituitary-adrenal axis d. Most potent glucocorticoid and accounts for 90% of total

glucocorticoid activity e. Released within 2-3 minutes of ACTH stimulation; t1/2 ~ 2 hrs

2. Methodology: Immunoassay 3. Specimen collection/handling: Due to diurnal variation and convenience,

routine draw times are 8am and 4pm

C. Urinary free cortisol (UFC) 1. Rationale/diagnostic value:

a. Useful for initial screening in patients suspected of having adrenal hyperfunction (Cushings syndrome)

b. Also used to evaluate patients with 1) adrenal insufficiency 2) congenital adrenal hyperplasia 3) other disorders of adrenal or pituitary function

c. Conditions which raise or lower plasma cortisol will affect UFC in a similar manner

d. Renal disease will cause lower UFC values


8

e. UFC is the non-protein bound, non-conjugated cortisol excreted in urine, reflecting the free cortisol in plasma. Approximately 2% of circulating unbound cortisol is found in urine.

f. Has replaced measurement (colorimetric reactions) of urinary 17-OH corticosteroids (17OHCS) and 17 ketogenic steroids (17KGS)

2. Methodology: Immunoassay (FPIA, RIA), HPLC 3. Specimen collection/handling:

a. 24 hr collection, acidified with glacial acetic acid or boric acid b. Multiple specimens often collected because cortisol excretion can

vary from day to day or be cyclic

D. Plasma ACTH (AdrenoCorticoTrophicHormone) 1. Rationale/diagnostic value:

a. Secreted by anterior pituitary and regulates production of steroid hormones by adrenal cortex

b. Controlled by negative feedback with cortisol, and a CNS-stress mediated control system

c. Used to differentiate primary from secondary hyper/hypo function of adrenal gland

2. Methodology: Immunoassay (FPIA, RIA, chemiluminescence) 3. Specimen collection/handling: follow method protocol closely since

ACTH is very labile

E. 11-deoxycortisol (Compound S) 1. Rationale/diagnostic value:

a. 11-deoxycortisol is produced in the biosynthesis pathway just prior to cortisol. For completion of pathway to cortisol formation, 11-beta hydroxylase enzyme is needed

b. Useful in the work-up of patients with possible 11-beta hydroxylase enzyme deficiency (causing congenital adrenal hyperplasia) using the metyrapone stimulation test

2. Methodology: HPLC, Immunoassay (RIA) 3. Specimen collection/handling: plasma, follow method protocol

11-beta- hydroxylase


9

F. DHEAs 1. Rationale/diagnostic value:

a. Used primarily to document an adrenal source of androgen in the work-up of female patients with hirsutism. A direct indicator of adrenal androgen output.

b. Has replaced measurement of urinary 17-ketosteroids (17KS) for adrenal androgen assessment.

c. Unlike cortisol, does not exhibit significant diurnal variation d. Unlike testosterone, does not circulate protein bound

2. Methodology: Immunoassay (RIA), HPLC 3. Specimen collection/handling: serum, follow method protocol

G. 17-OH progesterone 1. Rationale/diagnostic value:

a. 17-OH progesterone is produced in the biosynthesis pathway following progesterone and preceding 11-deoxycortisol

b. Useful in evaluating congenital adrenal hyperplasia syndromes Elevated levels: 21-hydroxylase and 11-hydroxylase deficiencies Decreased levels: 17-hydroxylase enzyme deficiency.

2 Methodology: Immunoassay (chemiluminescence, RIA), HPLC 3 Specimen collection/handling: serum, follow method protocol


10

H. Plasma aldosterone 1. Rationale/diagnostic value: Useful in evaluating patients with

hypertension caused by a suspected adrenal adenoma (Conn’s syndrome) 2. Methodology: Immunoassay (RIA), HPLC 3. Specimen collection/handling: follow method protocol. Renin is often

obtained at same time

I. Plasma renin activity (PRA) 1 Rationale/diagnostic value:

a. Useful in differentiating primary from secondary hyperaldosteronism.

b. Primary: increased aldosterone, decreased renin Secondary: increased aldosterone, increased renin

2 Methodology: a. Current methods today do not measure renin directly; instead they

measure plasma renin activity (PRA). b. PRA is defined as the rate of angiotensin-I production from the

substrate angiotensinogen 3. Specimen collection/handling: EDTA plasma, strict requirements, follow

method protocol closely. Documentation of left or right renal vein catheterization, supine or upright, ambulatory status, fasting, regular or salt restricted diet.

