2/12/22

DEPARTMENT OF MEDICAL TECHNOLOGY

INSTITUTE OF ARTS AND SCIENCES

ELECTROLYTES
FAR EASTERN UNIVERSITY

• -ions (minerals) capable of carrying an electric charge

• -classified as
ELECTROLYTES 1. Anions: Carry (-) charge and move toward the anode
Prepared by: Charlene Princess Salvador Tolenada, RMT, MSMT
• E.g. Cl-, HCO3-, PO4-

2. Cations: Carry (+) charge and move toward the cathode

• E.g. Na+, K+, Mg2+, Ca2+

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Functions of Electrolytes Water

• -40-75% : average water content
• Blood coagulation of the human body Location:
• Volume and osmotic regulation (Na+, Cl-, K+) -Extracellular fluid (ECF): 1/3 (16 L)
- Women have lower average
• Myocardial rhythm and contractility (K+ , Mg2+, Ca2+) -Intracellular fluid (ICF): 2/3 (24 L)
water content (higher fat content)
-Intravascular ECF (Plasma) : 25 %
• Neuromuscular Excitability (K+ , Mg2+, Ca2+)
-Interstitial cell fluid:75%
• Important Cofactors in enzyme activation (Mg2+, Ca2+, Zn2+) • Function:
• Regulation of ATPase ion pumps (Mg2+) - plasma : 93% water ; Remaining
-Transport nutrients to the cells
• Acid-base balance (HCO3-, K+, Cl-) volume: lipids and proteins.
-Removes waste products
• Production and use of ATP from glucose
- acting as the body's coolant by way
• Replication of DNA and translation of mRNA
of sweating
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Concentration of ions is maintained

Distribution of Body Water in Adult
by:
Compartment (%) of Body (%) of Total Body Osmolality
ü Passive Transport/ diffusion:
Weight H2O Passive movement of ions across
- based on the concentration of a membrane
Extracellular 20 33 solutes (expressed as millimoles) ü Active Transport: Requires
per kilogram of solvent (w/w).
Plasma 5 8 energy to move ions across a
(Intravascular) - (millimoles/kg) membrane

Interstitial 15 25 -maintaining a high intracellular

concentration of K+ and a high extracellular
Intracellular 40 67 (plasma) concentration of Na+ requires use
of energy
Total Body Water 60 100
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Osmolality is regulated by: Regulation of Blood Volume

1. Thirst Sensation: Response to 3. Renin-angiotensin-aldosterone
consume more fluids system (RAAS)
-Prevents water deficit
4. ANP: ↑ Na+ & H2O excretion
2. Arginine vasopressin hormone (AVP) in the kidney
- Antidiuretic Hormone (ADH)
-↑ reabsorption of water in kidneys
5. GFR: ↑ w/ vol. expansion & ↓
w/ vol. depletion
-Suppressed in excess H2O load
-Activated in H2O deficit

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Clinical Significance of Osmolality Determination of Osmolality

• IMPORTANCE: parameter to which the hypothalamus responds!!!!
• SPECIMEN: serum or urine.
WATER LOAD • -sodium, chloride, and bicarbonate:largest contribution to the
-↑WATER INTAKE (POLYDIPSIA)= ↓PLASMA OSMOLALITY= AVP AND THIRST SUPPRESSED osmolality value of serum.
-↓AVP=H2O NOT REABSORBED= ↑DILUTE URINE TO BE EXCRETED
• -Plasma is not recommended!

