8.

05 CLINICAL
EVALUATION
PATHOLOGY OF ENDOCRINE
Dr. John Tawasil
PITUITARY FUNCTIONS
PITUITARY GLAND HYPOPROLACTENEMIA
 Located within the sella tursica  Inability to lactate postpartum
 Contiguous to vascular and neurologic structures  No milk letdown
o Cavernous sinuses  Often manifested as Sheehan’s syndrome
o Cranial nerves (postpartum blues)
o Optic chiasm o Can lead to psychosis and first manifestation
of prolactin deficiency.
ANTERIOR PITUITARY GLAND  GnRH release is decreased in direct response to
 Secrete various trophic hormones elevated prolactin, leading to decreased production of
 Disease in this region may result in syndromes of LH and FSH
hormone excess or deficiency  In women: Amenorrhea (present earlier), Hirsutism
 Production of important hormones  In men: Impotence, tend to present later, large
tumors, sign of mass effect
POSTERIOR PITUITARY GLAND  Reference value for serum PRL is 1–25 ng/mL (1–25
 More of a terminus of axons of neurons in the μg/L) for women and 1–20 ng/mL (1–20 μg/L) for men
supraoptic and paraventricular nuclei of the  PRL deficiency can be seen with pituitary necrosis or
hypothalamus infarction and in some cases of
 Storehouse for the hormones pseudohypoparathyroidism
 The main consequence of disease in this area is
disordered water homeostasis GROWTH HORMONE DEFICIENCY
 Does not synthesize hormone – storage only SCREENING TEST

PITUITARY TUMOR
 Classifications:
o microadenomas (<1 cm in greatest diameter
and confined to the sella)
o macroadenomas (≥1 cm in greatest diameter)
 subcategorized into secretory and nonsecretory
varieties
 All tumors have the potential to grow
o It can compress the optic chiasm, resulting in
visual field defects
 bitemporal hemianopia is the most
frequent presentation
 Invasion into the cavernous sinus can lead to:
o compression of cranial nerves III, IV, VI, V1,
and V2 and the intracavernous portion of the
internal carotid artery
o hydrocephalus caused by obstruction of the
third ventricle
o hormonal deficiency due to compression of
other cell lineages within the pituitary gland
PROLACTIN
 Produced by lactotrophs
 For initiation and maintenance of lactation
 Secreted in a circadian fashion
o Highest during sleep
o Nadir between 10am-noon
 Half-life: 26-47 minutes
 It is recommended that when a patient is screened for
hyperprolactinemia, three specimens should be
obtained at 20 to 30 minute intervals
 Major circulating form is the non-glycosylated
monomer
 PRL is measured by immunometric assay (5ug/L)
 PRL acts on breast tissue, where, in the setting of
estrogen priming, it
 stimulates lactation.
 PRL also acts at the hypothalamus to inhibit the
secretion of gonadotropin-releasing hormone (GnRH)
 Quantitate first if deficient or excess - GH
measurements
 Pharmacologic stimulation and GH suppression –
Oral Glucose Load
CONFIRMATORY: INSULIN TOLERANCE TEST (ITT)
 “gold standard” for diagnosing GHD
 Needs attendance of a physician
 Contraindicated with seizures or cardiac or
cerebrovascular disease
SECOND CONFIRMATORY TEST: ARGININE
STIMULATION
 Combination testing with GHRH + arginine
 Adults will fail stimulation with arginine alone
GROWTH HORMONE EXCESSS
 Screened by failure of GH suppression ff. An oral
glucose load.
 If the condition develops before closure of the
epiphyses, these individuals may be exceedingly tall -
GIGANTISM.
 Goal is to normalize IGF-I suppress the GH response
to OGTT to <1 ng/mL (<1 μg/L).
 Growth hormone overproduction can result in the
condition called acromegaly.
 Characteristic features include prognathism,frontal
bossing, and spadelike hands.
 The screening test for clinically suspected acromegaly
is a randomly collected IGF-1.
 If the level of IGF-1 is elevated or borderline with
respect to the appropriate age- and gender-related
reference range, it becomes necessary to confirm the
diagnosis using an oral glucose tolerance test
(OGTT).
ORAL GLUCOSE TOLERANCE TEST
 Administering 75 g of glucose

