2ND SEMESTER

Cchm 312: clinical chemistry 2| Lecture

Instructor: PROF Kimberly ann pulga rmt,mph WEEK 11

ENDOCRINOLOGY 3  Zona Reticularis (15%)

(Midterm, 5th topic) o DHEA- DHEAS
(Dehydroepiandosterone)
hormones
adrenal glands
Adrenal Cortex – outer region
 Also known as the
suprarenal glands a. Cortisol
 They produce steroid b. Aldosterone
hormones and c. Weak androgens
neuropeptides that
help regulate Adrenal Medulla – inner region
metabolism, a. Norepinephrine
immune system, b. Epinephrine
blood pressure and c. Dopamine
also response to
stress and other
essential functions Adrenal cortex
 Pyramid-like shaped located above the kidneys
 Composed of distinct but conjoined glands, the  The outer region of the adrenal gland secreting
outer adrenal cortex (yellow) and the inner the steroid hormone
adrenal medulla (dark mahogany)  Major site of steroid hormone production
o Cortex includes mesenchymal cells o Cholesterol – parent cell of all our
which is differentiated into 3 steroid hormone
structurally and functionally distinct  The secretion of adrenal glucocorticoids and
zones
androgens is regulated by
o Medulla: neural crest cells
Adrenocorticotropic Hormone (ACTH), which
 It has prime effects on blood pressure
is under the control of the hypothalamic
corticotropin-releasing hormone (CRH)
 Mineralocorticoids secretion is controlled by
the Renin Angiotensin Aldosterone System
(RAAS)
 Cortical hormones are composed of CPPP
(cyclopentanoperhydrophenanthrene)

3 layers of adrenal cortex

 Cortex has 3 zones: Zona Glomerulosa (outermost layer) – 10%
 Zona Glomerulosa (10%)
 Principal source of mineralocorticoids
o Mineralocorticoids, aldosterone
o Sodium retention, potassium, acid-base o Responsible for salt regulation
homeostasis
 Zona Fasciculata (75%) Zona Fasciculata (middlemost layer) – 75%
o Glucocorticoids  Site of glucocorticoid synthesis
o Cortisol and corticotisone  Also synthesize unsulfated DHEA

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CCHM 312 | BSMLS 2023-2024 | TRANSCRIBER: SANTOS, S.V | FOR STUDENTS USED ONLY NOT INTENDED FOR SELLING
 2ND SEMESTER
Cchm 312: clinical chemistry 2| Lecture
Instructor: PROF Kimberly ann pulga rmt,mph WEEK 11

Zona Reticularis (innermost layer) – 15% Cortisol

 Produces androstenedione and  Principal glucocorticoid
dehydroepiandrosterone (weak androgens)  Synthesis is regulated by ACTH
 Critical to hemodynamic and glucose
Aldosterone
hemostasis
 Low cytoplasm to nuclear ratio and small o Glucocorticoids maintain blood glucose
nuclei with dense chromatin with by inducing lipolysis and
intermediate lipid inclusions. gluconeogenesis
o They also have anti-inflammatory and
Cortisol immunosuppressive action
 High cytoplasm to nuclear ratio, lipid laden  Mostly bound to glycoprotein (transcortin)
with “foamy” cytoplasm  Stimulates lipolysis and gluconeogenesis in
the liver
DHEAS  The only adrenal hormone that inhibits the
 Lipid deficient but has lipofuscin deposit secretion of ACTH
sharply demarcated cells  Therapeutic agent for Rheumatoid Arthritis
(RA), Systemic Lupus Erythematosus (SLE),
Multiple Sclerosis (MS)
 Secretion is diurnal and is associated with
sleep-wake cycle (highest at 8-9am and lowest
at 10pm-12am)

 Remember that the conversion of cholesterol

to pregnenolone will happen with the help of
CYP 450

 Serum, plasma, saliva and urine may be used,

blood samples should be drawn at 8am

2
CCHM 312 | BSMLS 2023-2024 | TRANSCRIBER: SANTOS, S.V | FOR STUDENTS USED ONLY NOT INTENDED FOR SELLING
 2ND SEMESTER
Cchm 312: clinical chemistry 2| Lecture
Instructor: PROF Kimberly ann pulga rmt,mph WEEK 11

