ENDOCRINE GLAND:

ADRENAL GLAND
ADRENAL GLAND
• also known as “suprarenal gland”
• resembles as “admiral’s cocked hat”
• pyramidal structures located above
each kidney, each weighing
approximately 4 to 6 g.

• divided into two distinct parts:

• the medulla (inner layer)
• the cortex (outer layer)
• produces hormones which give an impact on blood pressure &
electrolyte balance.
• By weight, they have the highest perfusion of blood per gram of
tissue — a feature that ensures rapid dissemination of hormones
throughout the body in response to stress.
CORTEX
STEROIDOGENESIS
• All adrenal steroids are
derived by sequential
enzymatic conversion of a
cholesterol.
• Corticotropin-releasing
hormone (CRH) is secreted
from the hypothalamus in
response to circadian signals, serum cortisol, and stress, causing
release of stored ACTH, which stimulates transport of free
cholesterol into adrenal mitochondria, initiating steroid
production.
• Only free cholesterol can enter steroidogenic path- ways in
response to ACTH. The availability of free intracellular
cholesterol is metabolically regulated by LDL negatively and
ACTH positively through multiple mechanisms.
EXPLANATION
• Corticotropin-releasing hormone (CRH) secretes from
the hypothalamus in response to circadian signals,
serum cortisol and stress, and will eventually cause the
release of stored ACTH and then stimulate transport of
free cholesterol to adrenal mitochondria. The
convertion of cholesterol into pregnenolone is rate-
limiting step in this steroid biosynthesis. 6 carbons are
removed from cholesterol by mitochondrial membrane.
Within mitochondria, cholesterol will be converted into
pregnenolone by an enzyme in the inner membrane
which is called P-11 (?). Pregnenolone is not a hormone
itself but it is an immediate precursor for the synthesis
of all the steroid hormones.
3β-hydroxysteroid
dehydrogenase
EXPLANATION
• This is the steroidogenic pathway of the adrenal cortex, major
cortex hormone - aldosterone, cortisol,
dehydroepiandrosteronesulfate, they are uniquely synsthesized
from a common precursor cholesterol by cells located in one of
the three functionally distinct zonal layers of the adrenal
cortex.
• Under G-zone, there are mineralocorticoids. In this zone, there
will be synthesization of mineralocorticoids especially
aldosterone which is critical in sodium retention, potassium
and acid base homeostasis.
• While in F-zone, there are glucocorticoids. In this zone, there
will be synthesization of glucocorticoids such as ---cortisone
which is critical in blood glucose homeostasis and blood
pressure.
• Within R-zone, there are androgens. In this zone, this is where
our sex hormones will be categorized.
 CONGENITAL ADRENAL
HYPERPLASIA (CAH)
• an inherited family of enzyme disorders affecting
cortisol, aldosterone, and sex steroid production
• there are enzymes that are needed in cortex
steroidogenesis, they are either missing or mutated and
that is what we call CAH
• CAH is part of the newbron screening here in the
Philippines, together with G6PD, barbs (?), hemoglobin-
A (anything hemoglobin-related missing on the
newborn)
• Virilization is when a woman develops masculine traits
such as male pattern baldness and excessive facial and
body hair
• 3B-Hydroxysteroid dehydrogenase – it results to
elevated ratio of 17A-Hydroxypregnenolone to 17-
Hydroxyprogesterone and there will be increase ratio of
DHEA to androstenedione
• This is characterized by pseudohermatophroditism in
female infants or what we call virilization and
incomplete musculinization in male infants
• Laboratory findings: elevated serum, 17-OHP,
pregnenolone, DHEA and increase urinary 17-OHP
3-β-HYDROXYSTEROID
DEHYDROGENASE DEFICIENCY
• leads to a block in the conversion of pregnenolone, 17-OH
pregnenolone, and dihydroepiandrosterone to
progesterone, 17-OHP, androstenedione
• associated with increased blood levels of pregnenolone,
17-OH pregnenolone, and dehydroepiandrosterone.
• characterized by manifestations of glucocorticoid
deficiency, with or without the accompaniment of salt
wasting
• affected males have incomplete masculinization, and
females may be normal or may have ambiguous genitalia.
 17-HYDROXYLASE DEFICIENCY
• 17-Hydroxylase (CYP17) is expressed in the adrenal
glands and the gonads and encodes two enzymes —
17α-hydroxylase and 17,20-lyase.
• 17α-hydroxylase catalyzes the conversion of
pregnenolone and progesterone to their respective
17-OH derivatives.
• 17,20-lyase converts 17-OH pregnenolone to
dihydroepiandosterone, and 17-OH progesterone to
androstenedione.
• CYP17 (defective gene) deficiency blocks the conversion
of pregnenolone and progesterone to the 17-hydroxy
derivatives, causing shunting from testosterone and
cortisol synthesis to aldosterone.
17-HYDROXYLASE DEFICIENCY
• This is characterized by hypertension and
hypokalemic alkalosis in association with incomplete
masculinization (in the male) and decreased
testosterone and cortisol levels.
Laboratory Tests:
• Blood DOC (deoxycorticosterone), pregnenolone,
progesterone determination: (+) high DOC,
pregnenolone, and progesterone levels
• Determination of urinary metabolites:
(+) decreased urinary 17-ketosteroids and 17-
hydroxycorticosteroids

