ADRENAL MEDULLARY ENDOCRINOLOGY

I. Adrenal Medulla – middle region of adrenal gland

− made of chromaffin cells (Pheochromocytes) that originate from the Neural crest (same part as the
sympathetic NS)
a. Neural Crest cells – embryonic cells; separate fr neuroectoderm to become peripheral & autonomic
nerves of CNS
b. Chromaffin cells – contain vesicles/storage granules containing catecholamines: a) Epinephrine &
b) Norepinephrine
◊ vesicles containing norepinephrine(10%) are darker than those containing epinephrine(90%)

− Epinephrine – 90% of all catecholamines; bec of presence of enzyme PNMT (Phenylethanolamine N-

methyltransferase) that converts norepinephrine → epinephrine
• PNMT – found only in tissues that produce epinephrine
◊ induced by high conc of glucocorticoids in the medulla

− Adrenal Medulla – highly innervated tiss w/ many neurons that are in direct contact w/ the chromaffin cells
• Neurons  stimulate catecholamine release

II. Adrenergic Receptors – mediate the action of catecholamine hormones

− 2 α -receptors & 2 β-receptors
− found in most tissues in the body; may have both or only 1 type
a. α 1-adrenergic receptor uses Calcium & Phosphatidylinositol for its 2nd messengers
• Both epinephrine(adrenaline) & norepinephrine (noradrenaline) are strong stimulants of α 1-

b. α 2-β1- & β2-adrenergic receptors – use cAMP as 2nd messenger

Myocardial cells – have many β1 adrenergic receptors; stimulates ↑ rate & force of contraction of heart

*Blocking receptors (w/ β-blockers) - ↓strength of contraction

B-blockers-used to treat patients with heart problems

III. Synthesis
− Catecholamine hormones: modified amines that have a catechol nucleus (catechol/benzene ring) w/ 2 hydroxyl
(OH -) side groups & a side chain amine
− synthesized from amino acid L-tyrosine  derived fr om food/synthesis from phenylalanine in the liver

a. Conversion of Tyrosine to L-DOPA

• Hydroxylation
• by enzyme tyrosine hydroxylase
• L-DOPA (L-dihydroxyphenylalanine)

*in the original formula, Tyrosine has 1 hydroxyl group. In L-dopa: 2.

b. Conversion of DOPA to Dopamine

• Decarboxylation
• By the enzyme L-aromatic amino acid decarboxylase (aka DOPA decarboxylase)
◊ nonspecific decarboxylase found in most tissues
◊ e.g. ↑conc in liver, kidney, brain & vas deferens

c. Conversion of Dopamine to Norepinephrine

• Dopamine enters storage vesicle
• Hydroxylation
• enzyme DBH (Dopamine-B-Hydroxylase)
◊ adds a OH- to dopamine to form norepinephrine
• DBH is found w/in the vesicle membrane
• Norepinephrine is the stored in vesicle

d. Conversion of Norepinephrine to Epinephrine

• Norepinephrine diffuses into the cytoplasm from vesicle & converted to epinephrine
 • Methylation (addition of methyl group to Norepinephrine)
• enzyme PNMT adds a methyl grp to norepinephrine to form epinephrine

− Once synthesized, the hormones can either be secreted or stored w/in the Chromaffin cells in
storage granules
− Active forms: dopamine, norepinephrine and epinephrine; can be stored in the chromaffin cells
Tyrosine
↓ Tyrosine hydroxylase
Dopamine
↓ Dopa decarboxylase
Dopamine
↓ Dopamine β-hydroxylase
Norepinephrine
↓ PNMT
Epinephrine
IV. Metabolism (breakdown/inactivation)
− Catecholamines secreted into plasma = short half-life (1-2mins)
− rapidly removed from circulation by liver & kidneys/taken up by sympathetic neurons & activate the target
organs

2 Enzyme systems responsible for metabolism

a. Monoamine oxidase (MAO)
◊ present in many tissues, throughout the body
◊ high concentration found: platelets, brain & liver
◊ deaminates the catecholamines will remove the amine from the catecholamine

b. Catechol-O-methyltransferase (COMT)
• highest concentration: kidney & liver; also present in red blood cells
• methylates a OH group on the aromatic ring structure
• methylation produces Metanephrine & Normetanephrine

The metabolism will require the 2 enzymes.