 Urine free cortisol levels are sensitive Signs and Symptoms

indicators of adrenal hyperfunction  Weight gain but with thin extremities “buffalo
(endogenous corticolism – 24-hour urine hump”, hyperglycemia, thinning of the skin,
collection) poor wound healing, hypertension,
hypercholesterolemia, decreased WBC
Urinary Metabolites
 The liver degrades all glucocorticoids to Screening Test
metabolites excreted in urine  24-hour urinary free cortisol (inc >120 ug/day)
 Overnight dexamethasone suppression test –
2 metabolites that we can measure: most widely used salivary cortisol
1. 17-hydroxycorticosteroid
o Measured by Porter-Silber Method Confirmatory Test
o Reagents: Phenylhydrazine in H2SO4 +  Low-dose dexamethasone suppression test
alcohol  Midnight plasma cortisol (>5.0 ug/dL)
 CRH stimulation test
2. 17-ketogenic steroids
 Measured by Zimmermann Reaction (reddish
purple)
 Reagent: Meta-dinitrobenzene
 Oxidation Procedure: Norymberski (Na
bismuthate)

Clinical disorders
 Hypercortisolism
 Hypocortisolism
o Primary Hypocortisolism
o Secondary Hypocortisolism
 Congenital Adrenal Hyperplasia (deficiencies
in enzymes needed for hormone conversion)
o 21-hydroxylase deficiency
o 11β-hydroxylase deficiency
o 3β-hydroxysteroid dehydrogenase
isomerase deficiency
o C-17, 20-lyase/17α-hydroxilase
deficiency

hyperCortisolism
Cushing’s syndrome
 Primarily caused by excessive production of
cortisol and ACTH
 Caused by overuse of corticosteroid

3
CCHM 312 | BSMLS 2023-2024 | TRANSCRIBER: SANTOS, S.V | FOR STUDENTS USED ONLY NOT INTENDED FOR SELLING
 2ND SEMESTER
Cchm 312: clinical chemistry 2| Lecture
Instructor: PROF Kimberly ann pulga rmt,mph WEEK 11

Procedure of Overnight/Rapid Dexamethasone  Synthetic stimulator of cortisol and

suppression test aldosterone secretion is present
 1mg of dexamethasone is orally given to
patient between 11pm to 12 midnight Primary hypoCortisolism
 Blood is collected the following day 8am to  Due to decreased cortisol production – 90%
9am and urine may be tested for 17OHCS destruction of the adrenal cortex; aldosterone
 Normal patient w/o Cushing Syndrome has a deficiency; excess ACTH release
cortisol value <5 ug/dL and 17 OHCS of <4 mg Disorder
Creatinine after the test
 Addison’s Disease
 (+) result: all results not suppressed (increase
 Hypotension, hyponatremia, hyperkalemia,
in cortisol levels)
weight loss, hyperpigmentation
 Due to autoimmune adrenalitis, tuberculosis,
Procedure for Low- Dose Dexamethasone
hemorrhage, HIV/AIDS infection
suppression test
 0.5 mg oral dexamethasone every 6 hours for Screening Test: ACTH Stimulation Test
2 days
 Increased ACTH and decreased cortisol and
 24-hour urine and serum samples are
aldosterone
collected
 (+) result: elevated cortisol levels secondary hypoCortisolism

Adrenal insufficiency  Also known as the secondary adrenal

insufficiency
 Low baseline corticol levels (8:00 am, supine)
 Due to hypothalamic-pituitary insufficiency
and an elevated ACTH greater than 200 pg/mL
with loss of ACTH
(PRIMARY)
 No problem with mineralocorticoid secretion;
 Lower serum concentrations of ACTH and
absence of hyperpigmentation
cortisol (SECONDARY)
 Lab test: ACTH Stimulation Test
o Suggested in patients with near normal
o (+) result- delayed in response to
response to cosyntropin but abnormal
stimulation test
response to metyrapone test.
o Decreased ACTH and cortisol

Procedure of ACTH Stimulation Test

Cosyntropin Test
 Also known as Cosyntropin Stimulation Test
 Determines the capacity of the adrenal gland
 Also differentiates pituitary/secondary
to increase hormone production in response
adrenal insufficiency (decreased or no ACTH
to stimulation
response) from hypothalamic/tertiary
 Synthetic stimulator of cortisol and
adrenal insufficiency (increased ACTH
aldosterone secretion is present
response)
 Cosyntropin is a synthetic cortisol and
Metyrapone Test
aldosterone stimulator
 Block 11β-hydroxylase, increasing 11-
 It requires administration of 250 µg of
deoxycortisol (>7 µg/dL) while cortisol
Cosyntropin IV or IM
decreases (<5 µg/dL).
4
CCHM 312 | BSMLS 2023-2024 | TRANSCRIBER: SANTOS, S.V | FOR STUDENTS USED ONLY NOT INTENDED FOR SELLING
 2ND SEMESTER
Cchm 312: clinical chemistry 2| Lecture
Instructor: PROF Kimberly ann pulga rmt,mph WEEK 11