V. Testing the Functional Status of the Adrenal Cortex

A. General information and considerations 1. Relying on basal hormone concentrations for confirming adrenal cortisol

disorders can be problematic due to episodic and circadian output 2. Dynamic testing of hypothalamus-pituitary-adrenal axis helps define

abnormalities; differentiate primary from secondary disease a. Stimulation tests are useful in documenting cause of hyposecretion

of adrenocortical hormones b. Suppression tests are useful in documenting cause of

hypersecretion of adrenocortical hormones

3. General test protocol a. Collect baseline hormone levels b. Administer drug: stimulate a hypo-functioning gland or suppress a

hyper-functioning gland c. Monitor the response by measuring the release of a given/specific

hormone. Usually multiple samples obtained at specific time intervals: example 30, 60, 90 minutes post dose


11

B. Rapid ACTH Stimulation Test 1. Rationale: Synthetic ACTH (cortrosyn, cosyntropin) when given to

normal subjects results in a rapid rise in plasma cortisol. Used to differentiate primary from secondary adrenal insufficiency.

2. Protocol: baseline plasma cortisol and aldosterone; administer synthetic ACTH intramuscularly or intravenously; obtain plasma cortisol 30 and 60 minutes post dose.

3. Interpretation: a. A significant increase in plasma cortisol excludes primary adrenal

insufficiency. (Normal response) b. Patients with adrenal destruction (Addisons disease) show no

change in plasma cortisol over baseline. Confirm with Prolonged ACTH Stimulation Test.

c. Patients with atrophied adrenal cortex due to pituitary or hypothalamus dysfunction, or exogenous glucocorticoid treatment, may also show no change in plasma cortisol or may show a slight rise in plasma cortisol (subnormal response) Follow up with Prolonged ACTH Stimulation Test

C. Prolonged ACTH Stimulation Test

1. Rationale: Used to evaluate adrenal cortex function. Performed when secondary adrenal insufficiency is suspected; used to differentiate primary from secondary adrenal insufficiency.

2. Protocol: ACTH gel is injected for 3 days, followed by an 8 hour infusion of ACTH. Urinary free cortisol and plasma cortisol are measured daily

3. Interpretation: a. A significant increase in plasma cortisol excludes primary adrenal

insufficiency. (Normal response) b. No change in cortisol levels over baseline confirms primary

adrenal insufficiency (Addison’s disease) c. A progressive staircase rise in cortisol levels over 2-3 days is seen

in patients with atrophied adrenal cortex due to pituitary or hypothalamus dysfunction, or exogenous glucocorticoid treatment.

D. Metyrapone stimulation test

1. Rationale: Various uses: differentiate causes of Cushing’s syndrome, measure pituitary ACTH reserve and useful in the work-up of patients suspected of having Addison’s. Metyrapone inhibits the 11-beta hydroxylase enzyme that converts 11-deoxycortisol to cortisol. When given to normal patients, 11- deoxycortisol will increase 40- to 80-fold within 3 hr after metyrapone dose. As the blood cortisol levels fall, the negative feedback is diminished, causing ACTH release from the pituitary. The stimulatory effect of ACTH to the adrenal cortex leads to a rise in 11-deoxycortisol, the compound immediately preceding cortisol in the biosynthetic pathway


12

2. Protocol: baseline plasma cortisol, 11-deoxycortisol and ACTH; metyrapone given orally at midnight.; 8am blood is drawn for 11-deoxycortisol, cortisol and ACTH levels

3. Interpretation:

a. A significant increase in 11-deoxycortisol and ACTH and a fall in cortisol compared to baseline is seen in Cushing’s disease due to pituitary adenoma, excludes primary adrenal failure, indicates adequate pituitary ACTH reserve. (Normal response)

b. No change in steroid production over baseline (cortisol baseline level high) suggest Cushing’s syndrome due to adrenal adenoma or ectopic ACTH secreting tumor

c. No change in cortisol, 11-deoxycortisol and ACTH levels over baseline (cortisol, 11-deoxycortisol and ACTH baseline levels low) suggest pituitary ACTH deficiency or hypothalamic dysfunction

E. Overnight Dexamethasone Suppression Test

1. Rationale: Screening test used to determine the cause of elevated cortisol levels. Dexamethasone is a potent (30X) cortisol analog (look alike) that suppresses secretion of ACTH from the pituitary gland resulting in decreased cortisol levels.