WATER DEFICIT
-↓ WATER INTAKE=↑ PLASMA OSMOLALITY=AVP AND THIRST ARE ACTIVATED
-DIABETES INSIPIDUS: NO AVP PRODUCTION OR NO ABILITY TO RESPOND TO AVP IN
CIRCULATION

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Determination of Osmolality Determination of Osmolality

• based on the colligative properties -Any substance dissolve in a solvent will:
• ↑osmolality=↓both the freezing point temperature and the vapor • ↓ freezing point by 1.858°C
pressure.
• ↑ boiling point by 0.52°C
• Samples must be free of particulate matter
• ↓ vapor pressure (dew point) by 0.3 mmHg
• Turbid serum and urine samples should be centrifuged before analysis
• ↑ osmotic pressure by 17,000 mmHg
• If reusable sample cups are used should be thoroughly cleaned,
disinfected, and dried
• Osmometers that operate by freezing point depression are -Main contributors are Na+, Cl-, Urea & Glucose
standardized using sodium chloride reference solutions.

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Determination of Osmolality

THE ELECTROLYTES

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ELECTROLYTES SODIUM
Reference Intervals -Natrium
Cation Extracellular Intracellular Anion Extracellular Intracellular
-major extracellular cation
(mmol/L) (mmol/L) (mmol/L) (mmol/L) -most abundant cation in the ECF
Na+ 136-145 15 HCO3- 23-29 10 -depends greatly on the intake and excretion of water
K+ 3.5-5.1 150 Cl- 98-107 1 -involved in ATPase ion pump (active transport)
Ca2+ 2.15-2.5 1 HPO42- 0.78-1.42 50 - 2 K in; 3 Na out; ATP converted to ADP

Mg2+ 0.63-1 13.5 SO42- 0.5 10 Reference value: 135-145 mmol/L

Threshold critical value:
160 mmol/ L (Hypernatremia)
ctolenada@ feu.edu.ph 120 mmol/ (Hyponatremia) ctolenada@ feu.edu.ph

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HORMONES AFFECTING SODIUM LEVELS Sodium Plasma concentration depends in:

• ALDOSTERONE: -Intake of water : Thirst
-it promotes absorption of sodium in the distal tubule -Excretion of water : AVP (↑ H2O reabsorption)
-It promotes Na retention and K excretion -The blood volume status
-Angiotensin II (↑ aldosterone)
• ATRIAL NATRIURETIC FACTOR (ANF)
-Aldosterone (↑ Na+ reabsorption in kidney)
-endogenous antihypertensive agent secreted from the cardiac atria.
-ANP (↑ Na + Excretion in the kidney )
-It blocks aldosterone and renin secretion, and inhibit the action of
angiotensin II and vasopressin
-It causes natriuresis

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Sodium Clinical Application Sodium Clinical Application

HYPONATREMIA HYPERNATREMIA
-Most common electrolyte disorder
-less than 135 mmol/L
-If renal failure occurs, the kidney ultimately fail to concentrate the
urine -Increased Na concentration in plasma
-decreased levels may be caused by increased Na+ loss, increased
water retention, or water imbalance water
-Increased Na+ loss in the urine can occur with decreased aldosterone
production
-serum sodium level >145 mmol/L
-K+ deficiency also causes Na+ loss -caused by loss of water, gain of
-Accumulation of glucose or mannitol in the ECF is caused of
hyponatremia sodium or both
-SIADH causes an increase in water retention
-Barterr’s syndrome, hyponatremia is not corrected with fluid -result from excessive water loss
restriction

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Determination of Sodium POTASSIUM

-Kalium
• Specimen -Major intracellular cation
-Serum, Plasma (heparin and oxalate) REFERENCE RANGE -In the ascending limb of Henle’s loop, K+ is reabsorbed together with Na+ and Cl by the
sodium potassium chloride cotransporter
False ↓ w/ marked hemolysis
-Considered as immediately life threatening
-FUNCTION: Heart contraction, neuromuscular excitability, ICF volume regulation, and
• Methods hydrogen ion concentration
-FES
-ISE (Glass aluminum silicate): most ↑ K+, ↑ cell excitability (muscle weakness)
commonly used method ↓ K+, ↓ cell excitability (arrhythmia/paralysis)
-AAS
Reference value: 3.5 -5.2 mmol/ L
-Colorimetry Threshold critical value: 6.5 mmol/L (hyperkalemia)
2.5 mmol/L (hypokalemia) ctolenada@ feu.edu.ph