Metyrapone Test  An overnight dexamethasone suppression test

 Measures the ability of the pituitary gland to is commonly used to screen patients for
respond to declining levels of circulating autonomous overproduction of cortisol
cortisol, thereby secretes ACTH
 Is used as an alternative diagnostic or
confirmatory test for tertiary adrenal
insufficiency
 500-750 mg of metyrapone is orally
administered every 4 hours for 24 hours
 Performed only if the ACTH stimulation test
gives normal result
 (+) result: decreased ACTH (24-hour urine)
 Metyrapone: inhibitor of 11β-hydroxylase,
therefore it can block cortisol formation

Things to consider
 24-hour urine free cortisol is the most
Congenital adrenal hyperplasia (cah)
sensitive and specific screening test for excess
cortisol production using HPLC or GC-MS-  It results from deficiency of enzymes such as
because plasma cortisol is affected by diurnal 21-hydroxylase deficiency, 11β hydroxylase
variation deficiency, 3β hydroxysteroid dehydrogenase
 Urinary free cortisol is the only portion of isomerase deficiency (necessary for the
cortisol that passes through glomerular secretion of cortisol)
filtration  This will result to decreased plasma cortisol,
 HPLC-MS: the current reference method for increased ACTH and increased levels of
measuring urinary free cortisol androgens
 Insulin Tolerance Test is the gold standard for  24-hour urinary free cortisol is not consistent
secondary and tertiary hypercortisolism, with CAH
confirms borderline response to ACTH
stimulation Definitive Tests:
 17-OHP measurement in amniotic fluid
standard assessment tests for diagnosing Cushing’s  Genotyping cells from chorionic villous
syndrome sampling-most preferred
determine loss of normal cortisol suppression by
dexamethasone
 Use of Dexamethasone
o Acts as an exogenous cortisol
substitute
o Suppresses ACTH if pituitary gland
is normal and cortisol secretion in
the adrenal gland is normal

5
CCHM 312 | BSMLS 2023-2024 | TRANSCRIBER: SANTOS, S.V | FOR STUDENTS USED ONLY NOT INTENDED FOR SELLING
 2ND SEMESTER
Cchm 312: clinical chemistry 2| Lecture
Instructor: PROF Kimberly ann pulga rmt,mph WEEK 11

11β hydroxylase deficiency

 The 2nd most common form of CAH

 Increased level of 11-deoxycortisol is
HTN: Hypertension indicative of this disorder
o Because it cannot be converted to
21-hydroxylase deficiency cortisol anymore due to 11B
 The most common form of CAH deficiency
 Elevated levels of 17α-hydroxyprogesterone  Associated with virilization and hypertension
are indicative of this disorder and urinary
excretion of pregnanetriol 3β hydroxysteroid dehydrogenase isomerase deficiency
o Elevated 17a because it cannot be
converted to 11-Deoxycortisol due
to 21B deficiency
 Leads to hirsutism in women and other
symptoms caused by excess androgen levels
such as virilization; infertility and amenorrhea
o Hirsutism: abnormal excessive
growing of terminal hair which is
common in females (androgen
excess)
o Ferryman-gallway scale: has 9
areas of assessment, used to
measure hirsutism
o 8/9 areas are checked= hirsutism  Results to elevated ratio of 17α-
 ACTH stimulation and 17α- hydroxypregenenolone to 17α-
hydroxyprogesterone tests are used for hydroxyprogesterone and increased ratio of
testing DHEA to androstenedione
 It is characterized by pseudohermaphrodism
in female infants and incomplete
masculinization in male infants

6
CCHM 312 | BSMLS 2023-2024 | TRANSCRIBER: SANTOS, S.V | FOR STUDENTS USED ONLY NOT INTENDED FOR SELLING
 2ND SEMESTER
Cchm 312: clinical chemistry 2| Lecture
Instructor: PROF Kimberly ann pulga rmt,mph WEEK 11

C-17, 20-LYASE/17α- hydroxylase deficiency
 ANP, intracellular calcium, and certain drugs
are aldosterone suppressors, including
ketoconazole, ACE inhibitors, nonsteroidal
anti-inflammatory drugs, and heparin
 The synthesis of this hormone is primarily
controlled by the RAAS
 18-hydroxysteroid dehydrogenase – an
enzyme needed for the synthesis of
aldosterone

