CLINICAL CHEMISTRY III

THE ADRENAL GLAND

Chapter 3: The Adrenal Gland

ADRENAL GLANDS

The adrenal gland lies at the upper pole of each kidney. Both glands
are pyramidal in shape, each measuring approximately 2-3 cm in
width, 4-6 cm in length, and 1 cm thick. Regardless of age, weight,
or sex, each gland weighs approximately 4 g and consists of a
yellow, outer cortex and a gray, inner medulla.

Composed of two embryologically distinct, but conjoined, glands-the

outer adrenal cortex (derived from mesenchymal cells) and inner
adrenal medulla (from neural crest cells).

ADRENAL CORTEX

LAYERS:
 Zona glomerulosa cells G-zone (outer) 10%: synthesize mineralocorticoids critical for sodium
(volume), potassium, and acid-base homeostasis. Low cytoplasmic-nuclear ratio, small nuclei, dense
chromatin, intermediate lipid inclusions.

 Zona fasciculate cells F zone (middle) 75%: synthesize glucocorticoids critical to blood glucose
homeostasis. Cords of clear cells, high cytoplasmic-nuclear ratio, lipids laden with “foamy”
cytoplasm.

 Zona reticularis R zone (inner) 10%: sulfate DHEA to dehydroepiandrosterone sulfate

(DHEAS), a precursor for adrenal sex hormones. Sharply demarcated with lipid-deficient cords of
irregular, dense cells with lipofuscin deposits.

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FIGURE 6: The Adrenal Cortex

STEROIDOGENESIS

 All adrenal steroids are derived by sequential enzymatic conversion of a common substrate,
CHOLESTEROL containing a cyclopentanoperhydrophenanthrene nucleus as the basic structure.

1. The 3 6-sided rings (A,B, & C): phenantrene nucleus

2. D: cyclopentane ring
3. Steroid hormones contain up to 21 Carbon atoms (C21)

 The adrenal gland can also synthesize additional cholesterol using acetyl-CoA, assuring adrenal
streoidogenesis remains normal in patients with variable lipid disorders and in patients on lipid
lowering agents.
 Only free cholesterol can enter steroidogenic pathways after transport to the inner mitochondrial
membrane in response to adenocorticotropic hormone (ACTH).
 The availability of intracellular cholesterol is metabolically regulated by LDL negatively and ACTH
positively through multiple mechanisms.
 Corticotropin-releasing hormone (CRH) is secreted from the hypothalamus to circadian signals,
serum cortisol, and stress, causing release of stored ACTH which stimulates transport of free
cholesterol into adrenal mitochondria, initiating steroid production.

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 Decrease activity of any enzymes required for biosynthesis can occur as an acquired or inherited
(autosomal recessive) trait. Defects that decrease production of cortisol cause increases in ACTH
and CRH secretion in an attempt to stimulate Cortisol levels and lead to adrenal hyperplasia or
overproduction of androgens, depending on the affected enzyme.

Synthesis of Cortical hormones:

- enzymatic steps involving cleavage of part of side of cholesterol

- dehydrogenation (conversion of the hydroxyl group to a keto group or formation of double bond in one
of the rings)
- hydroxylation
o adrenal cortex is the only gland containing the enzymes with the ability to hydroxylate steroid
molecules in the C-21 (21 hydroxylase) and C-11 (11 alpha-hydroxylase) position
o Change in the proportions or activities of key enzymes in the adrenal cortex lead to overproduction
of some hormones and underproduction of others. This imbalance may be caused by disease
processes, radiation damage, genetic defects or drugs that in some of the key enzymes.
o Congenital deficiency or absence of the 21-hydroxylase enzyme - most commonly encountered
defect.

FIGURE 7:
Pathways of
Major Steroid
Synthesis in the
Adrenal Gland
and their
Enzymes.

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Transport Proteins:

1. Albumin - nonspecific, carries many steroids

2. CBG (cortisol-binding globulin)
- carries cortisol and derivatives; progesterone
- a glycoprotein, contains sialic acid and other CHO, migrates in the alpha-1-globulin region
3. SHBG (sex hormone binding globulin)
- carries testosterone; estradiol
- a glycoprotein, contains sialic acid and other CHO, migrates in the beta-globulin region.