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Potassium : Regulation Potassium : Regulation

• Aldosterone
-↑ K + excretion in urine • Hyperosmolality
-Uncontrolled diabetes mellitus: causes water to diffuse from the cells,
• Na+, K+ -ATPase pump
carrying K+ with the water
-↓ function ↓ cellular entry
-↑ function ↑ cellular entry
• Cellular Breakdown
-Cellular breakdown releases K+ into the ECF.
• Exercise
- severe trauma, tumor lysis syndrome, and massive blood transfusions.
-increase plasma K+ (reversible after several minutes)
-0.3 to 1.2 mmol/L: mild to moderate exercise
-2 to 3 mmol/L: exhaustive exercise.
-Forearm exercise during venipuncture can cause erroneously high plasma K concentration
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Potassium: Clinical Application Potassium: Clinical Application

Hyperkalemia Hypokalemia
-due to impaired renal excretion

3 major mechanism of diminished renal

potassium excretion: -mild hypokalemia:3.0-3.4 mmol/L
-reduced aldosterone -Hypomagnesemia lead to hypokalemia
-renal failure -If plasma K level are low, it will be
-reduced distal delivery of sodium retained, and NH4 ions will be secreted
if balancing cations are needed.
-Elevation of serum K can directly stimulate
the adrenal cortex to release aldosterone

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Determination of Potassium Chloride

REFERENCE RANGE -major extracellular anion-chief counter ion of sodium in ECF
• Specimen
-Serum, Plasma (heparin) -maintenance of water balance and osmotic pressure
-False ↑ with hemolysis
-enzyme activator
-24 hour urine (diurnal variation)
-hemolysis must be avoided -excreted in the urine and sweat
-Increase platelet: Increase K+ conc (plasma is preferred) -disorders of chloride are the same as sodium
• Methods -FUNCTION: maintains osmolality, blood volume and electric neutrality
-FES (Chloride shift)
-AAS
-ISE (Use valinomycin membrane); current method of choice
-Colorimetry (Lockhead and Purcell)

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Chloride Clinical Application Determination of Chloride

• Hyperchloremia • Hypochloremia • Specimen
-Renal tubular acidosis -Prolonged vomiting -Serum or plasma (lithium heparin)
-Diabetes insipidus -Aldosterone deficiency -Hemolysis: decreased level of Cl
-Salicylate intoxication -metabolic alkalosis -24 hr collection (spx of choice in urine Cl)
-Primary hyperparathyroidism -salt-losing nephritis -sweat is suitable
-Metabolic acidosis
-Prolonged diarrhea

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BICARBONATE
Determination of Chloride
• Methods -Is the second most abundant anion in the ECF
-ISE (Use ion exchange membrane); most commonly used method -accounts for 90% of the total CO2 at physiologic pH
-Amperometric-coulometric (Cotlove Chloridometer): coulometric -it is composed of undissociated NaHCO3, carbonate and carbamate
generation of silver ions (Ag+), which combine with Cl− to quantitate -It buffers excess hydrogen ion
the Cl− concentration.
-maintenance of high plasma bicarbonate concentration occurs in
-Schales and Schales (Mercurimetric Titration) advanced renal failure, or when the renal threshold for bicarbonate is
-Diphenyl benzoate: indicator increased.
-HgCl2 (blue violet): end product
-Skeggs and Hochestrasser (Colorimetric): reddish color end product -FUNCTION: major component of the buffering system of the blood
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BICARBONATE BICARBONATE: REGULATION

•pH imbalance: -alkalosis =increase excretion of HCO − into the urine

-acidosis (increased excretion of H+ into the urine) : HCO −

-it diffuses out of the cell in exchange for chloride to maintain ionic reabsorption is virtually complete, with 90% of the filtered HCO −
charge neutrality within the cell (chloride shift) reabsorbed in the proximal tubule and the remainder in the distal
tubule