 Is characterized by inability to convert 17α-

hydroxypregnolone to DHEA and 17α-
hydroxyprogesterone to androstenedione.
 It will result to decrease androgen, cortisol,
and estrogen synthesis; decrease
progesterone synthesis
 For females, absence of menstruation and
defective ovarian maturation
 For males, pseudohermaphrodism

Aldosterone
 Major electroregulating hormone
 Most potent mineralocorticoid
 A steroid hormone that helps regulate water
and electrolytes and blood levels (Na level in
the serum depends almost completely on the
interplay between aldosterone and ADH)
 Main determinant of renal excretion of
Makitandaan daw po to sabi ni maam :>
potassium
PA: Plasma Aldosterone
 Acts on renal tubular epithelium to increase
PRA: Plasma Renin Activity
retention of Na and Cl, and excretion of K and
H- promotes 1:1 exchange of Na for K or H
Clinical disorders
Stimulators of aldosterone 1. Primary Hyperaldosteronism (Conn’s Disease)
 Angiotensin II, ACTH, elevated serum 2. Secondary Hyperaldosteronism
potassium 3. Hypoaldosteronism
4. Others:
Inhibitors/Suppressor of Aldosterone a. Liddle’s syndrome
 Progesterone and dopamine b. Bartter’s syndrome

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CCHM 312 | BSMLS 2023-2024 | TRANSCRIBER: SANTOS, S.V | FOR STUDENTS USED ONLY NOT INTENDED FOR SELLING
 2ND SEMESTER
Cchm 312: clinical chemistry 2| Lecture
Instructor: PROF Kimberly ann pulga rmt,mph WEEK 11

c. Gitelman’s syndrome  Secondary Hyperaldosteronism that results in

hypokalemia:
Primary hyperaldosteronism (conn’s disease) o Renal artery stenosis, diuretic
therapy, malignant hypertension,
 Caused by aldosterone-secreting adrenal
and congenital defects in renal salt
adenoma
transport such as Bartter’s
 Associated with elevated plasma aldosterone
Syndrome and Gitelman’s
and low plasma renin
syndrome
 In this condition, distal delivery of Na is
increased because of increased NaCl
Liddle’s syndrome (pseudohyperaldosteronism)
reabsorption in the CD by the action of
aldosterone inhibits salt reabsorption in the  Congenital disorders that are characterized by
PCT as the result of volume expansion increased Epiethelial Sodium Channel (ENaC)
 Symptoms: hypertension, hypokalemia, mild activity in the collecting ducts in the absence
hypernatremia and metabolic alkalosis of increased aldosterone
 Resemble primary aldosteronism clinically,
Screening test but aldosterone level is low and with absence
 Plasma aldosterone concentration/plasma of hypertension
renin activity ratio (PAC/PRA)
o (+) result: >25 ratio bartter’s syndrome
 Bumetanide-sensitive chloride channel
Confirmatory Test mutation
 Saline Suppression Test  A rare potassium-losing autosomal recessive
o (+) result: >5 ng/dL aldosterone disorder, caused by defective NaCl
 Procedure: reabsorption in the thick Ascending Loop of
o Involves infusing 2L of 0/9% saline Henle (ALH)
over 4 hours, or by administering  Accompanied by elevated concentrations of
10-12 mg NaCl tablet daily for 3 aldosterone and renin
days
 Captopril suppression gitelman’s syndrome
o 3 hours of taking 50mg of captopril
 Thiazide sensitive transporter mutation
o Plasma aldosterone remains high in
primary hyperaldosteronism  Associated with the defect in NaCl
reabsorption that occurs in the distal
secondary hyperaldosteronism convoluted tubule
 Accompanied by elevated aldosterone
 Occurs as a result of excessive production of
renin Hypoaldosteronism
 Accompanied by elevated plasma levels of
aldosterone and renin  Due to the destruction of the adrenal glands
and deficiency of glucocorticoid
 In this condition, hyperkalemia occurs only in
conditions that are accompanied by increased  Associated with enzyme 21-hydroxylase
distal Na delivery deficiency

8
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 2ND SEMESTER
Cchm 312: clinical chemistry 2| Lecture
Instructor: PROF Kimberly ann pulga rmt,mph WEEK 11

 Symptoms: hyperkalemia and metabolic  Excessive production can be confirmed by

acidosis measuring total and free testosterone and
DHEAS
test
Furosemide Stimulation Test or Upright Posture
 (+) result: low aldosterone level