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 Obtaining blood samples at baseline and every 30
minutes over the next 2 hours for glucose and GH.
 Patient with gigantism has a normal suppression of
GH to <1 ng/mL (1 μg/L) at any time during the OGTT
process.
 If GH fails to drop to below 1 ng/mL (1 μg/L), the
patient is diagnosed having acromegaly
POSTERIOR PITUITARY HORMONES
NEPHROGENIC DI
NEUROGENIC DI
OXYTOCIN
 Contributes to uterine contractions late in labor by
direct action and by stimulation of prostaglandin
secretion
 Plays a role in hemostasis at the placental site
following delivery
 Stimulates the myoepithelial cells surrounding the
mammary glands and lactiferous ducts to contract,
resulting in milk ejection
 Its function in males remains unknown.
 Fergusson reflex - stimulation of oxytocin by
stretching of the cervix and vagina during parturition
ARGININE VASOPRESSIN (AVP)
 Anti Diuretic Hormone
 synthesized within the paraventricular and supraoptic
nuclei of the hypothalamus
 Major function: to maintain osmotic homeostasis by
regulating water balance
o by stimulating theV2 receptors on principal
epithelial cells that line the cortical collecting
ducts of the kidney
 Other Functions:
 induces an increase in the production of cyclic
adenosine monophosphate (cAMP), increasing water
permeability and reabsorption.
 a potent pressor by causing vasoconstriction (V1
receptor)
 stimulates ACTH secretion from the anterior pituitary
gland
 stimulates the production of clotting factor VIII.
 Deficiency: Diabetic Insipidus – large amounts of
dilute urine
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 Excessive or inappropriate production predisposes to
hyponatremia if water intake is not reduced in parallel
with urine output
DIABETES INSIPIDUS
 2 CLASSIFICATIONS
o Central – absent or decreased ADH from the
hypothalamus or neurophysis
o Nephrogenic – due to renal resistance to the
actions of ADH
 both variants are characterized by the passage of
large volumes of dilute urine (>2.5 L/day) in the face
of inappropriately elevated plasma osmolality
 Water deprivation test – diagnostic test for DI.
o In neurogenic DI, ADH levels are low and the
kidney rapidly acts to conserve water in
response to exogenous ADH administration
o In nephrogenic DI, the ADH levels are normal or
increased and exogenous ADH administration
has little or no effect on renal water absorption
 most common congenital etiology: X-linked receptor
defect
 most common acquired etiology: medications
(Lithium, demeclocycline, methoxyflurane)
 congenital: autosomal dominant mutation in the ADH
signal peptide or in the exons that code for
neurophysins
 acquired: (more common) due to lesions affecting the
hypothalamus (metastatic tumor, trauma or
granulomatous disorders) or medications (phenytoin,
chlorpromazine, a-adrenergic agonists)
 idiopathic: presence of autoantibodies directed
against ADH-producing cells of the hypothalamus
SIADH – DYNDROME OF INAPPROPRIATE ADH
 euvolemic hypoosmolar hyponatremia associated with
hyperosmolar urine
 cannot be diagnosed until nonosmotic stimuli for ADH
secretion and other pathologies interfere with free
water clearance have been excluded
 Water load test – in the absence of medication or
other conditions that might impair diuresis, failure to
excrete 80 – 90% of the administered water load
within 4 hours and to suppress Uosm to <100
mOsm/kg is indicative of SIADH
 In the right clinical setting, a spot urine Na less than
30 mmol/L can generally distinguish those with
hyponatremia due to volume depletion from those
with SIADH in whom urine Na is greates than
30mmol/L.
 Cause of renal salt wasting (2-fold process):
1. At the onset of disease, volume expansion
inhibits RAAS axis and leads to greater
improved delivery to the distal tubules
2. Continued sodium loss due to secretion of
Atrial natriuretic peptide
THYROID FUNCTION
REVIEW OF THE THYROID
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 Has 2 lobes connected by a narrow isthmus
 Lobes are divided into lobules composed of 20 to 40
follicles each
 Follicles are ring-shaped, in which a single band of
follicular cells surrounds a closed cavity containing
colloid, thyroid hormone (TH), and thyroglobulin (Tg)
 Follicular cells rest on a basement membrane that is
rich in glycoprotein
 Apex of the follicular cells have microvilli that extend
into the colloid where initial phase of hormone
secretion occurs
 Also contains parafollicular cells (C cells) which
synthesize calcitonin
THYROID FUNCTION
 Normal function requires: intact hypothalamic-
pituitary-thyroid (HPT) axis and a ready source of
iodide
o Hypothalamus secretes TRH → TRH stimulates
thyrotrophs of anterior pituitary to secrete
thyrotropin (TSH) → TSH stimulates the thyroid
to synthesize and secrete hormones
 Thyroid hormones exerts negative feedback on the
hypothalamus and pituitary gland to maintain a
minimal TSH concentration; it also mediates various
metabolic activities
 Through TSH, inorganic iodine enters the follicular
cells, undergoes a series of metabolic steps and is
transformed into thyroid hormones, thyroxine (T4) and
3,5,3’-triiodothyronine (T3)
 100% of circulating T4 is of thyroidal origin, while 20%
of T3 is of thyroidal origin.
o Approximately 110 nmol (85µg) of T4 and 10
nmol (8.5 µg) of T3 are produced daily by the
thyroid
 Thyroid hormones circulate while attached to plasma
proteins
o T4 - 70% is bound to thyroxine-binding globulin
(TBG), 20% to transthyretin, 10% to albumin
o T3 - most are bound to TBG, but has a 10-fold
reduced affinity compared to T4
o Small percentages of both T4 (0.03%) and T3
(0.3%) remain unbound to plasma
 Thyroid diseases are functionally classified into 3:
o Hyperthyroidism
o Hypothyroidism
o Euthyroidism
THYROID HORMONE SYNTHESIS AND METABOLISM
 Iodine is a major component of thyroid hormone; the
main source of iodine is dietary intake.
 Inorganic iodide is transported into the follicular cell
by the sodium-iodide symporter (NIS) located in the
basolateral membrane
 TSH modulates NIS activity: An increase in TSH
secretion augments the uptake of iodide into the
follicular cell
 Thyroglobulin is a glycoprotein synthesized by the
rough endoplasmic reticulum in the basal and
perinuclear regions of the follicular cell
 70% of T4 peripheral metabolism are from
monodeiodination of T4 to T3 and rT3; 30 % are from
conjugation of T4 to sulfate, deamination, and
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EVALUATION OF ENDOCRINE FUNCTION 8.05
decarboxylation to form acetic acid analog tetrac and
by ether link conjugated with sulfate to form T3-sulfate
and can be converted to acetic acid analog, triac.
 Enzymes responsible: phenolsulfotransferases and L-
aminotranferases
 After formation from T4, both T3 and rT3 undergo
deiodination to form 3,3’-diiodothyronine (T2)
 Protein-bound thyroid hormones that do not enter the
cells, are considered biologically inert and function as
a storage reservoirs for circulating thyroid hormones
 Free thyroid hormones, on the other hand, readily
enter the cell by a specific transport mechanisms to
exert their biological effects, which are mediated by
T3 receptors located in the nucleus of the cell
 4 isoforms of the T3 receptors: α1, α2, β1 and β2; α1
and β1 present in most tissues, which binds to the T3
to promote thyroid hormone action: increasing mRNA
and protein synthesis; In contrast, α2 is inhibitory and
acts as a negative regulator of thyroid hormone
action. On the other hand, β2 is unique in pituitary
gland and is the central in negative feedback
regulation of TSH by thyroid hormone.
 Mutations in the β2 receptors have been described in
the syndrome of thyroid hormone resistance,
characterized by growth and mental retardations of
varying degrees, as well as hypothyroidism.
*APPENDIX
HYPOTHALAMIC-PITUITARY-THYROID AXIS
 Intro
 TRH
 TSH
 Thyroxine
 Free Thyroxine
 Triiodothyronine
 Reverse Triiodothyronine
 Thyroglobulin
 Thyroxine Binding Globulin
 Thyroid Autoantibodies
 Physiologic regulators responsible for integrating the
function of the thyroid gland and the periphery
include:
o TRH- the hypothalamic hormone
o TRH enhances TSH synthesis
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o Stimulates the secretion of any pre- formed
TSH from the thyrotrophs
o Modulates the bioactivity of TSH
o Under the negative-feedback influence of
circulating thyroid hormones
 TSH- the pituitary hormone
 Serum free T3 and T4 concentrations
THYROTROPIN-RELEASING HORMONE (TRH)
 Found in the:
o hypothalamus
o brain
o C cells of the thyroid gland
o β cells of the pancreas
o myocardium
o prostate and testis
o spinal cord
 Thyroid hormones regulate their own production by
feedback inhibition to synthesis of TRH and TSH in
the hypothalamus and pituitary gland
 Acts also on the production of other pituitary
hormones, especially prolactin
 Leptin plays a significant role in the regulation of the
TRH gene which affects the individual’s appetite for
food intake.
 difficult to develop a specific antibody for TRH,assays
are not clinically useful
THYROID STIMULATING HORMONE (TSH)
 a glycoprotein consisting of two monocovalently
linked α and β subunits
 α subunit:
o has the same amino acid sequences as LH,
FSH, and human chorionic gonadotropin (hCG)
 β subunit:
o carries specific information to the binding
receptors for expression of hormonal activities
 Due to the improvements in the sensitivity of the TSH
assay, it can identify all instances of hyperthyroidism
and hypothyroidism, except when there is damage to
the hypothalamus or pituitary gland, thyroid hormone
resistance, or interference with normal functioning of
the HPT axis due to medication
 Most individuals with hypothyroidism, serum TSH
results are clearly elevated
 Subclinical Hypothyroidism:
o elevated TSH
o normal levels of T4, T3, and FT4
*APPENDIX
THYROXINE
 bind to various proteins in the blood (thyroid-binding
globulin, albumin, thyretin) after release from thyroid
follicles.
 measured by immunoassay
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 reference range is 5 to 12.5 μg/dL in adults
 used along with TSH and can be important in
interpreting TSH results.
 Primary Hypothyroidism
o low T4 , increased TSH
 Primary Hyperthyroidism
o elevated T4 and T3, decreased TSH
 Hyperthyroidism
o Elevated serum T4 but with serum T3 levels
within the reference range or low.
 T4 thyrotoxicosis
o patients with iodine-induced thyrotoxicosis; in
patients on beta blockers, amiodarone, or large
doses of steroids; and in thyrotoxic patients
with NTI
 T3 thyrotoxicosis
o suppressed TSH level associated with a
normal to low-normal T4 and a high T3;
o more common in a toxic nodule
 Low T4 and T3 syndrome
o Severe nonthyroidal illness is associated with
low T4 and low T3; an adaptive response to
reduce metabolic demands and conserve
protein stores; arises from a maladjusted
central inhibition of TRH
 Euthyroid hyperthyroxinemia
o increased serum T4 level in association with a
normal TSH in an otherwise euthyroid
individual; caused by increased binding
Proteins as may be seen with certain drugs
(e.g., estrogen) or medical conditions (e.g.,
liver disease). It is also seen in patients who
are acutely hospitalized for psychiatric illness
and in patients with familial dysalbuminemia
 Free T4 correlates better with thyroid functional status
than does serum total T4 – because it is the free
hormone, not the protein-bound component, that is
bioactive.
 T3 uptake (T3-UP) test - classical method of adjusting
a total T4 measurement for alterations in binding
protein; relative measurement of the unoccupied
binding sites of all circulating proteins.
FREE THYROXINE
 Biologically active fraction of thyroxine in circulating
blood
 Traditional reference method for FT4 is equilibrium
dialysis – a method that is not affected by changes in
binding protein concentration
 More accurate measurement using: ultrafiltration and
symmetrical dialysis
 Mass spectrometry – well suited for thyroid hormone
measurement
 Increases or decreases in serum FT4 without
concomitant changes in metabolic state have been
reported in pregnancy with NTI, and in patients being
treated with certain drugs.
 Prolonged administration of phenytoin or
carbamazepine results in a 15% to 30% decrease in
both serum T4 and FT4
TRIIODOTHYRONINE (T3)
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 Generally measured by IMMUNOASSAY. Reference
interval of 60 to 160 µg/dL (0.9 to 2.46 nmol/L).
 Total T3 concentrations measured by competitive
immunoassay methods that are now mostly
nonisotopic and use enzymes, fluorescence, or
chemiluminescence molecules as signals
 Less bound to serum protein than T4; Relatively
greater proportion of T3 than T4 exists in the free,
diffusable state
 Serum total T3 measurement aids in confirming the
diagnosis of hyperthyroidism, especially in patients
with no or minimally elevated T4 or ambiguous clinical
manifestation
 Serum total T3 level can be in the reference range or
low in patients with hyperthyroidism if there is
coexistent NTI, or if patients are on drugs that
decrease the conversion of T4 to T3 (e.g.,
propranolol, amiodarone)
 T3 thyrotoxicosis - Hyperthyroidism with elevated
serum T3 but normal T4 and free T4.
 Generally, the measurement of serum T3 is not useful
in evaluating patients suspected of having
hypothyroidism because serum T3 levels are within
the reference interval in 15% to 30% of hypothyroid
patients
 Severe hypothyroidism - patients with serum T4
levels less than about 2 µg/dL (32 nmol/L), T3 serum
levels are also decreased
 Low serum T3 may occur in patients with a wide
variety of NTI, acute illness such as myocardial
infarction (occurs rapidly, declining to about 50% of
the reference value within days), cord blood (but
increase rapidly during the first few hours of life)
 Serum total T3 measurement aids in confirming the
diagnosis of hyperthyroidism, especially in patients
with no or minimally elevated T4 or ambiguous clinical
manifestation
 Patients receiving thyroid preparations containing T3
such as desiccated thyroid and synthetic T3 and T4
combinations and those treated with T3 will have
uninterpretable serum T3 results unless the time of
hormone administration is known.
 T3 (Ctyomel) administration - rapid rise in T3
concentration that peaks within 2 to 4 hours and then
begins to drop off
 T4 (levothyroxine) treatment - stable levels of T3,
which is derived from the peripheral conversion of T4,
are reached after only a few weeks of therapy
 Pharmacologic agents that are associated with low
levels of T3 and free T3, and with normal or even high
levels of serum T4 and FT4 (these patients usually
have normal TSH results):
o Glucocorticoids
o Amiodarone - can induce hyperthyroidism or
hypothyroidism (TSH values will be
suppressed or elevated, respectively)
o Propranolol (large doses)
 Low serum T3 may occur in patients with a wide
variety of NTI, acute illness such as myocardial
infarction (occurs rapidly, declining to about 50% of
the reference value within days), cord blood (but
increase rapidly during the first few hours of life)
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REVERSE TRIIODOTHYRONINE (rT3)
 Major metabolite of thyroxine, is produced by 5-
deiodination of T4
 Clinically, serum T3 and serum rT3 vary reciprocally
(serum rT3 measurements however is little clinical)
 Increased Serum rT3 in:
o Patients with NTI (whose ↓ serum total T3)
o Healthy newborns
o Patients with hyperthyroidism (including
factitious hyperthyroidism)
o Patients taking certain drugs, including
amiodarone and propranolol
THYROGLOBULIN
 Synthesized and secreted by the follicles
 Healthy indiviuals : 30 ng/mL
 Serum Tg concentration reflects
o Thyroid mass
o Thyroid injury
o TSH receptor stimulation
 Increased in Grave’s disease, thyroiditis, and nodular
goiter
 Measurement is to follow patients with thyroid
malignancy postoperatively
 Helpful in patients with well-differentiated thyroid
carcinoma but not in those who have undifferentiated
tumors or medullary thyroid cancer.
 Well-differentiated tumors typically display about a 10-
fold increase in Tg in response to a high TSH.
THYROXINE-BINDING GLOBULIN
Main serum carrier protein for both T3 and T4
 Measurement of TBG is helpful in patients who have
serum T3 and T4 levels that do not agree with other
lab parameters of thyroid function or are not
compatible with clinical findings.
 Inherited abnormalities include
o Complete
o Partial deficiency
o TBG variants with decreased affinity for T4
or T3, and TBG excess
 Acquired abnormalities include
o Medications - salicylates, phenytoin,
penicillin and heparin (displaces T4 from
binding to TBG)
o Medical conditions
 Measured by: IMMUNOASSAY (NV: 13-39 ug/dL)
THYROID ANTIBODIES
 Autoimmune thyroid disease causes cellular damage
and alters thyroid gland function.
o Cellular damage occurs when the
autoantibodies or sensitized T lymphocytes
bind so thyroid cell membranes, causing cell
lysis and inflammatory reactions.
 3 thyroid autoantigens responsible for the
autoimmune thyroid disorders:
1. TPO antibodies (TPOAb): involved in the
tissue destructive process associated with
hypothyroidism in Hashimoto’s and atrophic
thyroiditis
2. Tg antibodies (TgAb): used to detect
autoimmune thyroid disease in patients with
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goiter and to monitor iodide therapy in
endemic areas
3. TSH receptor antibodies (TRAbs): Grave’s
disease. Measurement has also been used
to predict the risk of thyroid dysfunction in
newborns of mothers with GD as a result of
trans-placental passage of maternal TRAb.
URINARY IODINE MEASUREMENT
 Iodine deficiency disorders affect 2.2 billion persons
throughout the world, including the United States
 Iodine measurements are used mainly to assess the
dietary iodine intake of certain populations.
 Iodine is required for normal thyroid hormone
production.
 Because most of the ingested iodine is excreted in the
urine, measurement of urinary iodine (UI) excretion
provides an accurate estimate of dietary iodine intake
SCREENING PROGRAMS FOR DETECTION OF
NEONATAL HYPOTHYROIDISM
 Prevalence in newborns id ~1 in 3000 to 1 in 5000
 Higher in certain ethincity and increased in iodine-
deficient regions
 Early detection of hypothyroidism: eliminate severe
mental retardation
 Using dry blood spots or cord serum: TSH and T4
measurement for screening
o False postive results= low T4 levels occuring
in premature infants and with congenital
absence of TBG
 Elevated TSH: the most sensitive test for the
diagnosis of congenital hypothyroidism
 For newborns:
o TSH value <10 mIU/L= no further action
needed
o TSH value 10-20 mIU/L= repeat test after 2-
6 weeks
o TSH value >20 mIU/L= endocrinologic
evaluation for hypothyroidism
NONTHYROID ILLNESS
 Severe Illness - total and free T3 rapidly decline
o Ex: Myocardial infarction and Sepsis
o Serum total T4 falls as the severity of the
illness increases due to disruption of T4-
binding protein by inhibitors in the circulation
 Euthyroid Sick Syndrome or Low T4 Syndrome -
thyroid function tests alterations seen in NTI
o Poor prognosis: patients with NTI whose T4
levels drop below 2ng/dL
o Patients with NTI: low or low-normal TSH,
normal or low-normal T4, very low T3 -- during
Acute Illness
 Certain drugs that are used for underlying illness can
suppress TSH and mask hypothyroid status
o Ex: glucagon, dopamine, and high doses of
corticosteroids
 Uremia - indoleacetic acid accumulation interferes
with thyroid hormone binding
PHYSIOLOGIC VARIABLES
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 Genetically determined FT4 setpoint for each
individual when the pituitary gland sense any
deviation from the setpoint, it will cause reciprocal
change in TSH secretion
 Serum TSH abnormality will precede the development
of an abnormal FT4 during early stages of developing
thyroid dysfunction
 Children: there is progressive maturation and
modulation of the hypothalamic-pituitary-thyroid axis
→ Higher TSH concentrations
 Midgestation to after puberty: continuous decline in
the TSH/FT4 ratio
 Pregnancy: increased estrogen production
progressively elevates TBG concentration which
results in increased total T4 and T3 reference range
to 1.5 times the nonpregnancy upper reference levely
by 16 weeks’ gestation. Serum FT4 and T3
concentrations remain unchanged
 Trimester-Specific Reference Range for TSH by
ATA:
o First trimester TSH: 0.1 - 2.5 miu/L
o Second trimester TSH: 0.2 - 2.5 miu/L
o Third trimester TSH: 0.3 - 3 miu/L
 Serum FT4 assessment during pregnancy: using
online extraction/liquid chromatography/tandem mass
spectrometry (LC/MS/MS), T4 is measured in the
dialysate or ultrafiltrate of the serum sample. If this is
not available, TSH is measured
 Elevation of TSH during pregnancy:
o Subclinical Hypothyroidism: normal FT4, TSH
is 2.5 - 10 miu/L
o Overt Hypothyroidism: TSH is >10 miu/L, low
FT4 or TSH 2.5 - 10 miu/L, low FT4
 Thyroxine - only used drug for treating hypothyroid
pregnant women
MEDICATIONS
 Estrogen-induced TBG elevation causes
abnormally high total T4 and total T3, but has no
effect on TSH or FT4 and free T3.
 Glucocorticoids in large doses can suppress TSH
and decrease the conversion of T4 to T3
 Dopamine suppresses TSH and can temper the
expected rise in TSH in hospitalized patients with
primary hypothyroidism
 Propranolol suppresses the conversion of T4 to T3
(this is one of the reasons it is used in the treatment
of thyrotoxicosis).
 Iodide, found in iodide-containing contrast media
used in CT scans and coronary angiography or in
solution to sterilize skin, and radiopaque dyes can
cause both hyperthyroidism and hypothyroidism in
susceptible patients.
 Iodine-containing drugs such as amiodarone (used
as an antiarrhythmic agent) have complex effects on
thyroid function and can cause hypothyroidism or
hyperthyroidism
 Lithium inhibits thyroid hormone synthesis and
release and can cause hypothyroidism; critical to the
development of hypothyroidism and goiter.
 Drugs such as phenytoin, carbamazepine,
salicylate, salsalate, and furosemide competitively
inhibit thyroid binding to serum proteins. Therapeutic
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doses of phenytoin can induce acceleration of T4
disposal and perhaps can directly suppress TSH
centrally, all of these mechanisms being responsible
for the reduced T4.
 Heparin can stimulate in vitro lipoprotein lipase and
can liberate free fatty acids, which inhibit T4 binding
to serum proteins and falsely elevate FT4.
 Medications such as phenobarbital, phenytoin,
rifampin, and carbam- azepine increase the rate of
thyroid hormone clearance by increasing the
deiodination of T4 and T3. Hypothyroid patients
treated with any of these medications need to have
their hormone levels monitored closely.
 Somatostatin or its analog, octreotide, which is
used to treat acromegaly, and dopamine inhibit TSH
synthesis to undetectable levels in patients with
severe NTI.
THYROID ILLNESS
 Hyperthyroid patients
o present with one or all of the following signs
and symptoms:weight loss, sweating, heat
intolerance, palpitations, insomnia, increased
bowel movement, tremors, infertility, or
amenorrhea
o Secondary Hyperthyroidism: TSH is elevated
along with T4 and T3; caused by TSH-producing
pituitary tumors, or other rare disorders such as
pituitary resistance to thyroid hormones.
o Subclinical Hyperthyroidism is defined as low
TSH (<0.1 µIU/mL) with normal levels of T4 and
T3
 Primary hypothyroidism patients:
o present with one or all of the following signs
and symptoms: cold intolerance, constipation,
water retention, hypercholesterolemia,
depression, pretibial myxedema, periorbittal
edema, elevated TSH with low T4 and T3
o In secondary hypothyroidism: TSH is low
along with T4 and T3; caused by pituitary or
hypothalamic disorders.
o subclinical hypothyroidism have elevated
TSH levels (>4.5 µU/mL), but both T4 and T3
are within the reference range.
SCREENING FOR THYROID DISEASE
 ATA guidelines recommend screening at 35 years of
age and every 5 years thereafter.
 Hashimoto’s thyroiditis
o High TSH
o Positive TPOAb
o Increasing prevalence with increasing age
 Incidence of low TSH is also increased in older adults
 Higher CV mortality rate in patients with chronically
low TSH
 Mild hypothyrodism in early pregnancy increases fetal
loss and impairs the IQ of offspring.
 Important to always confirm any TSH abnormality in a
fresh specimen drawn 3 weeks later before making
the diagnosis of mild abnormalities.
USES OF L-THYROXINE
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 An average replacement dose is 1.6 ug/kg body
weight/day for adults
 4.0 ug/kg body weight/ day for children
 1.0 ug/kg body weight/ day for older adults
 The requirement might increase during pregnancy
and in post-menopausal women starting hormonal
replacement
 A serum TSH between 0.5 to 2.0 mIU/ml is the
therapeutic goal level for L-T4 replacement in primary
hypothyroidism
 Serum FT4 concentration in the upper third of the
reference intervals is therapeutic range in central
hypothyroidism.
L-T4 is used to suppress TSH in patients with well-
differentiated thyroid carcinoma
CALCITONIN
 Medullary thyroid carcinoma (MTC) originates from
the C cells of the thyroid.
 25% of MTC cases are hereditary (multiple endocrine
neoplasia types 2A and 2B)
o Autosomal dominant inherited multiglandular
syndromes
o Recurring MTC genetic mutation = RET
oncogene located on the chromosome subband
10q11.2
o Recommended method of initial testing for
MEN2A is either a single or multi-tiered analysis
to detect RET mutation in exon 10 (codon 609,
611, 618, 620), exon 11 (condon 630, 634) and
exons 8, 13, 14, 15 and 16
 The current ATA risk categories for Hereditary MTC
are:
o Highest Risk (HST) - Patients with MEN2b and
RET codon M918T mutation
o High Risk (H) - Patients with MEN2a and RET
codon c634 mutation
 Moderate Risk - Patients with hereditary MTC and
RET codon mutations other than M918T and c634
 C cells secrete several hormones and biogenic
amines such as calcitonin and ACTH, B-melanocyte
stimulating hormone, somatostatin, chromogranin,
histaminase, neurotensin and carcinoembryonic
antigen (CEA)
 Calcitonin and CEA are valuable tumor markers for
patients with MTC
o Elevated level of calcitonin in circulating blood
indicates presence of MTC
o Measurement of calcitonin is done by two-site
immunometric assays using monoclonal
antibodies.
 One recognizes the N-terminal region
 The other one, recognizes the C-terminal
region
 This method is more specific and more
sensitive
 This method eliminates any cross-reactivity
with procalcitonin that can be elevated
during sepsis or inflammatory conditions
 The cutoff level in healthy adults is about 10 ng/L and
it is higher in children <3 years of age.
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 Some calcitonin measurements are used as tumor
markers for detecting residual thyroid tissue or
metastasis in patients with MTC.
 It should be measured before and 6 months after
surgery.
 Presence of residual tissue or a recurrence of MTC
can be ruled out only if both basal and
postpentagastrin or calcium-stimulating calcitonin are
undetectable.
 Provocative stimuli, such as calcium and pentagastrin
or omeprazole, have been used to detect C cell
abnormalities, as they increase calcitonin levels at all
stages of MTC
 Pg (pentagastrin) stimulation test for the diagnosis of
MTC
o An intravenous infusion of Pg (0.5 ug/kg body
weight) is given over 5 seconds.
o Blood samples are collected at baseline, 1, 2, 5
and 10 minutes after the start of the infusion.
 Calcitonin stimulation test
o Intravenous injection of 2.5 mg/kg of calcium
gluconate is given over 30 seconds.
o Blood samples are then collected at baseline, 1,
2 and 5 minutes.
o An increase in the plasma calcitonin level above
100 ng/L is an indication of C cell hyperplasia.
 Calcium infusion test is less sensitive than the Pg test
for the diagnosis of MTC, but if combined with the Pg
test, it enhances the sensitivity of the Pg test.
INTREPRETATION OF THE PENTAGASTRIN (Pg) TEST
ADRENAL FUNCTION
 The adrenal glands are pyramidal structures located
above the kidney.
 Anatomically divided into two parts: the medulla (inner
layer) and the cortex (outer layer)
 Medulla – of neural crest origin (ectoderm); stores
and secretes catecholamines
 Cortex – of mesenchymal origin; further divided into
three zones:
o Outermost zona glomerulosa – produces
mineralocorticoids
o Zona fasciculata – produces glucocorticoids
o Inner zona reticularis – produces
androgens
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 The adrenal glands have a very rich arterial supply
that forms a subcapsular plexus and empties into a
central vein.
 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.
HORMONES OF THE ADRENAL MEDULLA
 The adrenal medulla is part of the sympathoadrenal
axis. Being of neural crest origin, it possesses the
capability of synthesizing catecholamines through the
process of amine precursor uptake and
decarboxylation.
 The initial and rate-limiting step in catecholamine
synthesis is the conversion of tyrosine to 3,4-
dihydroxyphenyalanine (dopa) by the enzyme tyrosine
hydroxylase.
 Through a series of steps, L-dopa is converted to
dopamine, NE, and E.
 Epinephrine is almost exclusively produced and
secreted by the adrenal medulla.
 The catecholamines are either metabolized by either
catechol-O-methyl-transferase (COMT) or
monoamine oxidase (MAO).
 COMT – converts D to methoxytyramine, E to
metanephrine, and NE to normethanephrine, all of
which can be oxidized to vanillylmandelic acid
(VMA) by MAO.
 MAO – converts E and NE to 3,4-dihydroxymandelic
acid, which is acted upon by COMT to form VMA.
 3-Methoxy-4-hydroxyphenylacetic acid
(homovanillic acid [HVA]) is the final produce of
dopamine metabolism.
PHEOCHROMOCYTOMA
 Arises from the chromaffin cells of the adrenal
medulla and account for 90% of catecholamine-
producing tumors.
 Have an incidence of about 500 to 1600 per year and
account for <1% of all secondary causes of
hypertension.
 90% are benign
 Most pheochromocytomas are sporadic; however,
10% to 20% are familial
 Sustained or paroxysmal hypertension is the most
common manifestation
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 Other symptoms include orthostatic hypotension,
labile blood pressure, excessive sweating, anxiety,
nervousness, weight loss, fatigue, pallor, and tremor.
DIAGNOSIS
 The products of COMT, metanephrine and
normetanephrine, serve as specific markers of
chromaffin tumors
 Most recent guidelines recommend the measurement
of either plasma- free metanephrines or 24-hour urine
collection for fractionated metanephrines via high-
pressure liquid
 Plasma normetanephrines, and urine for fractionated
catecholamines and VMA may also be obtained.
 The plasma concentration of either free metanephrine
is normetanephrine that is about four times the
upper reference limit.
 Samples are recommended to be collected after the
patient has been supine for at least 30 minutes.
 In centers where this assay is not available the initial
test should be the chromatographic measurement of a
24 hr urine collection for
normetanephrine,metanephrine, fractionated free
catecholamines.
 Metanephrine is the most sensitive and specific of
these metabolites.
 Another method of diagnosis is by measuring plasma
catecholamines. But this method is only of used if
the sample is collected during paroxysm.
 The value from 24hr urine collection or plasma
catecholamines that are 2 to 3 times above the upper
limit of the normal are usually diagnostic of
pheochromocytoma.
SPORADIC PHEOCHROMOCYTOMA
 Choice and interpretation of the diagnostic tests
depends on the pretest level of suspicion for disease.
o A 24 hour urinary fractionated metanephrine and
fractionated catecholamine measurements are
clinically acceptable than fractionated plasma
free metanephrine values.
o Fractionated plasma free metanephrines are
considered the biochemical test of choice in
pediatric population.
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FACTORS THAT CAUSES FALSE POSITIVE RESULTS
 Patients should abstain from caffeinated beverages
and alcohol for 24 hours before testing.
 They should also avoid acetaminophen, tricyclic
antidepressants, phenoxybenzamine, α agonists (e.g.,
methyldopa [Aldomet]), and monoamine oxidase
inhibitors for at least 5 days before testing
 During testing for catecholamines, in addition to the
list of items to avoid when testing for metanephrines,
the patient should avoid nicotine, sympathomimetics
(theophylline, pseudoephedrine), α agonists (e.g.,
albuterol), and levodopa/carbidopa.
 Stressors such as an acute myocardial infarction,
congestive heart failure, surgery, and acute
cerebrovascular accident are all associated with
elevated levels of catecholamines.
 Plasma normetanephrine concentrations increase
with age; as a result, older adult patients are
particularly susceptible to having false-positive tests.
 10% to 20% are familial
 Genetic testing <50 y/o, physical traits suggestive of
one of the familial disorders, multifocal disease, or a
positive family history.
 Tests:
o Assaying for mutations in the gene for menin in
MEN 1
o RET oncogene in MEN 2A (Sipple’s disease)
and MEN 2B
o neurofibromin in NF-1
o VHL in von Hippel–Lindau syndrome
 Familial paragangliomas are associated with various
defects in the gene for succinate dehydrogenase
 Pretest and posttest counseling is mandatory
CHROMOGRANIN A (CGA)
 stored and secreted along with the catecholamines
from the adrenal medulla and sympathetic nervous
system.
 elevated in more than 80% of pheochromocytomas
 high sensitivity of 86%; poor diagnostic specificity
o kidneys play a major role in the clearance of
CgA from the circulation
o mild degrees of renal impairment can lead to
significant increases in serum concentration of
CgA
 Major use: Postoperative monitoring for recurrence of
these tumors.
NEUROBLASTOMA
 Neural Crest in origin, arising within adrenal glands/
sympathetic chain.
 Second most common solid malignant tumor in
childhood- (3-5yrs old)
 Symptoms relate primarily to tumor mass than
hypertension which is mild or absent.
 At time of diagnosis
o -70% have distant metastasis
o (about 90% have elevated urinary HVA
“homovanillic acid” levels)
o (almost 75% have increased urinary
vanilmandelic acid (VMA)levels)
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 Both test for HVA and VMA must be ordered for
screening.
o In healthy children until age of 15 VMA and
metanephrines usually higher than adults.
o Metanephrines also can be elevated in
neuroblastoma but not sensitive measure of
residual tumor.
o HVA is increased in familial dysautonomia
(Riley-Day syndrome) and pheochromocytoma
MINERALOCORTICOID AXIS
ALDOSTERONE
 Chief mineralocorticoid that promotes reabsorption of
Na+ and H2O by the kidney to help maintain blood
pressure and tonicity.
 May respond to acute changes in ACTH
 Under the control of the Renin-Angiotensin System
 Expression of the enzyme CYP11B2 (aldosterone
synthase) is site-specific to the zona glomerulosa.
 11-deoxycorticosterone (DOC) and 11-deoxycortisol
as well as precursor molecules to aldosterone can be
synthesized in both zona glomerulosa and zona
fasciculata.
 Zona fasciculata - responsible for the synthesis and
secretion of glucocorticoids
 Zona reticularis - produces androgens and estrogens
CORTISOL
 Key glucocorticoid that regulates its own secretion
through negative feedback from HPA axis
 Inhibits corticotropin-releasing hormone (CRH) from
hypothalamus
 Inhibits ACTH release from pituitary gland
 It is needed in times of stress to maintain blood
pressure and blood sugar, and to prevent shock.
 CORTICOSTERONE also possesses a glucocorticoid
activity
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CONGENITAL DISORDERS OF ADRENAL CORTICAL
ENZYME DEFICIENCIES
 The hormones of the adrenal cortex are steroid
derivatives, synthesized from low-density lipoprotein
(LDL) cholesterol.
 LDL is delivered to the adrenals where it is taken up
by LDL receptors.
 Steroid acute regulatory protein (StAR) shuttles LDL
across the mitochondrial membrane, where it begins
its journey down the steroidogenic pathway
 The enzymes that catalyze these synthetic reactions
are of four general types:
o Hydroxylases
o Dehydrogenases
o Desmolases
o Isomerases
 Because most of the inborn errors of metabolism
affecting steroid hormone synthesis in the adrenal
cortex involve de ciencies of hydroxylases, they
constitute the most clinically important group of
enzymes.
 At least eight different metabolic defects in the
synthesis of cortisol and aldosterone have been
described, each characterized by a deficiency of a
specific adrenal enzyme.
 A vast majority of these enzymatic deficiencies are
inherited as an autosomal recessive trait with variable
degrees of penetrance. Those enzymatic defects that
uniquely affect the biosynthesis of cortisol are
grouped together under the rubric of congenital
adrenal hyperplasia (CAH).
 These five enzymes are P450scc (defect in StAR), 3-
β-hydroxysteroid dehydrogenase, 21-hydroxylase, 11-
hydroxylase, and 17-hydroxylase
 In CAH, defects in the enzymes necessary for cortisol
production lead to cortisol deficiency; cortisol
deficiency results in disinhibition of negative feedback
on CRH and ACTH production.
 CRH and ACTH levels rise, inducing adrenal
hyperplasia and a forward push in steroidogenesis, as
the body tries to compensate and normalize cortisol
production.
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 Not only does this result in a buildup of hormonal
precursors directly preceding the enzymatic defect, it
also causes massive shunting of these precursors
down the remaining functional pathways.
 Symptoms range from shock, salt-wasting, and
anomalous sexual development in infancy, to
hirsutism and infertility in the adult.
*appendix
 The diagnosis is made by measuring the various
serum hormone levels and assessing which steroids
are produced in excess and which are deficient, and
by calculating the precursor/product ratio and
comparing these results to age- and sex-matched
normative data.
 If hormone levels return as borderline, but the clinical
suspicion for CAH remains high, the steroid levels
should be remeasured 60 minutes after intravenous
administration of 0.25 mg of ACTH. ACTH drives
steroidogenesis forward, accentuating the block.
 CAH is categorized according to severity of disease
into classic (neonatal, severe) and nonclassic (late-
onset, cryptogenic) forms.
 The classic form is further subdivided into salt-losing
and non–salt-losing (simple virilizing) variants.
21-HYDROXYLASE DEFICIENCY
 The most common cause of CAH (95% of all cases)
 21-hydroxylase (aka CYP21, CYP21A2, P450c21)
o Located in mitochondrial endoplasmic reticulum
 Screening:
o Capillary heel blood on paper filter disks
 Classic Form
o Detected in 1 in 16,000 live births
 Non-Classic Form
o 0.2% of the general Caucasian population
 Dysplasia of medulla
 Catecholamine hyposecretion
 Degree of medullary impairment: biomarker for CAH
severity
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Therefore, a deficiency in 21-OH leads to an increase in:
17-OHP in serum
Pregnanetriol in urine
Clinical symptoms depend on severity of the deficiency.
CLASSIC FORM VS NON-CLASSIC FORM
CLASSIC FORM (CYP21A1)
 Presents during newborn period or early childhood
 Adrenal insufficiency and virilization
 May or may not have salt wasting
 In a study of 38 children:
o 40%-80% lowering of plasma epinephrine,
metanephrine, and urinary epinephrine levels.
 In another study:
o Significant decrease in catecholamine response
to exercise that was unaffected by the
administration of stress doses of glucocorticoid
NON-CLASSIC FORM
 Late childhood = early adrenarche
 Young adulthood = hirsutism, amenorrhea, and
infertility
 In females: may present as PCOS
 In males: precocious puberty, adrenal rests within the
testes, and infertility
DIAGNOSIS
PRENATAL DIAGNOSIS
 Suppressive treatment with steroids can abrogate the
development of virilization of the female fetus.
 Measuring 17-OHP in amniotic fluid
 Genotyping cells obtained by chorionic villous
sampling
NEONATAL SCREENING
 Mandatory in the US
 Measuring 17-OHP (using automated time resolved
dissociation-enhanced lanthanide fluoroimmunoassay
[DELFIA])
 Genotyping blood from heel stick and collected on
filter paper
 CYP21 (CYP21A2) >>>> Located on
 CYP21P (CYP21A) >>>> Chromosome 6
 CYP21 is active
 Mutations in CYP21P interfere with normal gene
expression
 These mutations are identified by PCR and Southern
blotting on chorionic villous samples
 In the newborn with salt wasting, unstimulated 17-
OHP levels are typically greater than 8000 ng/dL,
rising to 100,000 ng/dL (3000 nmol/L) following
administration of ACTH
 Levels in the simple virilizing variant range from
10,000 to 30,000 ng/dL (300 to 1000 nmol/L).
 Those with nonclassic disease typically have 17-OHP
levels ranging from 1500 to 10,000 ng/dL (50 to 300
nmol/L) (New et al, 1983)
 The diagnosis can be confirmed by comparing serum
17-OHP levels before and 60 minutes after
administration of 0.25 mg ACTH (Cortrosyn).
DIFFERENT VALUES
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 Normal:
o Post ACTH 17-OHP <330 ng/dL
 Heterozygote Carrier
o 330-1000 ng/dL
 Non-Classic CAH
o >2000 ng/dL
MINERALOCORTICOID REPLACEMENT THERAPY
 In children:
o Attainment of normal growth
o Attainment of normal weight
o Attain normal pubertal development
o Optimization of final adult height
 In adults:
o Lessening of signs of virilization
o Resumption of fertility
 Normalization of 17-OHP should not be attempted
because it requires supraphysiologic levels if
glucocorticoids and may result in Cushing’s
Syndrome
 21-Oh causes varying degrees of hypoaldosteronism
 This is apparent due to elevated plasma renin activity
(PRA) and decreased aldosterone/PRA ration
 The goal of mineralocorticoid replacement therapy is
to normalize the plasma renin activity
11-β-HYDROXYLASE DEFICIENCY
 The second most common enzyme deficiency of the
adrenal cortex,
 A defect in this enzyme blocks the final conversion of
11-deoxycortisol to cortisol and DOC to corticosterone
 11-OH deficiency is an autosomal recessive disorder
caused by mutations of the genes CYP11B1 and
CYP11B2, on chromosome 8q21q22
DIAGNOSIS
 In the neonate, high basal and a high ACTH-
stimulated 11-deoxycortisol, Concentrations of urine
tetrahydro-11-deoxycortisol are also elevated
 Childhood and in young adults, the diagnosis of 11-
OH deficiency is made by the presence of elevated
early-morning and ACTH-stimulated serum levels of
11-deoxycortisol that are more than three times the
upper limit for age-matched normals.
 Levels of DOC and adrenal androgens
(androstenedione, dehydroepiandrosterone, and
dehydroepiandrosterone sulfate) are also elevated.
 Plasma renin activity and aldosterone are often
suppressed as a result of salt and water retention
induced by elevations of DOC.
 Prenatal diagnosis of 11-OH deficiency is made by
measuring levels of tetrahydro-11-deoxycortisol (THS)
in the maternal urine or amniotic fluid. These levels
begin to rise in the first trimester.
 Pathologically, the adrenal cortex shows marked
accumulation of cholesterol and other lipids, which is
the primary distinguishing feature from congenital
adrenal hypoplasi
3-β-HYDROXYSTEROID DEHYDROGENASE
DEFICIENCY
 3-B-HSD catalyze second enzymatic step in
steroidogenesis
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 Deficiency Leads to blockage in the conversion of Δ5
to Δ4 steroids
 Increase Δ5 steroid but Δ4 remains normal
 Manifestations:
 Glucocorticoid deficiency due with or without salt
wasting
 Incomplete masculinization for males
 Females may have ambiguous genitalia
 Late onset: polycystic ovarian disease
 Diagnosis
o Previous: examine basal and ACTH stimulated
Δ4/Δ5 ratios in urine or blood
o Revised: genotypic studies, ACTH stimulated
Δ5-17 P levels and Δ5-17 P/cotisol ratios( shown
best indices in diagnosing 3-B-HSD deficiency)
 Treatment:
o Glucocorticoids, mineralocorticoids as well as
sex steroids
17-HYDROXLASE DEFICIENCY
 17-Hydroxylase (CYP17, P450c17) - expressed in the
adrenal glands and the gonads and encodes two
enzymes:
o 17α-hydroxylase - catalyzes the conversion of
pregnenolone and progesterone to their
respective 17-OH derivatives
o 17,20-lyase - converts 17-OH pregnenolone to
dihydroepiandosterone, and 17-OH
progesterone to androstenedione
 CYP17 deficiency - blocks the conversion of
pregnenolone and progesterone to the 17-hydroxy
derivatives, causing shunting from testosterone and
cortisol synthesis to aldosterone.
 Patients develop hypertension and hypokalemic
alkalosis in association with incomplete
masculinization (in the male) and decreased
testosterone and cortisol levels.
 17-OH deficiency Diagnosis: high DOC,
pregnenolone, and progesterone levels, along with
decreased urinary 17-ketosteroids and 17-
hydroxycorticosteroids.
 Gene associated with this condition (CYP17) - located
on chromosome 10q, the same gene as 17,20-
desmolase
CONGENITAL LIPID ADRENAL HYPERPLASIA
 Most severe form of Congenital Adrenal hyperplasia
 Condition in which all the synthesis of all gonadal and
adrenal cortical steroids is markedly impaired.
 Maybe caused by a defect in the StAR or the P450
side chain cleavage (scc) which is located on
chromosome 8p11, controls the rate-limiting step in
steroidogenesis.
 It is responsible for the shuttling of cholesterol from
the outer to the inner mitochondrial membrane.
 20,22-Desmolase (CYP11A1, P450scc) converts
cholesterol to pregnenolone
 Pathologically, the adrenal cortex shows marked
accumulation of cholesterol and other lipids, which is
the primary distinguishing feature from congenital
adrenal hypoplasia.
 Clinical presentation:
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o Severe adrenal insufficiency with hypotension,
salt wasting, and feminization of external
genitalia in males.
o Occasionally, females may not present until
the onset of puberty.
 Diagnosis:
o Presence of extremely low cortisol and
aldosterone concentrations and elevated
ACTH and plasma renin activity.
ALDOSTERONE SYNTHETASE DEFICIENCY
 Aldosterone Synthetase (CYP11B2) =
 Final Step in Steroid synthetic pathway -->
Aldosterone
11-Hydroxylation of DOC → Corticosterone →
18-hydroxycortisone ⇏ Aldosterone
 Causes Salt wasting, hyperkalemia and metabolic
acidosis
 Dx=Demonstrating the presence of metabolites of
Corticosterone and 11-deoxycorticosterone in urine,
elevated DOC, and [↓corticosterone,18-
hydroxycorticosterone or aldosterone in the serum
CORTISOL AND GLUCOCORTICOIDS
 The adrenal cortex secretes cortisol in response to
ACTH, a diurnal rhythm, and stress.
 ACTH, synthesized in the adenohypophysis, is
formed from the cleavage of a much larger precursor
molecule: POMC.
o cleavage of POMC releases β-LPH, which in
turn is cleaved to yield γ-LPH and β-endorphin.
 Within the ACTH sequence are α-MSH and the
corticotropin-like intermediate lobe protein.
 Endorphins, which act on neurons in the brain,
constitute a distinct peptidergic system related to pain
perception. The significance of this remains unknown.
 Occasionally, POMC is incompletely processed;
leading to formation of other forms of ACTH that
usually have little biological activity.
 These forms may predominate in malignant
conditions such as ectopic production by primary or
metastatic lung cancer, and in some patients with
Nelson’s syndrome
 Defects in POMC cleavage enzymes may also be
responsible for the formation of rare forms of isolated
ACTH deficiency
 Nelson’s syndrome: a disorder characterized by the
occurrence of a pituitary tumor and skin
hyperpigmentation following bilateral adrenalectomy.
 ACTH secretion is mediated by CRH and AVP
(important)
o Other factors: atrial natriuretic factor (ANF),
angiotensin II, IL-6, IL-1, and tumor necrosis
factor-α.
 CRH is synthesized and released from the
hypothalamus
o acts to stimulate the synthesis and release of
ACTH from the pituitary gland.
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o CRH is released in a circadian pattern and in
response to physiologic stimuli such as stress
and hypoglycemia.
 The HPA axis consists of various feedback loops that
control cortisol synthesis and secretion. When plasma
cortisol increases, it suppresses the release of CRH,
ACTH, and AVP, which, in turn, leads to lowering of
the cortisol level.
 Conversely, when serum cortisol reaches a nadir, the
hypothalamus and pituitary gland respond by
increasing CRH and ACTH production, leading to
stimulation of cortisol formation and secretion.
o By this mechanism, ACTH and cortisol control
the concentration of each other within a very
narrow range, and a small change in one results
in a concomitant change in the other.
 When the adrenal gland is unable to respond to
ACTH
o cortisol levels are low and ACTH levels are high.
 Damage to the hypothalamus is also associated with
low ACTH and cortisol levels;
o Synthetic forms of CRH are used in testing the
anterior pituitary gland’s reserve of ACTH by
comparing plasma ACTH and cortisol before and
1 hour after CRH stimulation
 In those conditions in which the pituitary gland is
destroyed, ACTH is not formed and cortisol levels are
consequently low.
 testing with CRH may permit the distinction between
these two entities
 If the lesion is in the hypothalamus, after a time delay,
ACTH levels rise following CRH administration. If it is
in the pituitary gland, no significant ACTH response
occurs. With primary adrenal insufficiency,
administration of CRH causes a further rise in an
already elevated ACTH level, but little or no rise in
cortisol level.
 If the HPA axis is interrupted by the administration of
large quantities of exogenous glucocorticoids, they
will exert an inhibitory effect on the hypothalamus and
pituitary gland, suppressing CRH and ACTH
secretion.
 If this suppression continues = leads to atrophy of the
adrenal glands;
o the HPA axis unable to secrete cortisol in times
of stress.
o The HPA axis can fully recover after tapering off
of steroids.
 The second influence on plasma cortisol levels is the
diurnal pattern, which is due to the circadian pattern
of ACTH release.
o Major increases in secretion occur at between 4
am and 8 am, followed by a decrease in ACTH
during the rest of the day.
o the lowest ACTH concentrations are found
shortly after midnight.
 Sudden changes in sleep-wake patterns have little
effect on the diurnal pattern but permanent changes
in daily sleep habits result in a gradual change in
diurnal secretory patterns.
 Superimposed on the circadian periodicity is an
ultradian rhythm of 10 to 18 secretory bursts per 24
hours
L8