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BICARBONATE DETERMINATION BICARBONATE DETERMINATION

• Methods
• Specimen:
-ISE (pCO 2 electrode): measuring total CO2 uses an acid reagent to convert
-Serum or lithium heparin plasma all the forms of CO2 to CO2 gas and is measured by a pco2 electrode
-blood anaerobically collected -Enzyme Method: alkalinizes the sample to convert all forms of CO2 to HCO
-sample is analyzed immediately. −. HCO − is used to carboxylate phosphoenolpyruvate (PEP) in the 33
presence of PEP carboxylase, which catalyzes the formation of oxaloacetate
-left uncapped, CO2 escape (can decrease 6mmol/L/h)

• Reference Ranges
• CO2, venous 23 to 29 mmol/L (plasma, serum)

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MAGNESIUM
Physiology and Regulation of Magnesium
-second most abundant intracellular cation.
-Average human body: 70 kg=1mol (24 g) of Mg
• Parathyroid Hormone (PTH) - ↓ Mg2+
-Bone (53%), muscle (46%), serum and RBC (less than 1%)
• In serum: -Promotes Ca + renal reabsorption
-1/3 (33%) bound with albumin
-2/3 (61%): free or ionized state
-5%: complexed with other ions • Aldosterone & Thyroxine - ↓ Mg2+
-acts as cofactor in glycolysis; transcellular ion transport; neuromuscular
transmission; synthesis of carbohydrates, proteins, lipids, and nucleic acids; the -Promotes Na + renal reabsorption
release of and response to certain hormones; important in maintaining the
structure of DNA, RNA and ribosomes; regulates movements of potassium across
myocardium
-Mg loss leads to decreased intracellular K level
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Clinical Application of Magnesium Determination of Magnesium

Hypomagnesemia Hypermagnesemia

• Specimen
-Nonhemolyzed serum or lithium heparin plasma
-hemolysis must be avoided
-Oxalate, citrate, and ethylenediaminetetraacetic acid (EDTA)
anticoagulants are unacceptable.
-24-hour urine sample is preferred
-urine must be acidified with HCl

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Determination of Magnesium CALCIUM

1. Colorimetric method
-Calmagite Method
-Mg2+ + Calmagite à Reddish-violet (532nm) REFERENCE RANGE -present exclusively in plasma
-involved in blood coagulation, enzyme activity, excitability
- Formazen Dye Method
-Mg2+ + Dye à Colored complex (660 nm) of skeletal and cardiac muscle (myocardial contraction), and
-Magnesium Thymol Blue Method/ Methylthymol Blue Method maintenance of blood pressure
-Mg2+ + Chromogen à Colored complex
-absorbed in the duodenum
2.Dye-LakeMethod-Titan Yellow Dye (Clayton Yellow/ Thiazole yellow) -Decreased ionized Ca2+ concentrations : neuromuscular
-Mg2+ + Titan yellow à Red Compound
irritability (tetany).
3.Atomic Absorption Spectrophotometry (Reference Method)
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FACTORS AFFECTING SERUM CALCIUM IN THE

FORMS OF CALCIUM IN THE BLOOD BLOOD
• Ionized Ca (45%) : Active • Vitamin D
-unbound or free - Vitamin D3 is then converted in the liver to 25- hydroxycholecalciferol
(25-OH-D3)
- 25-OH-D3 à 1,25 Dihydroxycholecalciferol (1,25-(OH)2-D3)/ Vit D3
• Protein bound Calcium (40%)
-increases intestinal absorption of Ca
-bound to albumin
-increases reabsorption of Ca in the kidney
-increases mobilization of calcium from bones
• Complex Calcium (15%)
-bound to anions (HCO3- & PO4-)
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FACTORS AFFECTING SERUM CALCIUM IN THE FACTORS AFFECTING SERUM CALCIUM IN THE
BLOOD BLOOD
• Parathyroid Hormone (PTH) • Calcitonin
-conserve calcium -medullary cell
-increases the level by mobilizing BONE calcium -secreted by parafollicular C cells of the thyroid gland
-activates the process of bone resorption - secreted when the concentration of Ca2+ in blood increases
-It suppresses urinary loss of Calcium -inhibit PTH and Vit D3.
-stimulates the conversion of inactive Vit D to Active Vit D3 -inhibits bone resorption
-it promotes urinary excretion of Calcium