NOTE: (+) meaning positive for the deficiency

 11β-HYDROXYLASE
DEFICIENCY
• The second most common enzyme deficiency of the adrenal cortex,
accounting for about 7% of all cases of CAH
• This is associated with virilization and hypertension. This involves the
defective production of cortisol with accumulation of
mineralocorticoid precursor resulting in hypernatremia, hypokalemia
and hypertension (?)
• There is hypertension because there is excessive mineralocorticoid
effect and increase androgen level which leads to virilization
• A defect in this enzyme blocks the final conversion of 11-deoxycortisol
to cortisol and DOC to corticosterone.
• As with 21-OH deficiency, a compensatory increase in ACTH
secretion leads to adrenal hyperplasia and a mass action shunting of
precursor steroids toward testosterone synthesis, resulting in signs of
virilization.
• Diagnosis in the neonate is established by the presence of a high 11-
deoxycortisol and DOC.
21-HYDROXYLASE DEFICIENCY
• most common cause of CAH, accounting for about
95% of all cases
• 21-OH catalyzes the conversion of 17-
hydroxyprogesterone (17-OHP) to 11-deoxycortisol,
and progesterone to DOC.
• This affects how adrenal gland work and a missing
enzyme leads to overproduction of specific
hormone made by adrenal gland.
• Characterized by elevated levels of the steroid
precursors 17-OHP and pregnanetriol in the urine
and 17-OHP in the serum
• Laboratory test: 17-OHP test in amniotic fluid or
by genotyping cells obtained by chorionic villous
sampling
 Congenital Lipoid Adrenal
Hyperplasia
• The most severe form of CAH, in which the synthesis of all
gonadal and adrenal cortical steroids is markedly impaired
causing a marked accumulation of cholesterol and other lipids.
• There is a defect in the preparation to steroidogenesis.
• Characterized by severe adrenal insufficiency with hypotension,
salt wasting, and feminization of external genitalia in males.
Occasionally, females may not present until the onset of puberty.
• Diagnosis is made by the presence of extremely low cortisol and
aldosterone concentrations and elevated ACTH and plasma renin
activity.
• Adrenal gland is enlarged with cholesterol ester deposits at work.
Affect infants have low but measurable levels of steroid hormones.
ADRENAL GLAND
ADRENAL CORTEX
• makes up about 90% of the adrenal gland
• if we will have gross sectioning, the cortex will appear
yellow – this is the layer of the adrenal gland
responsible for the production and secreiton of
mineralocorticoid, glucocorticoids and weak
androgens
• the outer layer of the adrenal gland which is derived
from mesenchymal cells located near the urogenital
ridge that differentiate into three structurally and
functionally distinct zones:
• G-zone (Zona Glomerulosa)
• F-zone (Zona Fasciculata)
• R-zone (Zona Reticularis)
ADRENAL GLAND
ADRENAL MEDULLA
• the inner portion which arises from neural crest cells (ectoderm)
that invade the cortex during the second month of fetal development