2. Protocol: baseline plasma cortisol; 1 mg dexamethasone given orally at

midnight; plasma cortisol drawn at 8am 3. Interpretation: a. Cortisol suppression to <5 ug/dl (<50% of baseline value)

excludes Cushing’s syndrome. (Normal response) b. No change in cortisol over baseline (baseline levels high) suggests elevated cortisol due to Cushing’s syndrome, stress, obesity,

severe mental depression, alcoholism or non-compliance. Follow up with Low Dose Dexamethasone Suppression Test.


13

F. Low Dose Dexamethasone Suppression Test 1. Rationale: Performed on patients who have a positive overnight screening

test. Used to determine the cause of elevated cortisol levels and to differentiate primary from secondary Cushing’s syndrome.

2. Protocol: 24 hr urine samples are collected daily for 4 consecutive days. 0.5 mg dexamethasone given orally every 6 hr starting at 8am day 2 (for a total of 8 doses). Urinary Free Cortisol (UFC) and creatinine are measured on each 24 hr urine sample. Plasma cortisol is measured at 8am and 8pm on day 1 to look for diurnal variation.

3. Interpretation: a. Plasma cortisol and UFC suppression on day 4 to <50% of baseline

values excludes Cushing’s syndrome. (Normal response). Plasma cortisol on day 1 at 8pm should be 1/3 the value at 8am, indicating diurnal variation not lost. Suggests elevated cortisol levels due to stress, obesity, severe mental depression, alcoholism.

b. No change in cortisol levels over baseline (persistent elevation of cortisol levels) suggests primary or secondary Cushing’s syndrome. Follow up with High Dose Dexamethasone Suppression Test.

G. High Dose Dexamethasone Suppression Test

1. Rationale: Performed on patients who have a positive low dose dexamethasone test. Used to differentiate primary from secondary Cushing’s syndrome and to establish the differential diagnosis of an ACTH-secreting pituitary adenoma.

2. Protocol: 24 hr urine samples are collected daily for 4 consecutive days. 2.0 mg dexamethasone given orally every 6 hr starting at 8am day 2 (for a total of 8 doses). Urinary Free Cortisol (UFC) and creatinine are measured on each 24 hr urine sample. Plasma cortisol is measured at 8am and 8pm on day 1 to look for diurnal variation, and at 8am on day 5.

3. Interpretation: a. Patients with Cushing’s syndrome due to an ACTH producing

pituitary adenoma (usually) show suppression of UFC excretion >50% of baseline by day 4, lack diurnal variation in plasma cortisol on day 1, and show suppression of plasma cortisol

to <10 ug/dl at 8am on day 5. b. No change in cortisol levels over baseline (persistent elevation of

cortisol levels) with a lack of diurnal variation suggests Cushing’s syndrome due to adrenal cortical adenoma, adrenal cortical carcinoma, or ectopic production of ACTH.


14

VI. Pathophysiology of Adrenal Cortical Hormones A. Adrenal insufficiency

1. Adrenal hypofunction refers to decreased production of cortical hormones due to some type of disease process affecting the cortex. Hormone production/secretion may be insufficient to maintain life. Symptoms begin to appear after 90% of gland has been destroyed.

2. Addison’s Disease

a. Classified as primary disorder: cortisol, ACTH 1) autoimmune disease (~70%) 2) destruction of adrenal cortex (trauma, infection) 3) surgical removal of gland

b. Symptoms 1) insiduous onset, depends on extent of adrenal failure 2) fatigue, weakness, weight loss, gastrointestinal

disturbances, postprandial hypoglycemia, intolerance to stress, low blood pressure

3) hyperpigmentation of skin and mucus membranes through melanocyte stimulating hormone (MSH) action on melanocytes

4) if mineralcorticoid deficiency, dehydration with hypotension, hyponatremia, hyperkalemia

c. Laboratory assessment 1) baseline cortisol and ACTH: plasma cortisol, UFC, ACTH 2) ACTH stimulation test: no significant increase in plasma

cortisol levels over baseline.