Saline suppression Test

 (+) result: high aldosterone level

Things to consider
 Blood samples for aldosterone test should be
Virilization in boys: includes penal enlargement,
drawn in the morning before the patient has
androgen-dependent hair growth, secondary
gotten out of bed- to avoid markedly
sexual characteristics
increasing result
In Girls: hirsutism, acne, clitoromegaly
o fasting is not required
 Plasma samples are treated with extraction Adrenal medulla
agent to remove aldosterone from plasma
proteins  Composed primarily of chromaffin cells that
 Urine samples are assayed using acid secrete catecholamine
hydrolysis and extraction  L-tyrosine is the precursor of the
 Aldosterone levels are lower at night catecholamines
 Florinef – synthetic mineralocorticoid  Norepinephrine and epinephrine are
 Methods for Aldosterone Measurement: RIA metabolized by monoamine oxidase and
and Chromatography cathecol-0-methyl transferase to form
metanephrines and VMA
Weak androgens  Ratio of norepinephrine to epinephrine is
serum is 9:1
 Serves as precursor for the production of more
 The hormones are 50% protein bound
potent androgens and estrogens in tissues
 Produce as by-products of cortisol synthesis Hormones produced
that are regulated by ACTH
 Norepinephrine
 Precursors: Pregnenolone and 17-
 Epinephrine
hydroxypregnenolone
 Dopamine
 E.G. Dehydroepiandrosterone (DHEA) and
androstenedione
 DHEA- principal adrenal androgens are
converted to estrone
 They circulate bound to steroid hormone
binding globulin (SHBG)
 Excessive production of androgens results in
virilization

9
CCHM 312 | BSMLS 2023-2024 | TRANSCRIBER: SANTOS, S.V | FOR STUDENTS USED ONLY NOT INTENDED FOR SELLING
 2ND SEMESTER
Cchm 312: clinical chemistry 2| Lecture
Instructor: PROF Kimberly ann pulga rmt,mph WEEK 11

Norepinephrine  Major metabolite: HVA

 Primary amine
 Produced by sympathetic ganglia
 Highest concentration is found in the brain
 Acts as neurotransmitters in both CNS and
sympathetic nervous system
 Methoxy Hydroxyphenylgylcol (MHPG) is the
major metabolite in CNS
 Major metabolites:
o 3-methoxy-4-hydroxyphenylglycol
(CSF and urine)
o Vanillylmandelic Acid (VMA)

Epinephrine
 Adrenaline/secondary amine
Clinical disorders and methods
 Most abundant medullary hormone
 Produced from norepinephrine and comes Clinical Disorders
only from adrenal  Pheochromocytoma
 Called the “fight or flight hormone” because it  Neuroblastoma
is release in response to physiologic (injuries)
and psychological (stress, anxiety) threats Methods
 A form of stress that increases cortisol levels  Chromatography
stimulates its production  Radioimmunoassays
 Increases glucose concentration
(glycogenolysis) pheochromocytoma
 Best collected from indwelling catheter, since
 Tumors of the adrenal medulla or
venipuncture may cause levels of
sympathetic ganglia
catecholamines to rise
 Commonly seen in 3rd to 5th decade of life
 Major metabolites: VMA
 Due to overproduction of the catecholamine
 Other urinary metabolites
o Metanephrines,
Signs and Symptoms
normetanephrines, homovanillic
acid  Tachycardia, headache, tightness of chest,
sweating, hypertension
Dopamines
Screening test
 Primary amine
 High plasma metanephrines and
 Major intact hormone in urine
normetanephrines by HPLC (plasma-EDTA)
 A catecholamine produced in the body by the
decarboxylation of 3,4
Diagnostic Test
dihydroxyphenylalanine
 Present in highest concentration in the  High 24-hour urinary excretion of
regions of the brain metanephrines and normetanephrines (urine)

10
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 2ND SEMESTER
Cchm 312: clinical chemistry 2| Lecture
Instructor: PROF Kimberly ann pulga rmt,mph WEEK 11