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Calcium :CLINICAL APPLICATION

Calcium: Regulation
HYPOCALCEMIA HYPERCALCEMIA

Causes of Hypercalcemia: CHIMPS (Cancer, Hyperthyroidism,

iatrogenic causes, multiple myeloma, hyperparathyroidism
and Sarcoides)

Causes of Hypocalcemia: CHARD (Calcitonin,

Hypoparathyroidism, Alkalosis, Renal failure, Vit D deficit)
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DETERMINATION OF CALCIUM DETERMINATION OF CALCIUM

• Methods
• Specimen
• EDTA Titration Method
- serum (spx of choice) or lithium heparin plasma
• Precipitation and Redox Titration (Bachra, Dawer, and Sobel)
-collected anaerobically
-Clark Collip Precipitation • Ion Selective Electrode (Liquid
- EDTA or oxalate are unacceptable for use.
(End product : Oxalic acid -purple color) membrane)
-avoid prolonged contact of serum with cell clot (dec. Ca)
-Recumbent posture: dec. Ca -Ferro Ham Chloranilic Acid Precipitation • Atomic Absorption
(End product: Chloranilic acid- purple color)
-Venous occlusion: Inc. Ca (acidosis) Spectrophotometry
-Analysis of Ca in urine (24h urine) (reference method)
- acidified with 6 mol/L HCl, with approximately 1 mL of the acid added for each • Ortho- Cresolpthalein Complexone Dyes
(Colorimetric method) • Emission Flame Photometry
100 mL of urine.
-Hemolysis cause False Inc. -Dye: Arzeno III
-Mg inhibitor: 8 hydroxyquinoline (chelator)
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DETERMINATION OF CALCIUM
Inorganic Phosphorus
-major intracellular anion
-Component of phospholipid, nucleic acid, creatine phosphate, ATP and
2,3,BPG
-inveresely related to Ca
• REFERENCE RANGE:
-Absorbed in jejunum
-Phosphate is essential for the insulin mediated entry of glucose into
cells
-Most Phosphate in serum is in inorganic form

ctolenada@ feu.edu.ph ctolenada@ feu.edu.ph

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FACTORS AFFECTING PHOSPHATE

Inorganic Phosphorus CONCENTRATION
• Inorganic Phosphorus exist as: • PTH
-Organic phosphate: Principal anion within cells - lowers blood concentrations
-Inorganic phosphate: part of the blood buffer
• Vitamin D
- increases both phosphate absorption in the intestine and phosphate
• Distribution: Forms of Phosphorus reabsorption in the kidney.
-80% Bone -Free or unbound form: 55%
-20% Soft tissue -Complexed with ions:35% • GH
-Protein-bound: 10% - phosphate concentrations in the blood may increase because of decreased
-<1% serum /plasma
renal excretion of phosphate.