3. Hypocortisolism due to lack of pituitary ACTH a. Classified as a secondary disorder: cortisol, ACTH

1) pituitary disease or dysfunction affecting part or total (panhypopituitarism) pituitary gland

2) long term glucocorticoid treatment resulting in iatrogenic pituitary insufficiency

3) hypothalamic CRH deficiency (rare, tertiary) b. Symptoms: fatigue, weakness, weight loss, gastrointestinal

disturbances, postprandial hypoglycemia, intolerance to stress c. Laboratory assessment

1) baseline cortisol and ACTH: plasma cortisol,UFC,ACTH 2) ACTH stimulation test: a progressive staircase rise in

cortisol levels over 2-3 days is seen in patients with atrophied adrenal cortex due to pituitary or hypothalamus dysfunction, or exogenous glucocorticoid treatment


15

3) Metyrapone stimulation test: no change in cortisol, 11-deoxycortisol and ACTH levels over baseline (cortisol, 11-deoxycortisol and ACTH baseline levels low) suggest pituitary ACTH deficiency or hypothalamic dysfunction

4) aldosterone levels most often normal (not affected) due to primary control of zona glomerulosa by renin-angiotensin system

4. Addisonian crisis a. An acute adrenal insufficiency which is life threatening b. A patient with adrenal insufficiency on glucocorticoid replacement

therapy, undergoes a sudden decrease in cortisol levels such that the patient cannot respond to or recover from any additional stress such as acute/chronic illness, surgery, anesthesia, trauma, severe infection, personal life crisis. These patients will need occasional glucocorticoid supplements during periods of additional stress.

c. Symptoms: fever, dehydration, nausea, vomiting, hypotension; may rapidly evolve into circulatory shock, vascular collapse, coma and death.

B. Adrenal hyperfunction

1. Adrenal hyperfunction refers to increased production of cortical hormones due to some type of disease process affecting the cortex, producing clinical syndromes of glucocorticoid excess, mineralcorticoid excess and androgen excess. Cushing’s syndrome is a result of autonomous, excessive production of cortisol

2. Primary Hypercortisolism (Cushing’s syndrome): cortisol, ACTH

a. Caused by adrenal tumor: adenoma (benign), carcinoma (malignant) or nodular hyperplasia

b. Symptoms 1) physical appearance due to mobilization of fat stores:

truncal obesity, moon face, buffalo hump 2) muscle weakness, easy bruisability, abdominal striae 3) hypertension, hyperglycemia, decreased glucose tolerance 4) menstrual dysfunction, hirsutism, sexual dysfunction 5) psychiatric disturbances

c. Laboratory assessment 1) baseline cortisol, UFC and ACTH:

cortisol with loss of diurnal variation, UFC, ACTH 2) High dose dexamethasone suppression test: no change in

cortisol levels over baseline (persistent elevation of cortisol levels) with a lack of diurnal variation suggests Cushing’s syndrome due to adrenal cortical adenoma or adrenal cortical carcinoma (or ectopic ACTH)


16

3) Metyrapone stimulation test: no change in steroid production over baseline (cortisol baseline level high) suggest Cushing’s syndrome due to adrenal adenoma (or ectopic ACTH secreting tumor)

3 Cushing’s Disease

a. Caused by an ACTH-secreting pituitary adenoma and classified as a secondary disorder: cortisol, ACTH

b. Symptoms: same as for primary hypercortisolism, except hyperpigmentation of skin and mucus membranes through melanocyte stimulating hormone (MSH) action on melanocytes

c. Laboratory assessment 1) baseline cortisol, UFC and ACTH

cortisol with loss of diurnal variation, UFC, ACTH 2) High dose dexamethasone suppression test: shows

suppression of UFC excretion >50% of baseline by day 4, lacks diurnal variation in plasma cortisol levels on day 1, and shows suppression of plasma cortisol to <10 ug/dl at 8am on day 5.