• the inner layer of adrenal gland which is responsible for the

secretion of catecholamines. In gross sectioning, the medulla will be
seen as dark or mahogany in color or reddish-brown
ADRENAL CORTEX
G-ZONE (ZONA GLOMERULOSA)
• the outermost zone of the adrenal
cortex (10% of the adrenal
cortex) which has cells that
synthesize mineralocorticoids
(aldosterone) critical for sodium,
potassium, and acid–base
homeostasis.
• The glomerulosa cells have low
cytoplasmic-to-nuclear ratios and
small nuclei with dense chromatin
with intermediate lipid inclusions.
ALDOSTERONE
• the chief mineralocorticoid.
• under the control of the RAAS.
• expression of the enzyme CYP11B2 (aldosterone
synthetase) within the glomerulosa results to the
synthesis of aldosterone.
• promotes the reabsorption of sodium and water by the
kidney to help maintain blood pressure and tonicity
• stimulates H+ & K + excretion
• this is mainly regulated by the RAAS which functions to
regulate blood pressure and sodium balance
• NOTE: *Aldosterone will respond to acute changes in
ACTH but still it is mainly under the control of the RAAS*
• In RAAS, renin is a proteolytic enzyme made up by cells in the
juxtaglomerular apparatus and is secreted directly into the
circulation, that’s why we have the convertion of angiotensinogen to
angiotensin-1. Angiotensin-converting enzyme or what we call ACE, is
the one who will convert angiotensin-1 to angiotensin-2 which acts as
a powerful vasoconstrictor and stimulates aldosterone release. Sodium
retention leads to water retention. Where sodium goes, water follows.
The stimuli will be having a decreased blood sodium, decreased bleed
and decreased blood pressure.
• The action of RAAS dilates the afferent arteriole and constricts the
efferent arteriole and it also stimulates the sodium reabsorption in the
proximal convoluted tubule.
• This triggers the adrenal cortex to release sodium-retaining hormone
aldosterone to cause sodium reabsorption and potassium excretion in
the DCT and collecting duct and this also triggers ADH release by the
hypothalamus to stimulate water reabsorption in the collecting duct
and DCT
• The increase sodium retention results to increase potassium excretion
leading to decreased potassium levels in blood
 G-ZONE PATHOLOGY
•Main steroid: Aldosterone
•Main regulator: RAAS
•Major function: Blood pressure and K+
synthesis
Syndrome Causes Clinical Findings

Hypoaldosteronism adrenal destruction, Hyponatremia,

(Addison’s disease) chronic heparin hyperkalemia,
= low blood aldosterone therapy, G-layer metabolic acidosis
enzyme deficiencies
Hyperaldosteronism primary, second, Hypetension (HTN),
(Conn’s disease) pseudoaldosteronism hypernatremia,
= high blood hypokalemia,
aldosterone metabolic alkalosis
• Hyperaldosteronism (Conn’s Disease) – increase sodium and
chloride levels, low potassium and hydrogen ions which
leads to metabolic alkalosis
• This is a primary hyperaldosteronism caused by the
presence of aldosterone producing tumor or what we call the
Conn’s tumor

• Hypoaldosteronism (Addison’s Disease) – decrease sodium

and chloride levels, high potassium and hydrogen ions which
leads to metabolic acidosis
• This is a primary adrenal insufficiency disorder – hormones
produced by adrenal gland when having this condition are
expected to be low (example: aldosterone and cortisol – low)
• Treatment: dietary changes (florinef – enhances salt
retention and potassium and hydrogen secretion)
 HYPERALDOSTERONISM
PRIMARY ALDOSTERONISM
• low renin, high aldosterone – there is an autonomous
oversecretion of aldosterone
• Causes: aldosterone adenoma (Conn’s disease), adrenal
hyperplasia, glucocorticoid-remediable aldosteronism