 Liver is the site of synthesis for these proteins therefore changes in hepatic function can lead to altered
levels of these proteins.

I. GLUCOCORTICOIDS

CORTISOL

-Synthesis is critical to hemodynamics and glucose homeostasis.

-F-zone disorders manifest with blood pressure and glucose abnormalities.
-Glucocorticoids maintain blood sugar by inducing lipolysis and amino acid release from muscle breakdown
for conversion into glucose (gluconeogenesis) and storage as liver glycogen.
-Production is regulated by ACTH in the pituitary gland.
-ACTH and Cortisol are highest in the early morning (8am) and lowest at night (10pm to 12 midnight);
ACTH pulse amplitude (not frequency) rises between 2 and 4 am.
-Additional peaks of ACTH follow protein-rich meals, ADH stimulation, as well as CRH.
-Hypoglycemia, indirectly stimulates ACTH by increasing CRH and ADH release.
-Acute stress (physical and psychology) directly stimulates ACTH secretion, causing cortisol levels to rise.
-Elevated glucocorticoids (endogenous and exogenous), in turn, suppress ACTH through feedback inhibition,
decreasing POMC gene transcription in pituitary corticotroph cells and also by blocking the production,
secretion, and stimulatory effects of CRH on ACTH synthesis and release in the pituitary.

- A C21 steroid, principal glucocorticoid

- Acts on target cells by penetration and transport to the cell nucleus, binding to DNA, and altering
the transcription of RNA then alters various metabolic processes.

a. affect protein, carbohydrate and lipid metabolism

b. promote gluconeogenesis

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c. it mobilizes fat in adipose tissue for energy purposes

d. it inhibits the uptake of glucose by muscle (an insulin agonists)
e. reduces cellular reaction to inflammatory agents
f. it lessens immune response by inhibiting antibody production (used to treat rheumatoid arthritis)
g. actively released during stress

- Diurnal rhythm: peak: 8-9 am; trough: 10-11 pm (rise after meal); secreted approximately 25mg/day
- Other factors (higher than normal cortisol): stress (physical/emotional), pregnancy
- 3 forms in plasma:
1. bound to CBG, transcortin - 75% (83% Calbreath)
2. bound to albumin - 15% (12% Calbreath)
3. free - 10% (5% Calbreath)

- Inactivated by liver to tetrahydro-derivatives which are rapidly conjugated with glucoronic acid and
excreted in urine as 17-hydroxycorticosteroids(17-OHCS)

II. MINERALOCORTICOIDS

ALDOSTERONE
- The most potent mineralocorticoid, higher in the morning than afternoon.
- A C21 steroid, kidney is the target tissue, act on distal convoluted tubules to promote sodium retention.
- Decreases Na excretion and increases K excretion, maintains blood pressure, aids in regulating salts and
electrolytes, regulated to a great extent by RAS and less with ACTH concentration.
- Secreted at the rate of approximately 200 µg/day and synthesized exclusively by the G-zone.
- Stimuli to aldosterone secretion:
o Increased serum potassium
o Decreased serum sodium
o Increased ACTH level
o Low blood volume
o Rise in estrogen level
- Most are bound to alpha globulins
- Transport proteins:

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o 30% on CBG
o 42% on albumin
- Negative feedback to juxtaglomerular apparatus by long loop (aldosterone via volume expansion) and
short loop (angiotensin II via peripheral vasoconstriction).

FIGURE 8: Renin-Angiotensin-Aldosterone Axis

III. ADRENAL ANDROGENS

-Also secreted by the adrenal glands including progesterone and estrogen which are all produced by the
gonads as well.
-Produced as by-products of cortisol synthesis that is regulated by ACTH (not LH).
-Synthesized in the zona fasciculate and/or reticularis from the precursor substrate 17a-
hydroxypregnenolone.
-Principal Adrenal Androgens include dehydroepiandrosterone (DHEA), androstenedione, and 11-
hydroxyandrostenedione.