 The third important influence on cortisol secretion is
stress.
o Stimuli such as surgical trauma, pyrogens,
hypoglycemia, and hemorrhage are capable of
bringing about an acute increase in ACTH and
cortisol secretion.
 Response to stress may be absent or decreased in
magnitude in patients in whom large doses of steroids
have been administered for some time.
 The initiation of any stress response is dependent on
an intact nervous system.
 Evidence suggests that the stress response of cortisol
is mediated through excitatory and inhibitory inputs
that integrate at the level of the hypothalamus and
modulate CRH secretion.
o Cortisol levels also rise after meals, especially
those high in protein; it also rises in depression
 For example, trauma normally results in the acute
release of ACTH and cortisol; however, in patients
with spinal cord transections, the normal transmission
of neurologic stimuli is interrupted, and as a result, the
same trauma applied to an extremity will not elicit any
ACTH or cortisol response.
 Most disorders of cortisol secretion can be classified
by the patterns of response of the following three
hormones to suppression and stimulation: ACTH,
plasma cortisol, and urinary free cortisol
LABORATORY MANAGEMENT OF ACTH
 Plasma ACTH is measured using a two-site
immunoradiometric assay or an
immunochemiluminometric assay
 To prevent degradation of ACTH, it is best to collect
the sample in a pre-chilled EDTA lavender-top tube
 Specimen should be kept in an ice bath and
processed as soon as possible
 Following centrifugation in a refrigerated centrifuge,
the specimen should be separated, transferred to a
plastic tube, and kept frozen at −20° C until time of
analysis
 Normal: 2-12 pmol/L (9-52 pg/mL) between 7am and
10am
 Plasma ACTH is useful for distinguishing between
primary (adrenal), secondary (pituitary) or tertiary
(hypothalamic) adrenal insufficiency
 Primary adrenal insufficiency
o Low cortisol concentration
o Increased ACTH
 Secondary or Tertiary adrenal insufficiency
o Both ACTH and cortisol are low
 ACTH best discriminates between healthy individuals
and those with adrenal insufficiency when collected
between 8am and 10am
 ACTH is less useful in diseases of cortisol excess
 Measurement of ACTH by plasma extraction may be
useful in distinguishing patients with cancer-related
syndromes or ACTH-secreting tumors from those with
Cushing’s disease, as the former entities are more
likely to produce these other forms of ACTH
 ACTH measurement using selective venous sampling
has been proven useful in localization of the lesion
when ACTH-secreting neoplasms are occult
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 ACTH secretion is inhibited and levels are low or
undetectable in patients with increased levels of
circulating glucocorticoids due to an adrenal adenoma
or carcinoma, and those taking steroids
 In patients with pituitary-induced adrenal hyperplasia,
plasma ACTH may be at or above the upper
reference interval at 9 am, and fail to show the
expected fall after midnight
 Another use of ACTH assays is in the determination
of adequacy of cortisol replacement in congenital
adrenal hyperplasia
 When replacement therapy is optimal, ACTH values
are similar to those seen in a reference population
PLASMA CORTICOTROPIN-RELEASING HORMONE
 CRH measurements are performed by: liquid
chromatography and tandem mass spectrometry
 Two forms: free and bound to CRH-binging protein
 Reference range for men and pregnant women: <34
pg/ml
 CRH is increased in Cushing’s Syndrome due to
ectopic production of CRH
 CRH-binding protein: increases during pregnancy
o 1st trimester: <40 pg/ml
o 2nd trimester: <153 pg/ml
o 3rd trimester: <847 pg/ml
SERUM CORTISOL MEASUERMENTS
 90% of circulating cortisol is bound to serum protein
 Fluorometric assay: earliest and simplest method to
determine serum cortisol concentration
 Antibody-based Immunoassays: more specific
method for cortisol estimation
o Advantage: smaller specimen volume and more
rapid turnaround time
o Disadvantage: antibodies showed large degree
of cross reactivity resulting in elevated cortisol
concentration
 High performance liquid chromatography with tandem
mass spectrometry: has an ultimate specificity and it
is where serum cortisol is collected in a no-additive
tube.
 Reference values: M and F = 5-25 ug/dL at 8am to
10am (peak), dropping to about 3-12 ug/dL by 4pm
 Its diurnal and ultradian pattern of secretion are most
useful when evaluated in the context of dynamic
manipulation
SALIVARY CORTISOL
 Salivary cortisol is highly stable and easy to collect.
o Useful tool for screening and for diagnosing
instances of increased cortisol secretion.
 It does not appear to be affected by the rate of saliva
production.
o Reflect a change in serum cortisol in as rapid as
few minutes.
 Due to alteration in circadian rhythm: not an
appropriate test for shift workers or those w/ highly
variable bed times.
 Measurement of bedtime salivary cortisol was a
practical and accurate screening test for the diagnosis
of Cushing’s syndrome.
L8