SECONDARY ALDOSTERONISM
• high renin, high aldosterone – there is RAAS-activated
aldosterone secretion wherein the elevated renin results to
increased aldosterone levels
• Causes: Diuretics, Renin-producing tumor, Renal artery
stenosis, Hepatorenal syndrome, Congestive Heart
Failure
PSEUDOALDOSTERONISM
• variable renin and aldosterone levels; mimics the effects (laboratory
findings) of hyperaldosteronism
• Causes: Renal tubular disease (Liddle’s, Gitelman, and Bartter’s
syndromes), Congenital Adrenal Hyperplasia
• Liddle’s Syndrome – is one of the congenital disorder that is
characterized by increased epithelial sodium channel, activity in the
collecting duct and in the absence of increased aldosterone. This
resembles primary aldosteronism clinically, but aldosterone and
renin levels are low with absence of hypertension.
• Bartter’s Syndrome – this is what we call bumetanide sensitive
chloride channel mutation. This is a rare potassium-using autosomal
recessive disorder that causes defective sodium chloride
reabsorption and accompanied by elevated concentration of plasma
aldosterone and renin.
• Gitelmann’s Syndrome – is the thiozide sensitive transporter
mutation. It is associated with the defect in soduim chloride
reabsorption occuring in the DCT and also accompanied by elevated
aldosterone.
 DIAGNOSIS ALGORITHM FOR
ALDOSTERONISM
• Urinary K+ Excretion
• cost-effective screening test for aldosteronism
• >30 mmol/day strongly suggests hyperaldosteronism
(spot urine K+ > Na+ is also suggestive)
• Upright PA (Plasma Aldosterone) And RA (Renin
Activity) Ratio
• measured in a fluid-deprived patient (overnight
dehydration increases PRA)
• definitive test in distinguishing primary from other
causes of aldosteronism, particularly
• A PA/PRA ratio greater than 25 suggests primary
aldosteronism
 DIAGNOSIS ALGORITHM FOR
ALDOSTERONISM
• Captopril Suppression
• a confirmatory test for aldosteronism
• Within 3 hours of taking 50 mg of captopril (1 mg/kg), PA
remains high in primary aldosteronism (PA:PRA ratio >25
before and after test) but is suppressed in patients with other
forms of hypertension
• RA (renin activity) reflects the state of RAAS activation and this
varies with volume status, upright or supine position, dietary
sodium intake and PA (plasma aldosterone) is normally
suppressed by captopril. Captopril is a drug used for
hypertension which inhibits the ACE. If the patient has
aldosteronism, the PA remains to be elevated.
• Urine measurements for aldosteronism are superior to plasma
measurements.
 DIAGNOSIS ALGORITHM FOR
ALDOSTERONISM
• 18-hydroxycorticosterone
• not accurate
• 100 ng/dL suggest an aldosterone-producing adenoma (APA) and
levels less than this suggest idiopathic hyperaldosteronism (IHA)
• Adrenal Imaging (CT Or MRI)
• used to visualize adrenal gland anatomy
• Occasionally, aldosterone-secreting adenomas are missed
because they are too small to visualize or are obscured within
hyperplastic glands. If imaging is negative, the scan can be
repeated in 6 to 12 months.
• Scintigraphy / Adrenal Vein Sampling
• used to differentiate between unilateral adenoma and bilateral
hyperplasia. It is superior to adrenal CT. In one study, 50% of
patients diagnosed with APA by venous sampling had
hyperplasia by CT.
ADRENAL CORTEX
F-ZONE (ZONA FASCICULATA)
• the middle zone that makes up 75% of the cortex.
• containing fasciculata cells which synthesize
glucocorticoids, such as cortisol and cortisone critical to
blood glucose homeostasis and blood pressure.
• Fasciculata cells are cords of clear cells, with a high
cytoplasmic-to-nuclear ratio and lipids laden with
“foamy” cytoplasm. These cells also generate androgen
precursors such as dehydroepiandrosterone (DHEA),
which is sulfated in the innermost zona reticularis (R-
zone).
CORTISOL
• The major glucocorticoid, regulating its own secretion through
negative feedback on the hypothalamic- pituitary-adrenal (HPA)
axis and inhibiting corticotropin-releasing hormone (CRH) from
the hypothalamus and ACTH release from the pituitary gland.
• Known as the stress hormone, as it is needed in times of stress to
maintain blood pressure and blood sugar, and to prevent shock.
• A glucocorticoid which is responsible to maintaining blood
glucose by inducing lipolysis and causing amino acid release
from muscles in glucose and storage as liver glycogen.
• Other effects: increasing protein catabolism, reducing
inflammation, and suppressing antibody formation.
• Cortisol synthesis: 8 - 15 mg/day to regulate glucose homeostasis
and hemodynamics (blood flow).
CORTISOL
• This is the major glucocorticoid hormone produced
by zona fasciculata. Both secretion and production
of cortisol is regulated by the hypothalamus
pituitary adrenal axis. The loss of regulation of
cortisol can lead to disorder of cortisol excess such as
Cushing’s syndrome or cortisol deficiency such as
Addison’s disease.
• It is the only adrenal hormone that inhibits the
secretion of ACTH when plasma levels of cortisol is
elevated
• It is synthesized or regulated by ACTH, it is mostly
bound to glycoprotein which is the transportin.
 F-ZONE PATHOLOGY
• Main steroid: Cortisol
• Main regulator: ACTH
• Major function: Blood pressure and glucose
homeostasis
Syndrome Clinical Findings
Hypocortisolism / Adrenal Hypotension, hypoglycemia,
Insufficiency weight loss, weakness
= low cortisol
Hypercortisolism (Cushing’s HTN, hyperglycemia,
Syndrome) central obesity, weakness
= high cortisol
HYPOCORTISOLISM
•characterized by low cortisol
•can be:
•Primary hypocortisolism (primary
adrenal insufficiency)
•Secondary hypocortisolism
(secondary adrenal insufficiency)
•Tertiary hypocortisolism (tertiary
adrenal insufficiency)
• Primary Adrenal Insufficiency – if the cortex is damaged and it
doesn‘t produce enough adrenocortical hormones, thus the
condition is called PAI. This is most commonly the result of the
body attacking itself or what we call the autoimmune disease . For
unknown reasons, immune system views the adrenal cortex as
foreign or something to attack. There are other causes of adrenal
gland failure, this may include: spread of cancer to adrenal gland,
bleeding of adrenal gland. In this case, the patient may have
Addisonian crisis without any previous symptoms.
• Secondary Adrenal Insufficiency – pituitary gland makes a
hormone called the adrenocorticotropic hormone (ACTH). ACTH
in return, this stimulates the adrenal cortex to produce hormones.
Benign pituitary tumors, inflammation and prior pituitary
surgery are the common causes of not producing enough pituitary
hormones. If the patient has low levels of ACTH, it can also lead
to too little or too low level of glucocorticoid and androgens that
are normally produced by adrenal gland even though adrenal
gland themselves aren’t damaged.
 PRIMARY
HYPOCORTISOLISM
• Primary adrenal insufficiency
• Due to impairment of the adrenal gland: decreased cortisol
production, destruction of the adrenal cortex
• Autoimmune adrenalitis (70% to 90% of all cases),
tuberculosis (most common cause worldwide), adrenal
atrophy, granulomatous disorders, hemorrhage, HIV
infection/AIDS, other infections, CAH
Disorder: Addison’s disease
Laboratory test: ACTH Stimulation Test
• Low baseline cortisol levels which is 8 AM supine position and
there is also an elevated levels of ACTH greater than 200 are
suggestive of PAI
• Steroids from the G-zone and R-zone are replaced meaning
exogenous cortisol and aldosterone (example: florinef)
 ACTH STIMULATION TEST
• most convenient procedure for studying patients suspected of
having hypocortisolism.
• done by administrating 250 ug of Cosyntropin (a
commercially available ACTH analog) intravenously or
intramuscularly
1. Collect blood for baseline serum cortisol
2. Administer the ACTH analog
3. Collect blood for serum cortisol at 30 and 60 mins post-
Cosyntropin.