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-Proportion of total androgens (adrenal to gonadal) increases in old age.

-Primarily responsible for adrenarche (pubic and axillary hair)
-Peak concentrations in puberty.
-Stimulators: PROLACTIN, POMC PEPTIDES, T-LYMPHOCYTES

ADRENOCORTICAL INSUFFICIENCY

PRIMARY (ADDISON’S DISEASE)

-consequence of destruction or dysfunction of the cortex.
-idiopathic (autoimmune adrenal atrophy, autoimmune adrenalitis, lymphocytic adrenalitis) in 80%
of the cases.
Major causes: 80% autoimmune and 20% tuberculosis.
-usually diagnosed in the third to fifth decades.
-Conditions including infectious diseases such as Fungal (not candida) can also destroy the adrenal
gland. HIV
TB
Bilateral adrenal hemorrhage
Adrenoleukodystrophy
Infiltrative processes and metastasis
-tumors, hemorrhage, infiltrative processes, developmental abnormalities, and malignancies also
interfere with ACTH production by the pituitary gland.

SECONDARY
-Deficient pituitary ACTH secretion.
-Glucocorticoid therapy is the most common cause.

CLINICAL FEATURES:
Both primary and secondary
Weakness and fatigue
Postural hypotension
Nausea/vomiting, diarrhea, anorexia, and weight loss
Abdominal, Muscle, Joint pain

Primary
Hyperpigmentation of skin and mucous membranes
Hyperkalemia
Dehydration, salt craving

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Secondary
Low ACTH
Usually find normal K+, BUN/Crea

DIAGNOSIS

-low baseline levels (8am, supine) are suggestive but not reliable for establishing a diagnosis of insufficiency.
-Cosyntropin ([IV/IM]synthetic stimulator of cortisol and aldosterone secretion) tests the capacity of the
adrenal gland to increase hormone production in response to stimulation.
-Metyrapone (alternate diagnostic or confirmatory test for adrenal insufficiency

TREATMENT

-Primary: synthetic steroids from each cortex layer are replaced: G-Aldo (Florinef)
F-cortisol (hydrocortisone)
R-DHEA (controversial)
-Secondary: steroidogenesis from the non-ACTH regulated layers remains intact, so only cortisol is
replaced

HYPERCORTISOLISM
-caused by unregulated release of CRH, ACTH, adrenal glucocorticoid secretion and exogenous intake
-affects multiorgan systems including: Immune (suppression, poor healing)
Dermatologic (thin, friable tissue, wide purple striae)
Vascular (vessel fragility, ecchymoses)
Adipose (increased fat with redistribution to upper back and
central locations)
Muscle (wasting, proximal muscle weakness, heart failure)
Neurologic (peripheral neuropathy, autonomic dysregulation)
Bone (loss)
Renal (edema, HTN, calciuria)
Metabolic (hyperglycemia and insulin resistance)

-Cortisol also has CNS actions, influencing pain reception and sense of well-being.

CUSHING’S SYNDROME
-Regardless of etiology, all cases of endogenous Cushing syndrome are due to an increased production of
cortisol by the adrenal.
-all glucocorticoids, including synthetic, inhaled, and topical, can inhibit ACTH secretion; therefore, plasma
ACTH, serum cortisol, and cortisol excretion may all be low (unless cortisol or cortisone is used).

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ETIOLOGY:
 ACTH-dependent-bilateral adrenal hyperplasia secondary to:
a. Pituitary ACTH tumor (CUSHING’S DISEASE)
b. Ectopic ACTH-secreting tumor (e.g. small cell lung CA, bronchial carcinoid)
 ACTH-independent
a. Prolonged use of exogenous glucocorticoids (most common cause of Cushing Syndrome)
b. Primary adrenocortical hyperfunction: adrenal adenoma and carcinoma
c. Bilateral adrenal nodular hyperplasia

CLINICAL FEATURES:
Truncal (centripetal) obesity, thinning of extremities.
Supraclavicular fat pads, posterior cervical fat (“buffalo hump”), “moon facies”.
Hirsutism, Hypertension, Osteoporosis, Leukocytosis
Oligomenorrhea in women, Impotence in men.
Proximal muscle weakness.
Skin manifestations: thin skin, purple striae, easy bruising, poor wound healing,
mucocutaneous candidiasis, acne.
Psychiatric disturbances (depression, confusion, frank psychosis)
Impaired glucose intolerance common, but frank diabetes not very common.