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Inorganic Phosphorus : CLINICAL APPLICATION Determination of Inorganic Phosphorus

• Hypophosphatemia Hyperphophatemia • Specimen

-Hypoparathyroidism
-Alcohol abuse -Serum or lithium heparin plasma
-Renal failure (tubular failure)
-Primary hyperparathyroidism -Lymphoblastic leukemia -Oxalate, citrate, or EDTA anticoagulants should not be used
-Avitaminosis D -Hypervitaminosis D -Hemolysis should be avoided
-Myedema -Increase breakdown of cells -Circulating phosphate levels are subject to circadian rhythm
-Urine analysis for phosphate requires a 24-hour sample collection
-Fasting serum phosphate is controlled by the parathyroid gland

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55 56

Determination of Inorganic Phosphorus Determination of Inorganic Phosphorus

Methods
• Reference Range
• UV Method
-PO 4- + H 2SO 4 + Ammonium Molybdate à Ammonium Phosphomolydate
Complex (340nm)

• Fiske-Subbarow Method (Ammonium Molybdate method)

-Final Product: Molybdenum blue (600-700 nm): Reduced FORM
-Most commonly used method
-Most common reducing agent: Pictol (Amino naphthol Sulfonic Acid)
-Other reducing agents: Elon, ascorbic acid, and sanidine
-Unreduced complex at 340 nm is the most accurate measurement
-pH must be maintained in the acid range
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LACTATE
Lactate: Regulation
- by-product of an energy mechanism that produces a small amount of
ATP when oxygen delivery is severely diminished.
- The conversion of pyruvate to lactate is activated when a deficiency of
oxygen leads to an accumulation of excess NADH -O2 deprivation
(hypoxia)

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Lactate: Clinical Application Determination of Lactate

• Specimen REFERENCE RANGE

-Heparinized blood on ice
-Plasma must be quickly separated
-Avoid venous stasis during collection

• Method
-Lactate Oxidase
Lactate + O 2 –LOà Pyruvate + H 2O 2
H 2O 2 + H + + Chromogen –Peroxidaseà Colored dye + 2H 2O

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Anion Gap
Anion Gap
-the difference between the unmeasured cations (Sodium and • ↑ unmeasured anions ↓ unmeasured anions
Potassium) and unmeasured anions (chloride and bicarbonate) - Uremia (PO4 & SO4) -Hypoalbuminemia
-quality control for the analyzer used to measure this electrolytes -Ketoacidosis
-Used to monitor recovery from diabetic ketoacidosis -Lactic acidosis
-Abnormal anion gaps in sera from healthy person indicate an
instrument problem ↑ in unmeasured cations:
-Hypermagnesemia
• AG= Na+ – (Cl- + HCO3-) RV= 7-16 mEq/L -Hypercalcemia
• AG= (Na+ + K +) – (Cl- + HCO3-) RV= 10-20 mmol/L
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11
DEPARTMENT OF MEDICAL TECHNOLOGY
INSTITUTE OF ARTS AND SCIENCES
FAR EASTERN UNIVERSITY

Endocrinology
Prepared by: Charlene Princess S. Tolenada, RMT, MSMT
ENDOCRINE SYSTEM
• network of ductless glands that secrets hormones
• regulatory system of the body
 HORMONES
• chemical signals produced by specialized cells
• For growth and development of an individual
• regulated by the metabolic activity
MAJOR FUNCTION:
- maintain constancy of chemical
composition of extracellular and
intracellular fluids
- control metabolism
- Growth
- Fertility
- responses to stress
 Feedback mechanism
• Positive Feedback system:
- Is a system in which an increased in the product results to
elevation of the activity of the system and the production
rate
- Ex. Gonadal, thyroidal, adrenocortical hormone

• Negative Feedback system:

- Is a system in which an increased in the product results to
decreased activity of the system and the production rate
- Ex. Luteinizing hormone
 Types of Hormone Actions
• Endocrine
- secreted in one location and release into blood circulation

• Paracrine
- Is secreted in endocrine cells and released into interstitial space

• Autocrine
- Secreted in endocrine cells and sometimes released into interstitial space

• Juxtacrine
- Is secreted in endocrine cells and remains in relation to plasma membrane
 Types of Hormone Actions
• Intracrine
- Secreted in the endocrine cells and remained as well as function inside the synthesis of
origin