 Patient must avoid tobacco on the day of specimen
collection.
URINARY FREE CORTISOL MEASUREMENTS
 Only 1% of the total adrenal secretion appears in the
urine as cortisol, this fraction provides a valuable aid
in the diagnosis of adrenal disease.
 In the kidney, glomerular filtration of free cortisol
(unbound to CBG and albumin) is followed by passive
tubular reabsorption without a demonstrable
reabsorption maximum.
 Unlike serum cortisol, UFC is not affected by
conditions and medications that alter CBG and
albumin concentrations.
o Thus making it easier to differentiate patients
with adrenal hyperfunction from a reference
population.
 UFC is the best specimen to submit because it
provides an integrated profile of total cortisol secretion
over a 24-hour period, which is most helpful in those
with sporadic excess cortisol production.
 Reliability of the test may be further improved by
submitting urine collected over 2 or 3 days
 UFC is unreliable when CrCl is <20 mL/min, and is of
reduced reliability when CrCl is <60 mL/min
 Increased serum concentration of transcortin during
pregnancy and with estrogen therapy results in
increased serum cortisol levels.
 Conditions in which spuriously elevated levels occur
include starvation, use of topical steroids, and
hydration in the form of water loading.
 Method
o HPLC (High-performance liquid
chromatography) with mass spectrometry is
considered the current reference method for
measuring UFC; it has diagnostic sensitivity of
100% and specificity of 98% for distinguishing
patients with Cushing’s syndrome from normal
individuals (Rudd, 1985).
o Normal range varies according to the assay
technique used; values that are four times the
upper reference limit are diagnostic for
Cushing’s syndrome.
o A low urinary free cortisol value is suggestive of
adrenal hypofunction; however, because there
is great overlap with the normal reference
interval, this test is not used to make the
diagnosis of adrenal insufficiency.
HYPERCORTISOLISM: CUSHING’S SYNDROME
 Results from prolonged exposure to elevated
concentrations of glucocorticoids.
o Endogenous: secreted by adrenal zona
fasciculata.
o Exogenous: (Most common) Pharmacological
administration of steroids.
 Patients w/ severe forms of syndrome presents:
Striae, Facial plethora, Proximal muscle weakness,
Stereotypical accumulation of fat.
 Signs and Symptoms: fatigue, weight gain, obesity,
diabetes, hypertension.
 Laboratory findings:
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EVALUATION OF ENDOCRINE FUNCTION 8.05
o ↑ serum cortisol, urinary free cortisol, midnight
salivary cortisol
o Loss of circadian rhythm of ACTH and cortisol.
o Loss of suppression of cortisol production by
administration of dexamethasone.
 Most commonly iatrogenic in origin.
o May also be due to: primary adrenal malignancy,
ectopic production of CRH or ACTH by a tumor.
o CUSHING’S DISEASE: Hypercortisolism due to
an ACTH-secreting pituitary adenoma.
 CUSHING SYNDROME: Global term for
hypercortisolemia.
o Most commonly iatrogenic in origin.
o May also be due to: primary adrenal malignancy,
ectopic production of CRH or ACTH by a tumor.
24-HOUR URINARY FREE CORTISOL
 reflection of the unbound circulating cortisol that is
freely filtered by the glomerulus
 unaffected by the level of circulating CBG
 Best specificity - HPLC or gas chromatography
coupled with tandem mass spectrometry
 Values greater than four times the upper limit of
normal are diagnostic of Cushing’s syndrome
o From 10% to 15% of patients with Cushing’s
syndrome will have at least one in four 24-hour
urine collections for free cortisol return as normal
o If cortisol excretion is normal but clinical
suspicion is high, the study should be repeated
or a different screening method should be used
 Milder elevations of UFC can be seen in pseudo-
Cushing’s and during normal pregnancy
OVERNIGHT DEXAMETHASONE SUPPRESION TEST
patient
patient takes
takes 1
1 mg
mg of
of plasma
plasma cortisol
cortisol is
is drawn
drawn
dexamethasone orally the following morning
between
between the
the hours
hours of
of 11
11 between
between 8 8 am
am and
and 9 9 am,
am,
pm and 12 midnight respectively
 original criterion for an abnormal response was failure
to suppress the morning cortisol level to <5 μg/ dL
(138 nmol/L);
o revised downward to <1.8 μg/dL (50 nmol/L)
o provides a greater than 95 % sensitivity and 80%
specificity and minimize the number of false-
positive results
 False positivity can be due to:
o Dexamethasone was taken too early
o Patient was on phenobarbital, phenytoin, or
another medication known to accelerate the
metabolism of dexamethasone
o Malabsorption; alcoholism; or morbid obesity
o Pregnancy and drugs such as estrogen, which
increase serum transcortin (aka CBG), may also
result in elevated cortisol levels
MIDNIGHT SALIVARY CORTISOL
L8
 EVALUATION OF ENDOCRINE FUNCTION 8.05