NOTE: In ectopic ACTH, generally do not look Cushingoid, but characterized by severe hypokalemic
metabolic alkalosis and a rapid catabolic course with hyperpigmentation and muscle wasting.

TREATMENT

-options for primary and secondary Cushing’s are similar: surgery, radiation, &/or medications to suppress
adrenal cortisol production or actions.

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Testing Protocol:

Best screening test: urinary free cortisol in 24-hour urine sample (3 different samples)

1. first measure diurnal variation of cortisol in plasma:

Serum Cortisol determination: blood collected at 8 AM, measured by RIA or fluorescence polarization
immunoassay (FPIA) after displacing the cortisol from CBG (by lowering the pH, heat treatment or
displacing the cortisol by chemical means)

2. 24 hour urinary cortisol (reflects the serum free cortisol) - by RIA - elevated urine free cortisol is the
hallmark for the diagnosis of Cushing’s syndrome

3. 17-OH-corticosteroid - measured in 24 hour urine by Porter-Silber reaction:

17, 21-dihyroxy-20-ketone + 2,4-dinitrophenylhydrazine -----------> yellow pigment at 410 nm
o initial step include enzymatic hydrolysis (not acid) and extraction into organic solvent followed by
washing with NaOH
o lnterferences: acetone, fructose, vitamin C, phenothiazines, sulfa compounds, spironolactone and
propoxyphene. Caffeine consumption should be restricted or eliminated for several days prior to
specimen collection to minimize interference from this material

4. Dexamethasone Suppression Test

Dexamethasone - a synthetic steroid which act in the same manner as cortisol to induce feedback
inhibition at the level of pituitary. Use of high-dose dexamethasone (8 mg/day) is usually necessary for
suppression.

*also used as means of assessing cortisol production in depressed patients and as potential diagnostic
tool for identifying the possible cause of depression.

The standard protocol involves administration of 1.0 mg dexamethasone orally at 11 pm. Samples for
plasma cortisol measurement are collected the following day at 4 pm and 11 pm. There are no special
patient preparations, although individuals who are pregnant or have a major physical illness often have
altered cortisol production due to their physical condition should not have the DST administered.

Interpretation:
o either the 4 pm or the 11 pm sample shows a total cortisol level greater than 5.0 ug/dL - test is
presumed positive for the presence of endogenous depression
o A rise in the 4 pm value above 5 ug/dL cutoff is (termed early escape, implying a strong degree of
resistance to suppression of cortisol synthesis by dexamethasone

INHIBITION/SUPPRESSION OF CORTISOL SYNTHESIS

Clinical Situation Dexamethasone Metyrapone

Pituitary tumor or hyperplasia 50% suppression increased 17-OHCS
Ectopic ACTH production (e.g. oat cell CA) No suppression variable response
Adrenal tumor No suppression No response

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5. Metyrapone Inhibition Test:

Metyrapone - inhibits conversion of 11-deoxycortisol to cortisol therefore, less cortisol is formed,

increasing ACTH; decreased cortisol in plasma and urine; increased 11-deoxycortisol in plasma and
increase 17-OHCS excretion.
- useful in distinguishing pituitary malfunction from cancer in other tissue as reason for elevated cortisol
synthesis

HYPERALDOSTERONISM

Hyperaldosteronism
-State of hypersecretion of the mineralocorticoid aldosterone
-Increased aldosterone results in sodium retention, hydrogen and potassium loss, and high renin leading
to hypertension, alkalosis, polyuria, decreased urine specific gravity.
 Symptoms caused by low serum K:
-easy fatigalbility
-muscle weakness (paralysis)
-polyuria (loss of renal conc ability & renal cysts)
-palpitations (increased ventricular ectopy)
-autonomic dysregulation (hypotension w/o reflex tachycardia)
-impaired insulin secretion (decreased glucose tolerance)
-Aldosterone suppression