• Exocrine
- secreted in the endocrine cells and released into lumen of gut

• Neurocrine
- Secreted in neurons and released into extracellular space

• Neuroendocrine
- Secreted in neurons and released from nerve endings
Control of Hormones
• The majority of endocrine functions are regulated through the pituitary
gland, which in turns is controlled by secretions from the hypothalamus
Classification of Hormones According to
Composition or Structure
• Peptides and Proteins
- Synthesized and stored within the cell in the form of secretory granules
- cannot cross the cell membrane
- water soluble and not bound to carrier protein

1. Glycoprotein
• FSH, HCG, TSH, Erythropoietin

2. Polypeptides
• ACTH, ADH, GH, Angiotensin, cholecystokinin, gastrin, glucagon, insulin,
melanocyte stimulating hormone, oxytocin, PTH, prolactin, somatostatin
Classification of Hormones According to
Composition or Structure
• Steroids
- cholesterol as a common precursor
- Produced by adrenal glands, ovaries, testes, and placenta
- water insoluble and circulate bound to a carrier protein
- Ex. : aldosterone, cortisol, estradiol, progesterone, testosterone, and
activated Vit D3
• Amines
- Derived from an amino acid
- Intermediary between steroid and protein hormones
- Ex. Epinephrine, norepinephrine, triiodothyronine, and thyroxine
Hypothalamus and Pituitary Gland
 Hypothalamus
• Portion of the brain located in the walls and floor of the third
ventricle
• Above the pituitary gland and is connected to the posterior pituitary
by the infundibulum (pituitary stalk)
• It is the link between the nervous system and the endocrine system
Hypothalamus: Anterior Region
• Supraoptic region
• Supraoptic and paraventricular nuclei produce Vasopressin and
oxytocin
• Regulate body temperature through sweat
• maintains circadian rhythms
Hypothalamus: Anterior Region

• The neurons in the anterior portion release the following

hormones
• Thyrotropin releasing hormone (TRH)
• Gonadotropin releasing hormone (Gn-RH)
• Somatostatin aka Growth hormone inhibiting hormone (GH-IH)
• Growth hormone releasing hormone (GH-RH)
• Prolactin-inhibiting factor
Hypothalamus: Middle Region
- tuberal region
- arcuate nucleus and ventromedial nucleus
- The arcuate nucleus: appetite and releasing growth hormone-
releasing hormone (GHRH)
- ventromedial nucleus: regulate appetite and growth.
Hypothalamus: Posterior Region
• mammillary region
• posterior hypothalamic nucleus helps regulate body temperature
• mammillary nucleus is involved in memory function.
Endocrine Glands
Pineal Gland
• attached to the midbrain
• secretes melatonin
• secretion are controlled by the nerve stimuli
Pituitary Gland (Hypophysis)
• known as “Master Gland”
• located in small cavity in the sella turcica or Turkish saddle
• connected by the infundibular stalk to the median eminence of the
hypothalamus
• All pituitary hormones have circadian rhythm
Hypophysiotropic Hormones