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pledget for 2 The sample
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and then analyzed
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place the The sample is
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 High diagnostic sensitivity and specificity, and has
been demonstrated to have an excellent correlation
with the plasma free cortisol concentration
(Papanicolaou et al, 2002; Yaneva et al, 2004; Alwani
et al, 2014)
 Using a cutoff of 9.3 nmol/L on a commercial ELISA
kit, MSC had a sensitivity of 100% specificity of 83%,
PPV of 94%, and NPV of 100% (Alwani et al, 2014)
 It performs slightly better than the 24-hour UFC in
distinguishing patients with Cushing’s syndrome from
obese subjects (Yaneva et al, 2004)
 Late-night (11 pm to 12 am) salivary cortisol (LNSC)
 Falsely elevated - smokers and users of chewing
tobacco, refraining from use for 24 hours prior to
collection is recommended. PSEUDO-CUSHING’S SYNDROME
 Excess activity of the hypothalamic-pituitary axis,
SCREENING IN SPECIAL POPULATIONS similar to the seen in pituitary Cushing’s syndrome
 Pregnancy – UFC  Seen in some patients with alcoholism, major
o UFC levels normally rise during the second and depression, and obesity; resolution of the
third trimesters, therefore UFC values greater underlying problem results in the normalization of the
than three times the upper limit of normal are HPA axis
considered consistent with Cushing’s syndrome  Serum cortisol above 7.5 ug/dL at midnight
 Renal Failure – 1-mg DST discriminates Cushing’s syndrome from pseudo-
o Because of the decrease in cortisol filtration with Cushing’s with 100% specificity
declining renal function,  More definitive test: combined dexamethasone-
 Suspected of cyclic Cushing’s – serial UFC or MSC oCRH test
o diagnostic thresholds for MSC during pregnancy o o-CRH is infused 2 hours after a low-dose
and in renal disease remain to be established dexamethasone test
 For an incidentally discovered adrenal mass, the 1- o Plasma cortisol concentration > 38 nmol/L at 15
mg DST has been shown to be superior to the UFC minutes following administration of oCRH =
and MSC identifies all cases of Cushing’s syndrome and
all cases of pseudo-Cushing’s with specificity,
CONFIRMATORY TESTS sensitivity, and diagnostic accuracy of 100%
 Repeat testing in 3 months - equivocal screening ADRENAL INSUFFICIENCY
 Midnight plasma cortisol - 100%specificity  Categorized according to dysfunction with in HPA
o 48 Hour hospital admission axis: Primary (adrenal), Secondary (pituitary) and
Tertiary(hypothalamic)
o IV insertion before 10 PM
 Major dysfunction: mineralocorticoid deficiency
o Blood is drawn while patient is sleeping at
 Most common cause of central adrenal insufficiency:
midnight
o HPA axis suppression due to prolonged steroid
o Cortisol>7.5ug/dL - diagnostic of Cushing’s
 2-day low dose DST (LDDST) PRIMARY ADRENAL INSUFFICIENCY (ADDISON’S
o 2 baseline 24-hr urine UFC is measured DISEASE)
o Administration of 0.5mg oral dexamethasone  Most commonly due to : Autoimmune adrenalitis
every 6hrs for 2 days  Other causes:
o 24-hr urine UFC is measured o TB, Granulomatous disorders, Metastatic
o Normal: decrease in UFC to <10ug per 24hrs disease, hemorrhage, HIV, acquired
(97-100% specificity) & suppresion of plasma immunodeficiency and infection
cortisol to <1.8ug/dL (95% specificity)