 Causes of hypertension and unprovoked hypokalemia include:

o Primary aldosteronism (low renin)-autonomous oversecretion of Aldo
o Secondary aldosteronism (elevated renin)-RAS-activated Aldo secretion
o Pseudoaldosteronism (variable renin and Aldo levels) – renal tubular diseases
causing urinary potassium loss by by Aldo-independent mechanisms. In most
cases, Aldo is low; however, two syndromes are associated with high Aldo
levels: Bartter’s syndrome (bumetanide-sensitive Cl- channel mutation) and
Gitelman’s syndrome (thiazide-sensitive transporter mutation)
-Plasma Renin Activity (PRA) assessed relative to plasma aldosterone (PA) helps distinguish primary
from other forms of aldosteronism.

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Types of aldosteronism according to PA:PRA ratio (Bishop, p 417,FIGURE 18-7)

1. Primary aldosteronism (adosterone secreting adrenal adenoma/bilateral hyperplasia of adrenal

cortex) - diagnose by failure to increase potassium with supplements, increased aldosterone, decreased
renin

Primary
a. Conn’s Syndrome – treated by radiographic procedures and surgical therapy
b. Bilateral hyperplasia of adrenal cortex – therapy with angiotensin-converting enzyme
inhibitors that block aldosterone receptors in kidney

Classic picture: hypokalemia, hypernatremia, metabolic alkalosis, high 24-hr urinary or plasma
aldosterone, salt loading: unsuppressed aldosterone after 3 days of salt loading.

2. Secondary aldosteronism in non-adrenal conditions such as low sodium intake, potassium loading,
due to increased renal blood flow leading to renin release leading to high angiotensin (vasoconstrictor)
which stimulates adrenal gland to increase aldosterone, excess production of renin

Test: increased aldosterone in plasma or 24-hour urine

- aldosterone is measured by RIA, diurnal variation in plasma and increased with upright position

 Renin can’t be measured directly, so estimate by angiotensin 1 generation - draw in cold tube and freeze
immediately because renin is very unstable
>> Increased with: sodium depletion, decreased potassium, if patient is upright or taking diuretics
>> Decreased with: dopa, Conn’s, renal hypertension.

CONGENITAL ADRENAL HYPERPLASIA (CAH)

Pathophysiology

 Autosomal recessive pattern of transmission, leading to enzyme defects, which can range from
partial to total
 21-hydroxylase deficiency is the most common form
 results in decreased cortisol and aldosterone with shunting toward adrenal androgen pathway.
 deficiency of cortisol leads to elevated ACTH, which increases levels of unaffected steroids and
causes bilateral adrenal hyperplasia.

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Clinical Features

 depends on the degree and the specific deficiency

 infants may present as failure to thrive, salt-wasting (adrenal crisis due to lack of aldosterone),
clitoral hypertrophy, fused labia or sustained hypertension
 adult onset (11-hydroxylase variant) more insidious, may present as hirsutism
 female
o ambiguous genitalia to virilization
o amenorrhea
 precocious puberty, with early adrenarche
 accelerated linear bone growth in early years, but premature epiphyseal closure due to high
testosterone, resulting in short stature
 possible Addisonian picture (adrenal insufficiency) if adrenal output of cortisol severely
compromised

HIRSUTISM AND VIRILIZATION

 both terms refer to states of androgen excess

 hirsutism
o male pattern of hair growth in women: back, chest, upper abdomen
 virilization
o hirsutism, frontal balding
o clitoral enlargement
o deepening of voice
o acne
o increase in musculatute
 defeminization
o amenorrhea
o decreased breast size