methods
Pharmacological test
Chromatography
1. Clonidine
 HPLC or GC-MS – (VMA and metanephrines)
o Differentiates pheochromocytoma
(not suppressed) to neurogenic
Radioimmunoassay
hypertension (50% decreased
 Sensitive screening test for total plasma
catecholamines)
catecholamines
o 0.3 mg Clonidine is administered
which differentiates borderline  >2000 pg/mL of plasma catecholamines –
results from 1000-2000pg/mL diagnostic for pheochromocytoma
o Confirmatory test for
Pheochromocytoma Specimen Consideration
2. Glucagon Stimulation Test 1. Catheterization
o Used if it is highly suggestive of o Preferred method of blood
pheochromocytoma but blood collection to eliminate anxiety of
pressure is normal and venipuncture
catecholamines are modestly
elevated: 3 folds increased 2. Urine Preservation
 Urine samples with 10 mL of 6N HCl
Neuroblastoma (cathecolamines and metabolites are rapidly
oxidized at higher pH)
 A fatal malignant condition in children
resulting to excessive production of 3. 24-hour urine creatinine test
norepinephrine  To assess the quality of urine collection
 (+) high urinary excretion of Homovanillic (0.8g/day of urine creatinine is needed to
Acid (HVA) or VMA or both and dopamine validate the completeness of the urine
 Specimen: 24-hour urine and blood (plasma) collection)

Patient Preparation “To prevent catecholamine oxidation, blood samples must be

 The patient should undergo overnight fasting transported on ice.”
 Avoid smoking or drinking coffee at least 4
hours prior to blood collection
 The patient is placed in a reclining position in
quiet environment and a heparin lock is
inserted intravenously.
 After 20-30 mins blood is collected in a
prechilled EDTA tube
 Plasma concentrations of catecholamines are
affected body positioning and samples must
be collected after 30 mins in a stable position-
decreased value when supine

11
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 2ND SEMESTER
Cchm 312: clinical chemistry 2| Lecture
Instructor: PROF Kimberly ann pulga rmt,mph WEEK 11

REPRODUCTIVE HORMONES AND OTHER  Reference Values: 3.9 – 7.9 ng/mL (serum)
MISCELLANEOUS HORMONES  Transport proteins
(Midterm, 5th topic CONTINUED) o Sex hormone binding globulin
(SHBG) – 60%
Reproductive hormones o Albumin – 40%
o Concentration determines the level
 Testosterone
of testosterone
 Dehydroepiandrosterone (DHEA)
 Estrogen
 Progesterone Types of testicular infertility
1. Pretesticular Infertility (Secondary
other hormones Hypogonadism)
 Due to Hypothalamic/Pituitary lesions
Pancreas
 Testosterone, FSH, LH – normal/decreased
 Glucagon, Insulin, Somatostatin

2. Testicular Infertility (Primary Hypogonadism)

Other Miscellaneous Hormones  May be congenital (cryptochorchidism,
Klinefelter’s syndrome and 5-a-reductase
 Human Chorionic Gonadotropin deficiency) or acquired (variococele, tumor,
 Human Placental Lactogen orchitis)
 Gastrin  Decreased Testosterone levels and increased
 Serotonin FSH and LH
 Inhibin A

Testosterone
 Principal androgen hormone– most potent
 Synthesized by the Leydig Cells of the testis of
the male, derived from progesterone
 Controlled by FSH & LH
 Function: growth and development of the
reproductive system, prostate and external
genitalia
 Levels demonstrate a circadian pattern and
peak at (08:00 AM) and fall to their lowest
levels at 8PM
 There is gradual reduction of testosterone
after age 30, with an average decline of about
110 ng/dL every decade
 Test for male infertility: Semen analysis,
testosterone, FSH & LH tests
CCHM 312 | BSMLS 2023-2024 | TRANSCRIBER: SANTOS, S.V | FOR STUDENTS USED ONLY NOT INTENDED FOR SELLING
3. Posttesticular Infertility
 Due to disorders of sperm transport and
function
 Testosterone, FSH, and LH – normal
Other disorders of sexual development
1. Testicular Feminization Syndrome
 Most severe form of androgen resistance
syndrome, resulting in lack of testosterone
action in the target tissue
 Physical development pursues the female
phenotype, with fully developed breast and
female distribution of fat and hair
 No utility or response to administration of
exogenous testosterone
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 2ND SEMESTER
Cchm 312: clinical chemistry 2| Lecture
Instructor: PROF Kimberly ann pulga rmt,mph WEEK 11

 Lab tests: normal levels of testosterone with  Estrone and estriol are metabolites of
elevated FSH and LH intraovarian and extraglandular conversion

2. Sertoli-Cell only Syndrome Estrone

 Characterized by lack of Germ cells
 The most abundant estrogen in post-
 Men present with small testes, high FSH levels,
menopausal women
azoospermia and normal testosterone level
 Testicular biopsy Is the only procedure to
confirm the diagnosis
Estradiol