Enzymatic Metabolism of:

* Epinephrine & Norepinephrine require both enzymes
• Vanillylmandelic acid (VMA) = final metabolite formed from both

* Dopamine – can be catalyzed by either COMT/MAO

• Homovanillic acid (HVA or HMV) = final metabolite formed
Norepinephrine

Epinephrine Dihydroxymandelic acid COMT

MAO MAO
COMT COMT

Metanephrine Normetanephrine
MA
3-Methoxy-4-hydroxymandelic
O acid
MAO Oxidation

Vanillylmandelic Acid
(VMA)

Dopamine
MAO CCMT

Dihydroxyphenyl- 3-Methoxytyramine
acetic acid

CCMT MAO
Homovanillic Acid
V. Specific Analytes:
1. Dopamine – a catecholamine; highest concentration in CNS
− Functions primarily as neurotransmitter
− in hypothalamushelps to control synthesis & secretion of Prolactin from Anterior Pituitary
− large amounts is synthetic in CNS, an amt in adrenal medulla
− synthesized from amino acid L-tyrosine (like all catecholamines)
− enzymatic metabolism is either by COMT/MAO
− final metabolite: HVA/HMV

2. Norepinephrine
− highest conc: CNS, lesser  in SNS
− 1 func: neurotransmitter in both CNS & SNS
− found in hypothalamus
− produced in adrenal medulla; 10% of total catecholamines
− acts primarily through α -adrenergic receptors
− can cause varied responses: vasoconstriction of small BVs in skin or relaxation of smooth muscle in GIT
− metabolism begins w/ either COMT/MAO
− COMT action produces the metabolite: normetanephrine
− Final metabolite: VMA (Vanillymandelic Acid)

3. Epinephrine
− major catecholamine produced by chromaffin cells of AM
− axctions are mediated through both the α & β-adrenergic receptors
− “flight or fight” hormone
• release in response to physiologic/psychological threats (stress, anxiety)
− ↑ heart rate, BP, respiration & metabolic rate
• Release glucose
• Release of fatty acids from stored fat cells
• Target organs: liver, fat cells, heart, blood vessels
− stimulate glycogenolysis in liver & skeletal muscle
− metabolism begins w/ either COMT/MAO
− COMT action produces Metanephrine
− Final end product is VMA

VI. Lab Analysis:

SPECIMENS:
1. Plasma – for any catecholamine analysis
a. best to draw blood from an indwelling catheter
• venipuncture may cause ↑ catecholamine levels because of stress
• inserted 15-30mins before
b. Px should be lying down for @ least 30 mins
• Body positioning can also affect the levels of catecholamines
• levels will be 2-3x ↑ if sitting upright
c. fasting for at least 4 hrs
• should not have drunk coffee, tea & not used tobacco w/in 4 hrs
• Caffeine↑ heart rate; triggers release of catecholamines
 Inhibit action of adenosine
 Adenosine—inhibits release of catecholamines

d. Aldomet (a drug for hypertension) should be stopped for at least 1 week before testing
e. Blood drawing tubes should be prechilled & contain Na thiosulfite (antioxidant) & EDTA (anticoagulant)
• Antioxidant: prevent oxidation & degradation of catecholamines
f. Blood must be placed on ice ASAP after drawing
• separated using refrigerated centrifuge ASAP MAO is present of platelets & COMT is present in RBC
• frozen at -70C until analysis is performed
• Divide into aliquots Do not thaw and refreeze.
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2. 24-hour Urine
a. total urine excreted in 24 hrs into container w/ 10mL conc HCl
• conc HCl: prevent ? Of catecholamines in urine
b. refrigerate during collection time
c. total volume is recorded
d. Any aliquots for test must be adjusted to pH 3-4 using 6M HCl