HYPOALDOSTERONISM

Hypoaldosteronism – mostly due to destruction of adrenal glands

1. Addison’s disease
2. patient with congenital deficiency of 21-hydroxylase

Isolated Hypoaldosteronism
-seen with adrenal gland destruction, chronic heparin therapy following unilateral adrenalectomy (transient),
and with G-layer enzyme deficiencies.
-occurs in patients with mild renal insufficiency (diabetics with mild metabolic acidosis, high serum K +, low
urinary K+ excretion (urine K < urine Na), and low reninemia.
-treatment (with diet): bicarbonate; furosemide (K-wasting diuretic); or Florinef (synthetic mineralocorticoid
[enhances salt retention, K and acid secretion)

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ADRENAL MEDULLA
-secretes catecholamines
-primarily synthesized freom TYROSINE and stored in vesicles of chromaffin cell in adrenal medulla until
released in circulation
-other site of formation: symphathetic neuron (part of CNS)
-catecholamine products serve as first responders to stress by acting within seconds (cortisol takes 20
minutes) to promote fight-or-flight response, which increases cardiac output and blood pressure, diverts
blood toward muscle and brain, and mobilizes fuel from storage.
-predominant adrenal catecholamine = Epinephrine (adrenaline)
-predominant peripheral catecholamine = Norepinephrine (noradrenaline)

DEVELOPMENT

Sympathogonia  sympathetic cells (migrate out of the CNS to the aorta)  differentiate into
sympathoblasts (sympathetic ganglion cells) or pheochromoblasts (medulla chromaffin cells)

 tumors from sympathoblasts (malignant neuroblastomas & benign ganglioneuromas secrete

homovanillic acid (HVA) [rarely seen after adolescence])
 tumors of chromaffin cells (pheochromocytomas maintain the capacity to synthesize and store
catecholamines [norepinephrine NE and epinephrine EPI throughout life)

BIOSYNTHESIS AND STORAGE OF CATECHOLAMINES

-all reactions take place in the cytoplasm, except for NE production (outer mitochondrial membrane within
lipid vesicles)
-sequential conversion of phenylalanine  tyrosine  DOPA  Dopamine
-(in sympathetic neurons) dopamine in sequestered into vesicles, converted into NE, and stored until nerve
stimulation causes its release
-(in medulla chromaffin cells) NE can passively diffuse into the cytosol and converted to EPI by a cortisol-
dependent enzyme called PNMT (phenylethanolamine N-methyltransferase) [any form of stress that
increases cortisol levels stimulates EPI production]
-free cytosolic EPI (like dopamine) is actively transported into secretory vesicles by vesicle monoamine
transporters (VMAT) in pheochromocytes
-serum ration of NE:EPI is normally 9:1

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-in adrenal insufficiency (low cortisol), ratio increases to 45:1 (females) & 24:1 in males

CATECHOLAMINE DEGRADATION
-all catecholamines are rapidly eliminated from target cells and the circulation by 3 mechanisms:
1. reuptake into secretory cells
2. uptake in non-neuronal cells (mostly liver)
3. degradation
-Degradation relies on 2 enzymes to produce metabolites (METANEPHRINES AND VMA) from free
catecholamines:
 catechol methyltransferase (COMT) in non-neuronal cells
 monoamine oxidase (MAO) within neurons
-metabolites and free catecholamines are eliminated by infiltration into the urine and excreted as free NE
(5%), conjugated NE (8%), metanephrines (20%), and VMA (30%)
-Urine EPI (50%) is converted from NE by RENAL PMNT, not adrenal, before excretion

Metabolic Effects of Catecholamines

1. increase breakdown of triglyceride with increase in free fatty acid
2. decrease in protein synthesis
3. increase glucose level in blood
4. enhanced liver glycogen breakdown
5. enhanced synthesis of glucose from amino acid (gluconeogenesis)
6. inhibits release of insulin
*hypoglycemia
– stimulate catecholamine release, glucagons, cortisol, growth hormone increase to restore euglycemic
state