(+) for Hypocortisolism: Failure of cortisol to increase

(-) for Hypocortisolism: Increased cortisol levels
*ACTH levels can be determined in helping to distinguish
primary or secondary cause.
EXPLANATION
• This is the most convenient procedure for
studying patients suspected of having
hypocortisolism
• During the test, small amount of synthetic
ACTH is injected and the amount of cortisol
that the adrenals produce in response is
measured.
• In addition, this test can also distinguish
whether the cause is adrenal or the low
cortisol and aldosterone production or
pituitary with low ACTH production.
 SECONDARY HYPOCORTISOLISM
• Secondary adrenal insufficiency
• Due to impairment of the pituitary gland: decreased ACTH
production, destruction of the anterior pituitary gland
• Pituitary adenoma (suppressing ACTH production),
Sheehan’s disease
Laboratory test:
• ACTH Stimulation Test: (+) delayed response to stimulation
• Overnight Metyrapone test: a drug administered orally at
midnight which functions to inhibit the conversion of 11-
deoxycortisol to cortisol by blocking 11B-hydroxylase
(+) for 2ndary hypocortisolism: unresponsive ACTH level
(-) for 2ndary hypocortisolism: increased ACTH level
EXPLANATION
• Sheehan’s Disease – a hypopituitarism that
occurs following damage to pituitary gland due
to low levels of oxygen and this is through blood
loss. Often, during or after childbirth.
• Overnight Metyrapone Test – is usually done
after a near normal result in osintropin
administration
• Since steroidogenesis in G-zone remains intact,
steroids from the R-zone are replaced.
 TERTIARY
HYPOCORTISOLISM
• Tertiary adrenal insufficiency
• Due to impairment of the hypothalamus: decreased
CRH production, destruction of the hypothalamus
• CNS disorder, hypothalamic disease (tumors),
sudden withdrawal from long-term exogenous
steroid use (most common)
• Due to impaired hypothalamic release of
corticotropin-releasing hormone which results to
decrease production of ACTH by the pituitary
• The most common cause is suddenly stopping
corticosteroid after taking them in wrong time
Laboratory test:
• Insulin Tolerance Test (gold standard for
secondary and tertiary hypocortisolism)
• CRH Test
1. Draw blood for baseline levels (ACTH and
cortisol)
2. Administer 100 ug of exogenous CRH IV
3. Collect blood cortisol and ACTH levels every
15 minutes for 60 - 90 minutes
(+) : Unresponsive cortisol and ACTH levels
(-) : Increased cortisol and ACTH levels
 HYPERCORTISOLISM
• There is overproduction of CRH or ACTH, increased
adrenal glucocorticoid secretion & exogenous intake
hypercortisolism.
• Excess cortisol affects multiple systems including
immune (suppression), dermatologic (thin, friable skin,
wide purple striae, poor healing), 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 (osteopenia or osteoporosis), renal
(edema, HTN, calciuria), and metabolic (hyperglycemia
and insulin resistance).
EXPLANATION
• This is what we call Cushing’s syndrome – this
is a group of clinical and metabolic disorder
characterized by adrenocortical
hyperfunction. This is caused primarily by
excessive production of cortisol and ACTH
but has decreased the production of
aldosterone and renin. This is also caused by
the overuse of corticosteroid.
• NOTE: In this condition, there is lack of
cortisol diurnal variation (especially in
cushing’s syndrome)
 CUSHING’S SYNDROME
• composed of a group of clinical and metabolic disorders resulting from
prolonged exposure to elevated concentrations of glucocorticoids.
• The excessive levels of glucocorticoids may be of endogenous (secreted by
the adrenal zona fasciculata), or of exogenous (e.g. pharmacologically
administered steroids) origin.
• Laboratory findings:
• (1) excessive production of cortisol measured as elevated serum
cortisol, urinary free cortisol, or midnight salivary cortisol
• (2) loss of circadian rhythm of ACTH and cortisol,
• (3) loss of suppression of cortisol production by administration of the
synthetic glucocorticoid dexamethasone.