 Most potent estrogen secreted by the ovary,

3. Kallman’s Syndrome major estrogen
 A result of an inherited, X-linked recessive trait  Synthesized from testosterone, then diffuses
that manifests as hypogonadism during out of the cells of the ovaries of the female
puberty  Precursor of both E1 and E3 – serves as
negative feedback for FSH
 Used to assess Ovarian Function
Dehydroepiandrosterone  Transport proteins: Albumin (60%) and SHBG
(38%)
 The principal androgen formed by the adrenal
cortex; weak androgen  Free form of E2 is approximately 2%
 Derived from the adrenal gland
 Valuable in the assessment of adrenal cortical
Estriol
hormones
 Estrogen found in the Maternal urine
 Metabolite of estradiol
Estrogen  Major estrogen secreted by the placenta
 Arises through structural alteration of the during pregnancy- formation in pregnant
testosterone molecule women is dependent on fetal and placental
 Function: promotion of breast development, function
maturation of external genitalia, deposition of  Used to assess the fetoplacental unit
body fat (secondary sexual characteristic in (fetoplacental viability), postdate gestations
female) and intrauterine retardation
 In conjunction with progesterone, they  Used as marker for Down Syndrome
function in uterine growth and regulation of  Preferred specimen: plasma
menstrual cycle and maintenance of
pregnancy
 Deficiency: irregular and incomplete Progesterone
development of the endometrium  Produced by the granulose (lutein) cells of the
 Precursor: acetate, cholesterol, progesterone corpus luteum in the female
and testosterone  Prime secretory product of the ovary
 Forms: Estrone, Estradiol, Estriol
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CCHM 312 | BSMLS 2023-2024 | TRANSCRIBER: SANTOS, S.V | FOR STUDENTS USED ONLY NOT INTENDED FOR SELLING
 2ND SEMESTER
Cchm 312: clinical chemistry 2| Lecture
Instructor: PROF Kimberly ann pulga rmt,mph WEEK 11

 Dominant hormone responsible for the luteal  Responsible for the synthesis of digestive
phase cycle among females enzymes
 Single best hormone to determine whether  Acinus: functional secretory unit
ovulation has occurred
 Primarily for the evaluation of fertility in
female 2. Endocrine
 Serves to prepare the uterus for pregnancy  Responsible for the synthesis
and the lobules of the breast for lactation  Alpha cells (20-30%) – glucagon
 Deficiency: failure of implantation of the  Beta cells (60-70%) – insulin
embryo  Delta cells (2-8%) – somatostatin
 Metabolites: pregnanediols, pregnanediones,
pregnanelones
Human chorionic gonadotropin (hcg)
 Produced by the trophoblast cells of the
Test for Menstrual Cycle Dysfunction and
placenta
Anovulation
 Serves to maintain progesterone production
 Estrogen by the corpus luteum in the early pregnancy
 Progesterone  Can be detected 2-3 days after ovulation
 FSH  Qualitative test for urine samples has
 LH detection limit of about 50 mIU/mL
 Method: Immunometric (sandwich method)

Tests for Female Infertility

Human Placental lactogen
 HCG
 PRL  Functionally, structurally and immunologically
similar to Growth Hormone and Prolactin
 FT4
 In urine, serum & amniotic fluid
 TSH
 Stimulates the development of the mammary
 FSH
gland
 LH
 Increases maternal plasma glucose level and
 Estradiol
promotes positive nitrogen balance
 Progesterone
 Important in the diagnosis of intrauterine
growth retardation
Pancreas
 A digestive gland in the gastrointestinal system Gastrin
 A peptide secreted by the G cells of the antrum
of the stomach
Functions
 Released in response to vagal stimulation and
1. Exocrine food in the stomach
 Causes secretion of the HCl by parietal cells

14
CCHM 312 | BSMLS 2023-2024 | TRANSCRIBER: SANTOS, S.V | FOR STUDENTS USED ONLY NOT INTENDED FOR SELLING
 2ND SEMESTER
Cchm 312: clinical chemistry 2| Lecture
Instructor: PROF Kimberly ann pulga rmt,mph WEEK 11

 Diagnostic marker for Zollinger-Ellison Somatostatin

Syndrome
 Also called Growth Inhibiting Hormone
 Major stimulus: presence of amino acid
 Found in the GIT, hypothalamus and delta cells
 Increased levels: ZES, Achlorhydria, Chronic of the pancreas
Renal Failure
 An inhibitor of GH, Glucagon and Insulin

Gastric Fluid Acidity Inhibin A

 G cells → gastrin  A reproductive system hormone which inhibits
 Parietal cells (gastrin)→ HCl FSH activity
 Chief cells (pepsinogen) → pepsin