HYPERFUNCTION OF THE ADRENAL MEDULLA

CAUSES OF SYMPATHETIC HYPERACTIVITY

1. Autonomic dysfunction
2. Panic attack (emotions)
3. Stress responses: hypoglycemia, injury, infarction, infection, psychosis, and seizures
4. Drugs: decongestants, appetite suppressors, stimulants, bronchodilators, MAO inhibitors, thyroid
hormone, cortisol, nicotine withdrawal, or short-acting sympathetic antagonists (clonidine or
propanolol)
5. Foods containing tyramine: imported beer, red wine, soy sauce, overripe/fermented foods, smoked
or aged meats
6. Pheochromocytoma (catecholamine producing tumor)

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Pheochromocytoma

 Catecholamine producing tumors arising from chromaffin tissue

 Most commonly a single tumor of adrenal medulla and the tumor is not innervated but via unknown
mechanism, able to synthesize and relaease catecholamines.
 Rare cause of HTN (<0.1% of all HTN)
 Curable if recognized and properly treated, but fatal if not.

CLINICAL FEATURES:
-Symptoms are often paroxysmal
-Headache - the most common symptom of an attack
-Others: Sweating, palpitations, flushing, chest or abdominal pain, apprehension or anxiety.
-Severe hypertension during episodes; tachycardia
-Sustained HTN is more common and present between attacks in 60% of patients.

-increase catecholamine synthesis, limited degradation capacity, and limited storage for excess NE and
metabolites likely causes spillover into the blood, increasing circulating free NE and/or EPI along with other
active peptides that causes symptoms

DIAGNOSIS
-best test for diagnosis is still uncertain
-most sensitive screening profile: test both total plasma catecholamines (NE and EPI) and urine
metanephrines
-plasma catecholamine >2000 pg/mL in a rested, supine patient with an indwelling cannula is nearly
diagnostic of pheochormocytoma
-plasma metanephrine is measured by HPLC or RIA (most specific & sensitive diagnostic test) [normal,>1.2
mg/day]
-urine VMA by HPLC or FLUOROMETRY has the highest false-negative rate
-Chromogranin A is co-stored and secreted in quantum with catecholamines (80% of pheo patients have
plasma chromogranin levels)
-combined, resting plasma catecholamines >200 pg/mL and chromogranin A >20 pg/mL have a positive
predictive value of 97% when GRF is normal
-for localization of Pheo, either CT (without dye) or MRI of the abdomen and adrenal glands is performed

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TREATMENT
-Surgery (high-risk procedure)

OUTCOME AND PROGNOSIS

-in 114 patients, 14% recurred (48% of those were malignant)

-long term monitoring is indicated in all patients, even those who are apparently cured

INCIDENTALOMA

NEUROBLASTOMA
-similar to pheo (of neural crest origin)
-2nd most common solid malignant tumor in childhood usually occurring before the age of 3.
-Symptoms relate primarily to tumor mass rather than to hypertension, which is often mild or absent
-widely spread throughout the body wherever sympathetic nervous tissue is found
-tumor without enzyme responsible for conversion of norepinephrine to epinephrine

Test:
-About 90% of patients have elevated urinary homovanillic acid (HVA) levels at the time of
diagnosis, whereas almost 75% of patients have increased urinary VMA levels. Both tests should be
ordered when screening for the disease.

URINE AND PLASMA CATECHOLAMINE MEASUREMENTS

-catecholamines are hydrophilic

-circulate in low levels (50% bound to albumin)
-short half-lives (seconds to 2 minutes)
-wide, rapidly fluctuating plasma levels
-urine catecholamines (NE and EPI) are assayed using LIQUID CHROMATOGRAPHY or
FLUOROMETRICS (24-hr urine)
-interferences: most antihypertensive drugs, tetracyclines, ephedrine, a-methyldopa
-clonidine & thiazides preferred agents to control hypertension during evaluation

Collection:

Plasma
-patient lying down for 15-30 mins prior sample collection
-needle/catheter should be inserted 15 mins or prior collection and kept open with heparin lock or IV
drip
-after collection, it is cooled and centri to separate plasma and cells (prolonged contact with platelets and
cell will decrease catecholamine).