3) Metyrapone stimulation test: a significant increase in 11-deoxycortisol and ACTH and a fall in cortisol compared to baseline

4 Secondary hypercortisolism: cortisol, ACTH

a. Caused by 1) ectopic ACTH-secreting tumor (oat cell carcinoma of lung) 2) long term ACTH treatment resulting in iatrogenic

hypercortisolism b. Symptoms: same as for primary hypercortisolism, except

hyperpigmentation of skin and mucus membranes through melanocyte stimulating hormone (MSH) action on melanocytes

c. Laboratory assessment 1) baseline cortisol, UFC and ACTH:

cortisol with loss of diurnal variation, UFC, ACTH 2) High dose dexamethasone suppression test: no change in

cortisol levels over baseline (persistent elevation of cortisol levels) with a lack of diurnal variation suggests ectopic production of ACTH.

3) Metyrapone stimulation test: no change in steroid production over baseline (cortisol baseline level high) suggest Cushing’s syndrome due to ectopic ACTH secreting tumor


17

C. Congenital adrenal hyperplasia (CAH); Adrenogenital syndrome 1. Most common adrenal disorder encountered in pediatrics 2. Due to a genetic disorder causing a lack of a specific enzyme required in

the biosynthetic pathway of adrenal steroid hormones causing cortisol deficiency. Lack of cortisol in the negative feedback loop causes an increase in pituitary ACTH. Increased ACTH overstimulates the adrenal gland causing hyperplasia of the adrenal gland and increased output of steroids prior to the enzyme deficiency.

3. 21-hydroxylase deficiency

a. Most common enzyme deficiency b. Inhibits glucocorticoid (cortisol) and mineralcorticoid

(aldosterone) production c. Excess progesterone and 17-OH progesterone are ‘shunted’ toward

the androgen pathway causing an increase in adrenal androgens ( DHEAs, TST).

d. Increased adrenal androgens leads to virilization of external genitalia in female babies termed ambiguous genitalia. Male babies appear normal at birth and develop secondary male sex characteristics at an early age termed precocious puberty.

e. Laboratory assessment: decreased plasma cortisol and aldosterone; increased ACTH; increased plasma 17-OH progesterone is diagnostic; increased testosterone; DHEAs may appear normal due to peripheral tissue conversion to testosterone.


18

4. 11-beta-hydroxylase deficiency a. Second most common enzyme deficiency b. Inhibits glucocorticoid (cortisol) and mineralcorticoid

(aldosterone) production c. Excess deoxycorticosterone and 11-deoxycortisol are ‘shunted’

toward the androgen pathway causing an increase in adrenal androgens ( DHEAs, TST).

d. Increased adrenal androgens leads to virilization of external genitalia in female babies termed ambiguous genitalia. Male babies appear normal at birth and develop secondary male sex characteristics at an early age termed precocious puberty.

e. Laboratory assessment: decreased plasma cortisol and aldosterone; increased ACTH; increased plasma 11-deoxycortisol is diagnostic; increased testosterone; DHEAs may appear normal due to peripheral tissue conversion to testosterone.

5. 17-hydroxylase deficiency a. Third most common enzyme deficiency (rare) b. The conversion of progesterone to 17-OH progesterone and

pregnenolone to 17-OH pregnenolone are inhibited resulting in decreased glucocorticoid (cortisol), androgen and estrogen production.

c. Decreased androgens and estrogens leads to sexual infantilism: male babies are born with ambiguous genitalia, and may be identified incorrectly as a female baby.; female babies appear normal. At puberty, ovaries fail to secrete estrogen and testes fail to produce testosterone causing the pituitary gonadotropins to be elevated.

d. Laboratory assessment: decreased plasma cortisol and increased ACTH; decreased testosterone; decreased estrogen (estradiol); increased LH and FSH

D. Primary hyperaldosteronism (Conn’s syndrome): aldosterone, renin (PRA)

1. Caused by adrenocortical adenoma or hyperplasia affecting only the zona glomerulosa (outer layer) of the adrenal cortex

2. Symptoms: a. Hypertension, edema, muscle weakness, fatigue and cramping b. If chronic: cardiac dysrhythmias

3. Laboratory assessment: increased aldosterone, decreased renin (PRA), hypernatremia, hypokalemia, tendency toward metabolic alkalosis