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16 | 22 CERVANTES DIN
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PRIMARY ADRENAL INSUFFICIENCY (ADDISON’S
DISEASE)
 Basal Hormone Measurements
o Cortisol levels are diurnal
o Cortisol of less than 3 ug/dl at 8 am to 9 am
indicated adrenal insufficiency and needs further
testing.
ACTH
 Produced by the pituitary gland
 Stimulates the adrenal gland, which sit atop the
kidneys, to release two hormones: Cortisol and
Adrenaline (epinephrine)
ACTH STIMULATION TEST
 Aka cosyntropin test
 Done to test if the adrenal glands aren’t functioning
properly
 Measures how your adrenal glands react to ACTH in
your blood
 Used to diagnose adrenal insufficiency known as
Addison’s disease
 Requires to receive an injection of cosyntropin, a
synthetic portion of ACTH
 Two blood samples drawn: one before injection and
one after injection
HOW IS AN ACTH (COSYNTROPIN) STIMULATION
TEST PERFORMED?
 Take a blood sample. This blood sample will measure
your blood cortisol levels. You can use this sample as
a baseline against which to compare the results of the
second blood test.
 You will receive an injection of cosyntropin, a
synthetic portion of ACTH. This hormone should
trigger the adrenal glands to produce cortisol. You will
then wait for about an hour while your body reacts to
the cosyntropin injection.
 Take a second blood sample after this first hour is up.
This sample will reflect your cortisol levels after your
body has had time to react to the injection.
 Both of your blood samples will be tested for their
cortisol levels.
OVERNIGHT METYRAPONE TEST
 What is Metyrapone?
o Metyrapone inhibits 11-β-hydroxylase,
preventing the conversion of 11-deoxycortisol
(compound S) to cortisol.
o Under normal circumstances, the metyrapone-
induced drop in cortisol is detected at the level of
the hypothalamus and pituitary gland; a
compensatory increase in CRH and ACTH
secretion occurs, stimulating steroidogenesis
and leading to a buildup of 11-deoxycortisol, the
hormone preceding the block.
 Measure the ability of pituitary gland to release ACTH
in response to decrease cortisol levels
 It is done when Metyrapone 30 mg/kg is administered
orally at midnight; blood for cortisol and 11-
deoxycortisol is collected the following morning at 8
am.
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EVALUATION OF ENDOCRINE FUNCTION 8.05
 A rise in 11-deoxycortisol to greater than 7 μg/dL (200
nmol/L) is NORMAL.
 An ABNORMAL RESPONSE is defined by 11-
deoxycortisol less than 7 μg/dL accompanied by
cortisol less than 5 μg/dL.
 Risk in factor:
o Addisonian Crisis in those with Central
Hypocortisolism
o Metyrapone is currently only available on a
compassionate basis
CORTICOTROPIN-REALEASING HORMONE TEST
 Used to diagnose hypocortisolism and localize the
site of damage.
 Safe but expensive.
 After blood is drawn for baseline ACTH and cortisol,
100 µg of oCRH is administered intravenously.
 Blood is then collected for cortisol and ACTH every 15
minutes for 60 to 90 minutes.
 A normal response is seen as cortisol greater than 20
µg/dL.
 Basal ACTH levels and their change in response to
oCRH are used to localize the site of pathology.
 Plasma corticotropin usually peaks at 15 to 30
minutes, and cortisol usually peaks at around 30 to 40
minutes post oCRH injection.
 Both the bound and free cortisol concentration show a
progressive rise throughout pregnancy.
 It has been recommended that higher cutoffs be used
to properly interpret the ACTH stimulation results in
pregnancy.
RENIN-ALDOSTERONE AXIS
 Maintain blood pressure within normal limits
 Renin
o Released when there is a drop in renal
hydrostatic pressure, hyponatremia,
hypokalemia, or a decrease in cathecholamines,
angitensin II, or atrial natriuretic hormome
ISOENZYMES OF RENIN
 Renin acts on angiotensinogen, converting it to
angiotensin I.
 ACE converts angiotensin I to angiotensin II
 Angiotensin II
o Responsible for physiologic effects on target
tissue
o Very potent vasoconstrictor
RENIN RELEASE
 Rate limiting step in Renin-Aldosterone axis.
 Dependent on changes in effective plasma volume
 Dependent on tubular reabsorption of Na+
 *Renin secretion is inhibited by hyperkalemia
ALDOSTERONE AND GLUCOCORTICOIDS
 Mineralocorticoid receptor
 Cause reabsorption of Na+, leading to increase
intravascular volume
 Causes renal K+ loss
L8