CUSHING’S DISEASE
• refers to hypercortisolism due to an ACTH-
secreting pituitary adenoma
DIAGNOSIS OF CUSHING’S
SYNDROME
Screening test:
• Urinary free cortisol test: sensitive indicator of endogenous
cortisol production; reflection of the unbound circulating cortisol
that is freely filtered by the glomerulus
• When serum cortisol exceeds the binding capacity of its carrier
protein, free cortisol levels rise rapidly, increasing the free
cortisol filtered into the urine.
• Specimen: 24 hour urine or overnight urine (10 pm -8am)
• Method: Tandem mass spectroscopy – the most sensitive (95%
to 100%) and specific (98%) screen for excess cortisol
production.
*The creatinine should be measured in all collections to ensure
the adequacy of the specimen.

(+) for Cushing’s syndrome: Elevated (4x) urine cortisol

 DIAGNOSIS OF CUSHING’S
SYNDROME
Screening test:
• Overnight Dexamethasone Suppression Test
Dexamethasone acts as an exogenous cortisol substitute,
suppressing ACTH and cortisol secretion if the pituitary and
adrenal glands are normal, respectively. This is used to screen
patients for autonomous overproduction of cortisol.
1. The patient takes 1 mg of dexamethasone orally between the
hours of 11 pm and 12 midnight.
2. The plasma cortisol is drawn the following morning between
8 am and 9 am, respectively.
• This checks for a condition in which large amounts of cortisol
are produced by the adrenal glands.

(+) for Cushing’s syndrome: failure to suppress the morning

cortisol level
 DIAGNOSIS OF CUSHING’S
SYNDROME
Screening test:
• Overnight Dexamethasone Suppression Test
After the dose of dexamethasone, the cortisol levels will
often stay very high on patients with Cushing’s syndrome
and some times, other conditions can keep cortisol levels
high during this time. This includes major depression,
alchohol use disorder, renicity (?), kidney failure and also
pregnancy.
The night before the blood test, the patient will take
dexamethasone pill then the next morning, cortisol levels in
the blood will be measured. If the cortisol levels of the
patient remains high, then Cushing’s syndrome may be the
cause.
• NOTE: Values greater than four times the upper limit of
normal are diagnostic of Cushing’s syndrome.
DIAGNOSIS OF CUSHING’S
SYNDROME
Screening test:
• Midnight Salivary Cortisol test
• Saliva cortisol is stable at room temperature for days.
• Collection is not invasive
• Less sensitive than serum or urine (has 100% specificity)
• Can be performed at home in which patient will collect saliva at
around 11pm - 12am
• This can be done in two ways:
• patient can chew on a cotton pledget for 2 to 3 minutes and
then place the pledget into a plastic tube
• patient can passively drool directly into a test tube
Method: Immunoassay (RIA/Competitive protein binding assay)
or liquid chromatography–mass spectrometry (LC- MS/MS)

(+) for Cushing’s syndrome: Elevated saliva cortisol

DIAGNOSIS OF CUSHING’S
SYNDROME
Confirmatory test:
• Midnight Plasma Cortisol test
• This take into account the normal fluctuation of cortisol levels.
Cortisol peaks in the mornings and suddenly declines throughout
the day reaching its lowest levels ar midnight
• Plasma cortisol is highest between 6:00 - 8:00 AM and 50 - 80%
lower between 10:00 PM and 12:00 PM.
**Measuring at late night knowing that cortisol levels are low and
when measured high signifies a hypercortisolism.
1. Patient should be admitted for at least 48 hours
2. An IV access should be inserted before 10pm
3. Patient should be sleeping at time of collection

(+) for Cushing’s syndrome: Elevated plasma cortisol (>7.5 ug/dL)