E. Secondary hyperaldosteronism: aldosterone, renin (PRA) 1. Kidney disease or systemic disease causing increased renin release from

the juxtaglomerular cells in the kidney a. Excess sodium loss due to kidney disease b. Decreased blood pressure causing decreased renal perfusion c. Volume depletion d. Renin-secreting tumor (rare)


19

2. Symptoms: hypertension predominates 3. Laboratory assessment: increased aldosterone, increased renin (PRA),

hypernatremia, hypokalemia, tendency toward metabolic alkalosis VII. Hormones of the Adrenal Medulla: Catecholamines

A. Dopamine, norepinephrine, epinephrine 1. The main secretory products of the adrenal medulla are epinephrine

(~80%) and norepinephrine (~20%). Epinephrine is considered the ‘true’ medullary hormone since adrenal medulla is only site of synthesis.

2. Main sites of catecholamine production: central nervous system (CNS),

chromaffin cells (adrenal medulla) and peripheral nervous system. a. Dopamine: produced primarily in CNS b. Norepinephrine: produced primarily in nerve tissue and adrenal

medulla; principle product synthesized in CNS c. Epinephrine: produced exclusively in the adrenal medulla;

principle catecholamine produced by the adrenal glands critical enzyme (PNMT) for conversion of norepinephrine to epinephrine is located in adrenal tissue in adult humans, extra- adrenal chromaffin cells exist in and around sympathetic ganglia and as residual clusters found in regions of the abdomen and neck: lung, liver, heart, intestine and prostate

3. Neurogenic stimulus commands catecholamine release from storage particles in the CNS and adrenal medulla, and are released as a result of anxiety, stress, fear, pain, a fall in blood pressure or blood volume, hypoxia, exposure to cold, drugs, anesthesia, trauma, hypoglycemia, anorexia, congestive heart failure, cardiac arrhythmia, myocardial infarction, muscle exertion, emotional disturbances, hypothyroidism, etc


20

4. Rapidly cleared from circulation by liver metabolism (conjugation with glucuronic acid and sulfate, and deamination), kidney excretion, or taken up by sympathetic neurons a. Metabolism of dopamine to final product: homovanillic acid (HVA)

b. Metabolism of norepinephrine and epinephrine to final product:

vanillylmandelic acid (VMA)


21

B. Cell membrane receptors and biological action 1. Biological actions of catecholamines are initiated through their interaction

with two different types of specific cell membrane receptors: a. alpha-adrenergic receptors b. beta-adrenergic receptors c. Because epinephrine and norepinephrine are produced in the

adrenal gland, their receptors are termed ‘adrenergic’ 2. These receptors have different affinities for norepinephrine and

epinephrine and cause opposing physiological effects a. Norepinephrine: primarily interacts with alpha-adrenergic

receptors b. Epinephrine: interacts with both alpha- and beta-adrenergic receptors

3. Stimulation of alpha-adrenergic receptors results in a. Vasoconstriction b. Decrease in insulin secretion c. Sweating d. Piloerection (hair standing on end) e. Stimulation of glycogenolysis in the liver and skeletal muscle

(increase blood glucose) 4. Stimulation of beta-adrenergic receptors results in

a. Vasodilation b. Stimulation of insulin release c. Increased cardiac contraction rate d. Relaxation of smooth muscle in the intestinal tract e. Bronchodilatation by relaxation of smooth muscles in bronchi f. Stimulation of renin release (enhancing Na/H2O retention by kidney) g. Enhanced lipolysis

C. Function/physiologic effects of catecholamines 1. Physiologic actions are diverse; t1/2 ~ 1-2 minutes 2. Function as transmitters of hormonal or neuronal signals in a wide range

of physiological processes a. Neurotransmitters in the CNS (dopamine and norepinephrine) b. Peripheral neuro-hormonal transmitters in the sympathoadrenal

medullary system (epinephrine and norepinephrine) 3. Dopamine

a. Neurotransmitter in the CNS (brain and spinal cord) b. Found in highest concentration in CNS c. Site of release = site of action

4. Norepinephrine (noradrenaline): a. Neurotransmitter in the CNS, sympathetic nervous system, and

peripheral tissues b. Site of release = site of action c. Changes in norepinephrine levels in the brain can change many

regulatory functions of the hypothalamus: thirst, hunger and temperature regulation, blood pressure maintenance, reproduction and behavior.