 Development of vasculopathy
 Stimulates gene for collagen synthesis, TGF,
inflammatory factors, plasminogen activator inhibitor
type 1.
 Aldosterone synthetase(CYP11B2)
o Converts corticosterone to aldosterone
 Major stimuli for aldosterone secretion:
o Angiotensin II and hyperkalemia
 *ACTH much weaker stimulus
 Inhibits aldosterone synthesis:
o Somatostatin, atrial natriuretic hormone,
dopamine, and heparin
 Cortisol
o Circulates at much higher concentration than
aldosterone
o Does not normally function as a major
mineralocorticoid
11-B-HYDROXYSTEROID DEHYDROGENASE II
 11-HSD-2, aka CYP11B2
 Expressed by mineralocorticoid target cells in
collecting tubules
 Acts to convert cortisol to cortisone
 Cortisone
o An in active metabolite
RENIN AND HYPERTENSION
 Essential hypertension can be classified on the
basis of renin measurements as high, low, or normal
renin, and that drug selection
 Renin profiling has shown renin to be an independent
risk factor for myocardial infarction
 Increased aldosterone (secondary aldosteronism)
may contribute significantly to the symptoms and
course of high-renin hypertension
CAUSES OF HYPERTENSION SOOCIATED WITH HIGH
LEVELS OF PLASMA RENIN
1. Renin-secreting tumors
o extremely rare and can be difficult to diagnose.
o can be benign or malignant, renal or extrarenal.
o most common location is in the juxtaglomerular
apparatus
2. Malignant hypertension
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EVALUATION OF ENDOCRINE FUNCTION 8.05
o is also associated with high plasma renin activity
and aldosterone.
3. Renal vascular hypertension,
o the renin and aldosterone levels are also
elevated.
o in contradistinction to malignant hypertension,
treatment with ACEs usually results in further
deterioration of renal function.
4. Chronic renal failure
o almost any renin level can be expected. A small
number of hypertensive patients on dialysis
have intractable, accelerated hypertension.
a. Renal transplant patients
 elevated plasma renin may be
indicative of renal ischemia and
rejection.
5. Cushing’s syndrome
o Systemic hypertension occurs
o Some patients have increased plasma renin and
renin substrate.
o Other Cushing’s syndrome patients have
hypokalemia associated with secretion of a
minor mineralocorticoid (such as DOC or
corticosterone) causes hypertension
o other patients, severely elevated cortisol levels
overwhelm the capacity of 11-hydroxysteroid
dehydrogenase (11-HSD
6. Treatment with medications
o diuretics, vasodilators, or other antihypertensive.
o Hormonal agents such as glucocorticoids,
estrogen-containing oral contraceptives
o Even though plasma renin activity, aldosterone,
and urinary sodium excretion may be normal in
60% of hypertensive patients, evidence indicates
that renin-angiotensin plays a significant part in
normal-renin hypertension.
 Primary aldosteronism is uncommon compared with
renin hypertension. It is characterized by (1) systolic
and diastolic hypertension caused
L8