DIAGNOSIS OF CUSHING’S
SYNDROME
Confirmatory test:
Low-dose dexamethasone suppression test (LDDST)
requires the collection of two baseline 24-hour urine
samples for urinary free cortisol
1. The patient is given 0.5 mg dexamethasone orally
every 6 hours, starting at 9 am for 2 days.
2. On day 2 of dexamethasone, another 24-hour urine
sample is collected for urinary free cortisol.
*All 24-hour urine collections should include the
measurement of creatinine to assay for adequacy of the
collection.
DIAGNOSIS OF CUSHING’S
SYNDROME
Confirmatory test:
Low-dose dexamethasone suppression test (LDDST)
(+): fail to decrease in urinary free cortisol
per 24 hours on the second day of
dexamethasone.

For alternative, serum cortisol can be

measured at baseline (9 am day 1) and at 48
hours following the first dose of
dexamethasone. A normal response consists of
suppression of the plasma cortisol.
ADRENAL CORTEX
R-ZONE (ZONA RETICULARIS)
• the inner zone (15%) of the cortex
• sulfates DHEA to DHEAS (Sulfated),
which is the main adrenal androgen.
• The zone is sharply demarcated with
lipid-deficient cords of irregular, dense
cells with lipofuscin deposits
• This is the site of biosynthesis of
androgen which is the androgen
precursors such as DHEA from
cholesterol
• These androgens are released into the
bloodstream and transported to gonads
where they are converted to testosterone
and estradiol
 ADRENAL ANDROGENS
• produced as by-products of cortisol synthesis that are regulated by
ACTH.
• Main function: Sexual characteristics
• Androgens stimulate organ development, linear growth, and
epiphyseal fusion.
• The DHEA and DHEAS – binds mainly to albumin and they are the
precursors to more active androgens although both have minimal
adrogenic activity, their adverse effect are caused by conversion to active
androgen in the adrenal and peripheral tissues
• DHEA = dehydroepiandrosterone 
• DHEAS = dehydroepiandrosterone sulfate 
These weak androgens are the precursors of the more active
androgens (i.e. testosterone, estrogen)
R-cells primarily produce DHEA and multiple 19-carbon steroids
(androgens and estrogens) from 17α-hydroxylated pregnenolone and
progesterone. DHEA is sulfated to DHEAS by sulfotransferase, an
adrenal enzyme, and secreted daily.
 ANDROGEN EXCESS
• causes ambiguous genitalia in infants
• precocious puberty in children
MALE FEMALES
Manifestation • Infertility with feminizing • Infertility
of Androgen effects • Hirsutism
Excess • Inhibition of pituitary • Acne
gonadotropins • Male pattern
• Low testicular testosterone baldness
production • Menstrual
• loss of muscle mass irregularities
• decreased hair growth • Virility
• Decreased testes size (Masculinity)
• Low testicular testosterone
production
• Low spermatogenesis
DIAGNOSIS OF ANDROGEN EXCESS
• High DHEAS and DHEA production strongly
suggests adrenal hyperandrogenism, whereas
elevated testosterone values are seen with
either adrenal or gonadal hyperandrogenism.