22

5. Epinephrine (adrenaline): a. Epinephrine is released into the bloodstream and is carried to

target tissue to exert its effects on metabolic processes, especially carbohydrate metabolism

b. Responsible for the classic ‘fight or flight response’ 6. Epinephrine is critical (along with norepinephrine and dopamine) in

maintaining the body’s homeostasis and in responding to acute and chronic stress via cardiovascular, metabolic and visceral activities. Overall effects of catecholamines include: a. Increase alertness, wakefulness; erects body hair; gooseflesh b. Bronchodilatation c. Increase respiratory rate and depth of respirations d. Increase rate and force of contractions of the heart e. Increase oxygen consumption of tissues, basal metabolic rate (BMR) f. Vasoconstriction g. Vasodilation h. Stimulates glycogenolysis in liver and muscle i. Stimulates release of insulin j. Increases free fatty acid liberation from adipose tissue (lipolysis) k. Inhibits intestinal motility l. Smooth muscle relaxant

VIII. Laboratory Methodologies: Adrenal Medulla Hormones

The purpose of measuring catecholamines and their metabolites is to 1) check adrenal medulla function, 2) investigate possible causes of hypertension, and 3) investigate cause of an abdominal mass A. General information and considerations

1. Methods of catecholamine measurement are problematic: a. Catecholamines are easily oxidized and very sensitive to air and

light exposure; found in very low concentrations; very short t1/2 b. Synthesis is not continuous, but in response to neurogenic stimuli c. Catecholamine excretion influenced by diet (caffeine, nicotine),

medications and drugs d. Historical methods of measurement included fluorometric and

spectrophotometric assays, which were based on non-specific reactions with the biogenic amines. These assays were prone to many interferences, thus assay modifications have been suggested to improve their accuracy.

2. HPLC: most widely used method in clinical laboratories (highest sensitivity,

specificity and reliability) and is the preferred method because it can fractionate each catecholamine and metabolite. Other methodologies include fluorometric and spectrophotometric methods, GC/MS, and limited immunoassay (FPIA, only for VMA at this time)


23

3. One or more of the following procedural steps may be required before final quantitation: a. Pretreatment: preliminary extraction of plasma is required to

measure the very low levels of catecholamines found in normal subjects. Example: alumina (oxide) extraction or ion-exchange chromatography

b. Hydrolysis: usually done to measure both the catecholamine and its conjugates in urine or blood. Hydrolysis frees the catecholamine from its conjugate so you can measure the total amount of catecholamine present

c. All urinary methods require preliminary removal of protein and interfering substances: ion-exchange chromatography or adsorption method

4. Analysis of urine

a. Catecholamines can be excreted into the urine as free amines and/or as conjugates and/or as metabolites.

b. Antihypertensive medications should be withheld from the patient for at least 2 days before and during specimen collection

c. 24 hour urine collection, preservative (6M HCl) added, refrigerated during collection. Follow method protocol

d. Problem: incompleteness of urine collection, altered renal function

5. Analysis of blood a. Both free amine and conjugated forms circulate in blood b. Assay methods requires high sensitivity, specificity and reliability

in order to measure the normally very low concentrations of dopamine, norepinephrine and epinephrine

c. Strict specimen collection and handling requirements because catecholamines are unstable and easily oxidized: draw sample in prechilled EDTA or heparinized plasma with antioxidant added, transport to laboratory on ice, centrifuge at 4oC within 30 min of collection, store plasma at -70oC until analysis, avoid repeated freeze/thaw cycles. Follow method protocol.

d. Draw through catheter to eliminate false increase due to stress of venipuncture procedure

e. Catecholamine levels increase 2-3 fold when a supine subject assumes an upright position. Thus, recommend resting quietly 30 min in recumbent position after insertion of catheter

f. Refrain from eating, using tobacco or drinking coffee or tea for at 4 hr before venipuncture

g. Most antihypertensive drugs and many other drugs cause false positive results. Use of these drugs should be discontinued 3-7 days before obtaining sample