 by oversecretion of aldosterone by an adrenal
adenoma (aldosteroneproducing adenoma [APA]) or
hyperplasia (idiopathic hyperaldosteronism [IHA]), (2)
low renin or a high aldosterone/renin ratio (>50), (3)
renal potassium wasting, and (4) sodium retention.
 A low-salt diet (<2 g/day), stress, upright posture, and
diuretics all increase plasma aldosterone, whereas a
high-salt diet and lying in a supine position suppress
aldosterone secretion in healthy subjects.
PRIMARY HYPERALDOSTERONISM – SCREENING AND
CONFIRMATION TESTS
 Preferred screening test is “Plasma Aldosterone
Concentration/Plasma Renin Activity Ratio (PAC/PRA
ratio)”
o A ratio >30 is suggestive of primary
hyperaldosteronism, and a value of 50 can be
diagnostic.
o Special Considerations:
 4-6 weeks prior the test spironolactone and
eplerenone should be discontinued and
other diuretics for at least 2 weeks.
 Hypokalemia should be corrected, and
patient should not be on a sodium-
restricted diet.
 The diagnosis of hyperaldosteronism is confirmed by
one of the four ff confirmatory tests:
o Oral sodium loading test
o Saline infusion test
o Fludrocortisone suppression test
o Captopril challenge test
 The choice of test depends on several factors:
o Patient compliance
o Uncontrolled HPN
o Renal Insufficiency
o CHF
o Expertise
1. Oral sodium loading test
o Done by administering sodium chloride tablets
10-12g daily for 3 days
o On the last day of oral administration, a 24-hour
urine is collected for aldosterone, creatinine, and
sodium.
o An aldosterone >10mg/24hours ff the 3 days of
sodium loading confirms the diagnosis.
2. Saline infusion test
o Done by infusing 2L of 0.9%saline over 4 hours.
o PAC is measured at the end of infusion:
 <5 ng/dL = negative
 5-10ng/dL = considered intermediate and
may be seen with IHA.
 >10ng/dL = positive
3. Fludrocortisone suppression test
o Done by giving:
 0.1mg fludrocortisone orally every 6 hours for
4 days.
 Slow-release potassium chloride
supplements
 Slow-release NaCl and liberilization of
sodium intake.
o On the 4th day an upright plasma cortisol is
measured at 7am and 10am, then PAC and PRA
are measured at 10am.
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EVALUATION OF ENDOCRINE FUNCTION 8.05
o A PAC >6ng/dL with a PRA <1ng/mL/hour
confirms the diagnosis.
4. Captopril challenge test
o Baseline blood is collected for PRA,PAC, and
cortisol then a 25-50mg Captopril is given orally.
PRA, PAC, and cortisol is measured again after
1-2 hours. The patient is kept seated throughout
the entire test.
o In primary hyperaldosteronism, PAC remains
elevated and PRA is suppressed.
DIFFERENTIATING AMONG THE DIFFERENT CAUSES
OF PRIMARY HYPERALDOSTERONISM
 Difference of aldosterone-producing adenoma (APA)
and idiopathic hyperaldosteronism (IHA), is that APA:
o has more extreme blood pressures
o Greater potassium wasting
o Higher natriuretic peptide level
 (+) postural stimulation test (plasma aldosterone:
ambulating rising to less than 130% of the supine
level) has moderate sensitivity and specificity for
ADENOMA (but not for hyperplasia)
 The specificity of CT or MRI correctly categorize
patients as having APA versus IHA
 BILATERAL ADRENAL VEIN SAMPLING is
recommended for >40 yrs old who are contemplating
for surgery. This is bec. hyperplasia may present as a
unilateral adenoma.
o It entails risk but still it is the GOLD STANDARD
for distinguishing adenoma from hyperplasia
o Procedure: pt is administered with ACTH at
50u/hr IV, 30 mins before the test and is
continued throughout the procedure (to mitigate
any stress-induced fluctuations in aldosterone
and maximize the difference in secretion of
aldosterone bet. right and left adrenal glands in
instances of APA)
 CORTISOL-CORRECTED ALDOSTERONE: correct
dilutional effects.
o No gradient is seen in hyperplasia whereas in
APA, the ratio is 5-fold higher on the side with
the adenoma
 Syndrome of inapparent mineralocorticoid excess is
due to dysfunction of 11-B-hydroxysteroid
dehydrogenase type 2.
o Cortisol can function as a potent
mineralocorticoid, binding to the
mineralocorticoid receptor in the kidney with the
same affinity as ALDOSTERONE.
o ELEVATED CORTISOL/CORTISONE CONC.
(24-hr urine) confirms the Dx.
 There are 2 variants of familial hyperaldosteronism
(FH), FH-1 (aka glucocorticoid remedial
hyperaldosteronism-GRA) and it is characterized by 3
H’s: hyperaldosteronism, hyporeninemia and severe
hypertension) and FH-2
o Confirmatory dx for FH-1: normalization of
aldosterone (after 4-6 wks steroid suppression)
or Southern blot or long PCR.
o Unlike FH-1 aldosterone does not suppress in
response to glucocorticoid tx.
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 LIDDLE SYNDROME- rare autosomal dominant 1. Lecture Notes

disorder characterized by hypertension and 2. Lecturer’s PPT
hypokalemia.
o PRA and PAC are both low unlike in primary
hyperaldosteronism
o Dx test: Genetic testing or clinical response to
amiloride or triamterene, and by LACK of
response to dexamethasone or aldosterone
antagonist.

ALDOSTERONE MEASUREMENTS
 An isolated aldosterone measurement with no
attention to patient preparation is of “LITTLE
CLINICAL VALUE”.
 It is difficult to discriminate with certainty between
primary aldosteronism and other forms of
hypertension with the use of PLASMA
ALDOSTERONE MEASUREMENTS alone.
o Even when time of sampling, posture, and
dietary sodium and potassium are controlled.
 Interpretation of the aldosterone level must be done in
the context of a simultaneously collected plasma renin
and serum potassium.

ALDOSTERONE ASSAYS
 Performed on plasma using extraction to remove
aldosterone from plasma proteins,
o followed by chromatography and immunoassay
or, preferably, LC-MS/MS.
 Urine is assayed following acid hydrolysis and
extraction.
 Normative values exist for different age groups,
postures, and sodium intakes.
 HPLC with tandem mass spectrometry affords greater
consistency and is gradually replacing older methods.

RENIN MEASUREMENTS
 Renin is extremely labile and its release is controlled
by many physiologic and pharmacologic variables:
o upright posture
o Diuretic
o Low sodium diets (most potent)
 2 TYPES:
 PRA – most widely used method for determination of
renin.
o bioassay wherein a plasma specimen containing
renin is allowed to react with its substrate
(angiotensinogen) after a specfied period of
time, the reaction is terminated and generated
amgiotensin I is measured by RIA
o endogenous substrate is not eliminated
 PRC – immunoassay directed to the renin molecule
itself
o measured the effect of substrate eliminated

REFERENCES

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CERVANTES

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CERVANTES

22 | 22 CERVANTES DIN
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