TREATMENT
• Surgery: Adenomas/carcinomas
• Discontinue exogenous sources
• Antiandrogenic drugs (e.g., minoxidil,
spironolactone, birth control pills)
 ADRENAL MEDULLA
• The adrenal medulla is part of the sympathoadrenal
axis which functions as an atypical sympathetic
ganglion
• It possesses the capability of synthesizing
catecholamines through the process of amine
precursor uptake and decarboxylation.
• Contains chromaffin cells that produce and secrete
catecholamines (i.e. dopamine, epinephrine,
norepinephrine)
• The precursor of catecholamine is L-tyrosine
• NOTE: Chromaffin cells are called as chromaffin cells
because when they are treated with chromates they
oxidized and turned brown.
 CATECHOLAMINES
• Hormones synthesized from tyrosine.
• Serves as first responders to stress by acting within
seconds (cortisol takes 20 minutes) to promote the
fight-or-flight response, which increases cardiac output
and blood pressure, diverts blood toward muscle and
brain, and mobilizes fuel from storage.
• Epinephrine is almost exclusively produced and
secreted by the adrenal medulla, where the ratio of
NE/E is about 1:4. However, because all three
catecholamines are also synthesized within the central
and sympathetic nervous systems, the peripheral NE/E
ratio is more like 9:1.
CATECHOLAMINES
EPINEPHRINE
• a neurotransmitter which causes vasodilation, and increases
cardiac rate, oxygen consumption and hepatic glycogenolysis,
allowing the voluntary muscles to have greater work output.
• Released in response to low blood pressure, hypoxia, cold
exposure, muscle exertion and pain
• This is the secondary amine and is the most abundant medullary
hormone.
• Flight hormone because it is released in response to physiologic
injuries or psychologic conditions like stress and anxiety
• This is also increased in glucose concentration or the
glycogenolysis and any form of stres that increases cortisol level
stimulates the production of epinephrine
• Its main metabolite is the VanillylMandelic Acid (VMA)
CATECHOLAMINES
NOREPINEPHRINE
• A neurotransmitter affecting the vascular
smooth muscle and heart via vasoconstriction
• Released primarily by the postganglionic
sympathetic nerves
• This is the primary amine and it is produced by
the sympathetic ganglia. Highest concentration
is found in the brain especially in the CNS.
• This acts as a neurotransmitter both in the CNS
and sympathetic post (?) system
 CATECHOLAMINES
• All catecholamines are rapidly eliminated from target cells and the
circulation by three mechanisms:
1. Reuptake into secretory vesicles
2. Uptake in nonneuronal cells (mostly liver)
3. Degradation
Metabolized by either catechol-O-methyl- transferase (COMT) or
monoamine oxidase (MAO – within the neurons)
1. COMT converts D to methoxytyramine, E to metanephrine, and
NE to normetanephrine, all of which in turn can be oxidized to
vanillylmandelic acid (VMA) by MAO.
2. MAO can also convert E and NE to 3,4-dihydroxymandelic acid,
which is acted upon by COMT to form VMA.
3. 3-Methoxy-4-hydroxyphenylacetic acid (homovanillic acid
[HVA]) is the final product of dopamine metabolism.
 URINE & PLASMA CATECHOLAMINE
MEASUREMENTS
• Catecholamines are hydrophilic, circulate in low levels (50%
albumin bound), have short half-lives (seconds to 2 minutes),
and produce wide and rapidly fluctuating plasma levels.
• Urine catecholamines (free NE and EPI) are assayed using:
• liquid chromatography (LC)
• Fluorometry
• LC-tandem mass spectrometry (LC-MS/MS).
24-hour urine catecholamine and metabolite levels are more
reliable and are not altered by age or gender.
Vanillylmandelic acid (VMA) - major epinephrine metabolite
in urine
Homovanillic acid (HVA) - major dopamine metabolite
CAUSES OF SYMPATHETIC
HYPERACTIVITY
• Autonomic dysfunction
• Panic attack (emotions)
• Stress responses: hypoglycemia, injury, infarction,
• Infection, psychosis, and seizures
• Drugs: decongestants, appetite suppressants, stimulants,
bronchodilators, MAO inhibitors, thyroid hormone,
cortisol, nicotine withdrawal, or shortacting sympathetic
antagonists (clonidine or propranolol)
• Foods containing tyramine: imported beer, red wine, soy
sauce, overripe/fermented foods, smoked or aged meats
• Pheochromocytoma (catecholamine-producing tumor)
 PHEOCHROMOCYTOMA
• Catecholamine-producing tumors (has two categories:
pheochromocytomas and paragangliomas) arising from
chromaffin tissue that cause hypertension in association with
nonspecific clinical symptoms mimicking anxiety
• This is a tumor of adrenal medulla or sympathetic ganglia and
this is due to the overproduction of catecholamine. It is
commonly seen in the 3rd or 4th decade of life.
• Pheochromocytoma: arise from the chromaffin cells of the
adrenal medulla and account for 90% of these tumors.
• Paragangliomas: extra-adrenal in origin, arising in the
paravertebral sympathetic ganglia of the chest, abdomen,
and pelvis, and the parasympathetic chains along the vagus
and glossopharyngeal nerves.
 PHEOCHROMOCYTOMA
• Sustained or paroxysmal hypertension is the most
common manifestation of this disease and is present
in about 90% of patients.
• More than 90% will present with paroxysmal
attacks characterized by at least two of the three
following symptoms: headache associated with
palpitations and diaphoresis.
• Other symptoms include orthostatic
hypotension, labile blood pressure, excessive
sweating, anxiety, nervousness, weight loss,
fatigue, pallor, and tremor.
 DIAGNOSIS OF
PHEOCHROMOCYTOMA
• Measurement of fractionated metanephrines and
cateholamines in a 24-hour urine
• Best test for diagnosing pheochromocytoma (98% sensitivity
and specificity)
(+) Elevated levels
• Measurement of Plasma metanephrines using HPLC/RIA.
• (+) Plasma concentration of either free metanephrine or
normetanephrine is about four times the upper reference limit.
• Chromogranin A
• is a protein that is stored and secreted along with the
catecholamines from the adrenal medulla and sympathetic
nervous system
• 80% of pheochromocytoma patients have increased plasma
chromogranin levels.