6 Urinary measurements preferred due to complexity of plasma

methodologies and instability of plasma catecholamines


24

B. Plasma catecholamines 1. Useful in the diagnosis of catecholamine-secreting neurochromaffin

tumors: pheochromocytomas, neuroblastomas 2. Data indicate plasma catecholamine levels to be best indicator of

increased catecholamine production. 3. HPLC is the preferred method because this method can determine the

amount of each individual catecholamine present (can fractionate dopamine, norepinephrine, epinephrine)

C. Urine catecholamine

1. Useful in the diagnosis of catecholamine-secreting neurochromaffin tumors: pheochromocytomas, neuroblastomas

2. Free amine measurement relates more closely to the tumor load when compared to total amine measurement and are least affected by diet

3. Method: HPLC (historical tri-hydroxy-indole fluorometric reaction)

D. Urinary Total Metanephrines (metanephrine and normetanephrine) 1. Metanephrines are intermediary metabolites of norepinephrine and

epinephrine, are excreted in urine as free amines and conjugates, and are not significantly influenced by diet.

2. Useful in the evaluation of suspected pheochromocytoma; good screening method

3. Method: HPLC (historical Pisano’s colorimetric reaction)

E. Urinary Vanillylmandelic Acid (VMA) 1. VMA is the final urinary metabolite of norepinephrine and epinephrine

and represents ~60% of the excretion products derived from norepinephrine and epinephrine

2 Useful in the evaluation of suspected pheochromocytoma and neuroblastoma; good screening method

3 Method: HPLC (historical Pisano’s colorimetric reaction)

F. Urinary Homovanillic Acid (HVA) 1. HVA is the final urinary metabolite of dopamine. 2. Useful in the evaluation of suspected neuroblastoma 3. Method: HPLC

IX. Pathophysiology of Adrenal Medulla Hormones

A. General information 1. Adrenal medulla hypo-function is generally of little clinical interest 2. Hyperfunction of the medulla with consequent increase in catecholamines

and their effects is clinically significant. 3. Hypertension affects ~22 million people in North America, and <1% is

caused by a neurochromaffin tumor. This cause of hypertension can be cured with surgical removal of the tumor. 20% of individuals with a pheochromocytoma remain undiagnosed and deprived of a cure.


25

B. Pheochromocytoma 1. Tumor of chromaffin cells, often benign

a. Major source of chromaffin cells: adrenal medulla (90% of cases) b. Minor source: lung, heart, liver, intestine (anywhere from base of

skull to perineum, sympathetic nervous system); (10% of cases, and termed paragangliomas)

c. 30-50 year old age group most affected; no gender predilection

2. Symptoms: severity correlates with extent of catecholamine increase; can be episodic or persistent; episodes may last a few minutes to hours to days a. Hypertension: paroxysmal (sudden) or sustained b. Severe headache c. Palpitations, tremor; panic attacks d. Sweating; pallor e. Chest pain; rapid heart beat; rapid respirations; anxiety; nausea

3. Can be misdiagnosed because symptoms similar to those of other

disorders: hyperthyroidism, MI, heart failure, panic attack

4. Laboratory assessment: generally, adrenal tumors produce principally epinephrine; extra-adrenal tumors produce principally norepinephrine a. Plasma catecholamines, urinary catecholamines

( epinephrine, norepinephrine, normal dopamine) b. urinary total metanephrines, urinary VMA, normal HVA c. Fasting hyperglycemia

C. Neuroblastoma

1. Most common extra-cranial childhood solid tumor (75% occur in children <6yrs); malignant; accounts for 15% of all childhood cancer deaths

2. Of neural crest origin, neuroblastomas may arise anywhere in the sympathetic nervous system (lymph nodes, bone marrow, spleen, etc) a. ~75% arise in abdomen (2 adrenal glands, 2 abdominal

paravertebral autonomic ganglia) b. Tumors often large and metastatic to the liver

3. Symptoms: a. Weight loss b. Weakness c. Generalized malaise d. Cerebral encephalopathy, ataxia, jerking movement of eyes e. Hypertension less frequent as compared to pheochromocytoma

4 Laboratory assessment a. plasma catecholamines, urinary catecholamines

(norepinephrine, dopamine, normal epinephrine) b. urinary VMA, urinary HVA

ADRENAL FUNCTION, TESTING AND DISORDERS I. Physiology and Anatomy

Documents

Transcript of ADRENAL FUNCTION, TESTING AND DISORDERS I. Physiology and Anatomy