LAB METHODS:
1. Radioenzymatic Method – analysis of catecholamine in plasma
a. uses COMT to transfer a (tritiated) 3H-methyl group to the catecholamines
b. catecholamine are extracted from the sample & separated by TLC (thin layer chromatography)
c. Amount of radioactivity in each chromatographic spots is measured
d. the individual catecholamines can be quantitated
− Reference Range:
• Dopamine = 0-83 pg/dL
• Norepinephrine = 111-603 pg/mL
• Epinephrine = 0-62 pg/mL

2. Ethylenediamine (EDA) Fluorometric Method

− by Weil-Malherbe
− analysis of plasma epinephrine & norepinephrine

a. extracting the catecholamines from plasma 1st w/ an alumina column followed by an Amberlite CG-50
column
b. EDA is added to the collected eluate
c. 3 fluorescent products are obtained from norepinephrine & 1 from epinephrine
d. these products are measured in a Fluorometer at 2 diff wavelengths
 emission 510 & 580
 activation 420
 Difference between emission & activation/excitation wavelength reading: Stoke Shift?
e. concentrations of hormones are calculated

− Specimen: plasma
− RR:
• Norepinephrine = 360-800 pg/mL
• Epinephrine = 140-300 pg/mL

Substances that may interfere w/ the test:

a. Common drugs (Ampicillin)
b. Beverages (coffee & tea)increase level of catecholamines

2. HPLC (High Performance Liquid Chromatography)

− by Shoup & Kissinger
− analyzes plasma & 24-hr urine specimen
− more precise in measuring urinary catecholamine & metabolites in 24 hr urine
• highly specific, easy to use
a. urine is divided into aliquots; pH is adjusted to pH3-4
b. extraction of catecholamines by column chromatography
c. catecholamine in column eluate are separated on a reverse phase HPLC w/ an amperometric detector
d. individual catecholamines can be quantitated

− RR for Adults:
• Homovanillic acid = <15 mg/24 hrs
• Epinephrine = 0.5-20 μg/24 hrs
• Norepinephrine = 14-80 μg/24 hrs

*Homovanillic acid
o End product of metabolism of Dopamine
o stable in urine
o more specific than determination of dopamine in plasma
 Gas chromatography (GC) by Williams & Greer
o can also be used for analysis of urinary catecholamine & metabolites

2. Meas. of Total Metanephrines – by Colorimetric method

− cannot separate normetanephrine & metanephrine in urine
− COMT causes production of metanephrine

a.urine is hydrolyzed w/ conc HCl

b.metanephrines are removed using Amberlite CG-50 column
c.Periodate is added to oxidize metanephrine to Vanillin
d.Abs is measured in SPM at 360nm
− RR: 0.3-0.9 mg/24 hrs

 End product measured: Vanillin (after addition of Periodate)

3. Measurement of VMA in urine

a. VMA is removed from urine by means of a resin column
o (this column is specific for VMA & does not bind other phenolic acids present in urine that can
interfere w/ VMA measurement)
b. VMA is eluted from the column
c. Periodate is added to eluate to oxidize VMA to vanillin
d. Abs is measured at 360nm

− RR for Adults: 2-7 mg/24 hrs

− End product: Vanillin
− VMA is oxidized to Vanillin

Sources of Phenolic Acids that can interfere w/ VMA assay:

a. Coffee
b. Chocolate
• a&b: caffeine
c. Vanilla
d. Bananas
• ↑ tyrosine content: ↑ catecholamines
e. Citrus fruits
• ↑ tyrosine content

These will react with periodate producing colored compounds false high result of the test