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-repeated thawing and refreezing will cause significant loss of catecholamines

-reduced gluthatione stabilizes catecholamines against oxidation

Urine
-24 hour output of catecholamine – iced container with 10 ml of HCl
-refrigerate during collection
-after collection – pH is adjusted between pH 2-5 with 6N HCl
-no repeated thawing and freezing

Assay:
1. HPLC
2. Fluorometric Assay
3. Radiometric Assay
4. Colorimetric Assay

A. Urine Metanephrine

 (24-hr urine) 95% sensitive for pheo; BEST single test

PISANO Method – measure total metanephrine (metanephrine/norepinephrine)

1. hydrolysis with HCl breaks down all conjugates
2. solution is poured to ion-exchange column which loosely binds metanephrines but
allows other components to pass through and be removed from reaction mixture
3. elution with ammonium hydroxide washes metanephrine off the column
4. conversion to vanillin through periodate oxidation
*false +/increased – chlorpromazine, imipramine, phenothiazines, methyldopa, tetracycline
derivatives

B. Urine VMA

1. hydrolysis with HCl to destroy conjugates

2. extraction with ethyl acetate separates VMA into inorganic layer
3. purification: reextraction of VMA into aqueous potassium carbonate solution
4. treatment with sodium metaperiodate oxidizes VMA to vanillin
*false decrease – pH above 6, salicylates, clofibrate, L-dopa, clonidine, disulfiram, hydrazine
derivatives, inipramine, MAO inhibitors, morphine, phenothiazine, radiographic agents
*false increase – banana, coffee, tea, vanilla, chocolate, salicylates, disulfiram, nalidixic acid,
oxytetracycline, phthalein dyes, patients on insulin, isoproterenol, L-dopa, lithium, reserpine

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Physiological Factors

Release in circulation – greatest during waking hours, 3-5x higher than night, increase is due to response to
increase in physical activity
Epinephrine production – increased markedly during mental stress, pain, loud noises or stress
Increased catecholamine (norepinephrine) – upon standing

PHARMACOLOGIC TESTS:

 Clonidine Suppression Test

-If clonidine is administered, Px with primary hypertension (not associated with tumor) show
decrease in plasma catecholamine levels (or below) the reference range.
-px with pheochromocytoma show decline in values but still remain above reference range.

Plasma levels of normetanephrine less than 112 ng/liter (0.61 nmol/liter) and of metanephrine less than 61
ng/liter (0.31 nmol/liter) virtually exclude pheochromocytoma so that no immediate further testing for the
tumor should be necessary. With plasma concentrations of normetanephrine above 400 ng/liter (2.19
nmol/liter) or metanephrine above 236 ng/liter (1.20 nmol/liter), the probability of pheochromocytoma is so
high that the immediate task is to locate the tumor

Pharmacologic Tests for Diagnosing Pheochromocytoma

Clonidine suppression test
Indications Patients with hypertension and clinical findings or family history that is
highly suggestive of pheochromocytoma and the catecholamines are
elevated but not to the extent that is diagnostic of pheochromocytoma
Interpretation Normal: decrease in NE to below normal or a > 50% decline from baseline.
A decrease in normetanephrines to below normal or a 40% decline from
baseline
Pheochromocytoma: failure of NE to drop below normal or decrease
by more than 50% from baseline. Failure of normetanephrines to drop
below normal or decrease by more than 40% from baseline
Glucagon stimulation test
Indications When clinical findings or family history are highly suggestive of
pheochromocytoma but blood pressure is normal and catecholamines are
only modestly elevated
Interpretation Pheochromocytoma: A threefold or greater increase in plasma NE or a rise
in the level to > 2000 pg/mL

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 CLINICAL CHEMISTRY III
THE ADRENAL GLAND

HYPOFUNCTION OF THE ADRENAL MEDULLA

-occurs only in individuals receiving adrenocortical steroid replacement therapy following adrenalectomy.
-disorders associated with autonomic insufficiency:
Familial dysautonomia
Shy-Drager syndrome
Parkinson’s disease
Tabes dorsalis
Syringomyelia
Cerebrovascular disease
Peripheral neuropathy due to diabetes
Idiopathic orthostatic hypotension
Sympathectomy
Drugs: antihypertensives, antidepressants

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