BAO BAO/MAO
Methods
Normal 2.5 10% Sample for hormonal assay
Pernicious 0 0 1. Whole blood: LH and Testosterone
Anemia 2. Plasma
Gastric 1.0 25%
o EDTA: (ACTH, ADH, PTH)
Carcinoma
Duodenal 5.0 17% o Heparin: (Catecholamines, Cortisol,
Cancer Dopamine, FSH)
Zollinger- 18.0 72% 3. Serum
Ellison o Aldosterone, androstenedione,
Syndrome DHEA, estrogen, FSH, GH, HCG,
progesterone
Basal Acid Output/Maximal Acid Output 4. Urine: estriol
 Boric Acid (1 g/dL) preserves estriol and
Serotonin (5 hydroxytryptamine)
estrogen for 7 days
 An amine derived from hydroxylation and  10mL of 6N HCl Is added to 3-4L of container
decarboxylation of tryptophan (catecholamines, vanillylmandelic acid, 5-
 Synthesized by Argentaffin cells, primarily in GI HIAA)
tract
 Also found in high concentration in pineal
gland and CNS Classic Assay
 Binds to platelets and released during
 Bioassays
coagulation
 Competitive Protein Binding
 Urinary metabolites: 5-
Hydroxyindoleaceticacid (5-HIAA) Immunologic Assays
 5-HIAA Is a diagnostic marker for carcinoid
 Radioimmunoassays
tumor
 Immunoradiometric Assays
 Test for 5-HIAA: Ehrlich’s Aldehyde
 Enzyme-linked Immunosorbent Assays
 Enzyme Multiplied Immunosorbent Technique

15
CCHM 312 | BSMLS 2023-2024 | TRANSCRIBER: SANTOS, S.V | FOR STUDENTS USED ONLY NOT INTENDED FOR SELLING
 2ND SEMESTER
Cchm 312: clinical chemistry 2| Lecture
Instructor: PROF Kimberly ann pulga rmt,mph WEEK 11

 A radiolabeled substance is attached to the

antibody instead of the hormone; an AG-AB
Fluorescent Techniques
reaction
High Performance Liquid Chromatography
Colorimetry
ELISA

 An enzyme attached to an antibody in which

Bioassay the end product can be measured
spectrophotometrically
 Based on observation of physiologic responses
specific for the hormone being measured
 Involves injection of the test materials into
Enzyme Multiplied Immunosorbent Technique
animals
(EMIT)

 Enzyme tags are used; the enzyme is attached

Competitive Binding Protein to the hormone or drug being tested
 The rate of NADH produced is proportional to
 Based on competition for protein-binding sites
the amount of drug tested
between a known “tagged/labelled” hormone
 Requires no separation of bound free antigen
and the unlabeled hormone in the patient’s
 This method is widely used in Therapeutic Drug
sample
Monitoring (TDM)
 Measurement of total T4 is based on the
specific binding properties of TBG

Immunometric
Immunologic Assays  For TSH test; sensitive test
 Widely used to quantify hormones using
labeled-antibody with non-isotopic labels
Fluorescent techniques
Fluorescence Polarization Immunoassay
Radioimmunoassays
 Fluorescein-labeled drug, serum, and antibody
 Is a CBP technique that utilizes radiolabeled are mixed and placed in the light path of a
hormone as the tagged hormone and antisera fluorometer. Antibody bound conjugate is
prepared against the specific hormone as the inversely proportional to serum drug
binding site concentration

High Performance Liquid Chromatography (HPLC)

Immunoradiometric  Based on the differential partitioning of the

compounds between the mobile phase and
stationary phase

16
CCHM 312 | BSMLS 2023-2024 | TRANSCRIBER: SANTOS, S.V | FOR STUDENTS USED ONLY NOT INTENDED FOR SELLING
 2ND SEMESTER
Cchm 312: clinical chemistry 2| Lecture
Instructor: PROF Kimberly ann pulga rmt,mph WEEK 11

colorimetry
A. Porter-Silber Method: 17-OCHS
B. Zimmermann Reaction
o Measures those steroids with 17-KS
C. Pisano Method
o For quantifying metanephrines and
normetanephrines
D. Kober Reaction
o For estrogen
o H2SO4 + hydroquinone
o (+) reddish brown color

17
CCHM 312 | BSMLS 2023-2024 | TRANSCRIBER: SANTOS, S.V | FOR STUDENTS USED ONLY NOT INTENDED FOR SELLING