Hormone Action
Thyrotropin-releasing hormone (TRH) Releases TSH & prolactin
Gonadotrophin-releasing hormone Releases LH and FSH
(GnRH)
Corticotropin-releasing hormone (CRH) Releases ACTH
Growth hormone-releasing hormone Releases GH
(GHRH)
Somatostatin Inhibits GH & TSH release
Dopamine (Prolactin inhibitory factor) Inhibits prolactin release
Anterior Pituitary (Adenohypophysis)
Anterior Pituitary (Adenohypophysis)
• True endocrine gland
• Regulates the released of Prolactin, growth hormone, gonadotropins
(FSH, LH), TSH, and ACTH
Anterior Pituitary (Adenohypophysis)
5 Types of Cells by Immunochemical Test
1. Somatotrophs- growth hormone
2. Lactotroph or mammotrophs- prolactin
3. Thyrotrophs- TSH
4. Gonadotrophs- LH and FSH
5. Corticotrophs- proopiomelanocortin
Hormones Secreted by the Anterior
Pituitary Gland
Hormone Feedback Hormone
Luteinizing hormone (LH) Sex steroids (E2/T)
Follicle-stimulating hormone (FSH) Inhibin
Thyroid-stimulating hormone (TSH) Thyroid hormones (T4/T3)
Adrenocorticotropin hormone (ACTH) Cortisol
Growth Hormone Insulin-like growth factor, IGF-I
Prolactin Unknown
Growth Hormone (GH)/ Somatotropin
• Most abundant of all pituitary hormones
• Controlled by GH-RH and somatostatin
• secretion is eratic
• metabolize fat stores while conserving glucose
• Major Stimulus: Deep sleep
• Major inhibitor: somatostatin
• Physiologic stimuli : stress, fasting, high protein diet
• Pharmacological stimuli: sex steroid, apomorphine, and levodopa
• GH suppressor: Glucocorticoid and elevated fatty acid
• Method: Chemiluminescent immunoassay
• Reference Range (fasting): 7 ng/mL
Growth Hormone (GH)/ Somatotropin:
Disorder
Increased Decreased
Acromegally Hyperglycemia
Chronic Malnutrition Obesity
Renal disease Hypothyroidism
Cirrhosis
Sepsis
GH Deficiency (GHD)
• Idiopathic growth hormone deficiency
- Most common in children
- In children with pituitary dwarfism, normal proportion are retained
and show no intellectual abnormalities
• Pituitary Adenoma
- Most common etiology in adult-onset GH deficiency
Growth Hormone (GH)/ Somatotropin:
Diagnostic Test

•Patient Preparation: Complete rest 30 minutes

before blood collection
•Specimen requirement: preferably fasting serum
Growth Hormone (GH)/ Somatotropin:
Diagnostic Test
For GH deficiency

Screening test: Physical Activity Test (Exercise Test)

- Result of the test: elevated serum GH

Confirmatory Test
• Insulin Tolerance Test: Gold standard
• Arginine Stimulation test: 2nd confirmatory test
- Procedure: 24 hour or night time monitoring of GH
- Expected Value: >5ng/mL (adults)
>10ng/mL (child)
Growth Hormone (GH)/ Somatotropin:
Diagnostic Test
• Acromegaly
- Screening test: Somatomedin C or Insulin-like growth factor 1 (IGF-1)
-Increased in px with acromegaly
- Decreased in GHD
• Confirmatory Test: Glucose suppression test -OGTT (75 g glucose)
- Blood is collected every 30 minutes for 2 hrs; fasting samples is
required
Growth Hormone (GH)/ Somatotropin:
Diagnostic Test
• Interpretation of result (CONFIRMATORY TEST)
- normal response : suppression of GH less than 1 ng/mL
-ACROMEGALY: If GH fails to decline less than 1 ng/mL
- Failure of GH to be suppressed below 0.3 ug/L, accompanied by
elevated IGF-1
-EXCLUDE ACROMEGALY: Suppression of GH below0.3 ug/ L with
normal IGF-1
- FOR FOLLOW UP AND MONITORING: Suppression of GH but increased
IGF-1
Gonadotropins- Follicle Stimulating Hormone
(FSH) and Luteinizing Hormone (LH)
• fertility and menstrual cycle disorders
• Present in both male and female
• FSH : spermatogenesis (male)
• LH : testosterone (male)
• For ovulation and the final follicular growth (female)
• LH acts on thecal cells to cause the synthesis of androgens, estrogens
(estradiol and estrone) and progesterone
• Elevation of FSH : premature menopause
• Increased of FSH and LH after menopause is due to lack of estrogen
Thyroid Stimulating Hormone (TSH)
- Thyrotropin
- uptake of iodine by the thyroid gland
- Acts to increase the number and size of follicular cells
- Alpha subunit: has the same amino acid sequence of LH, FSH, HCG
- Beta subunit: carries specific information to the binding receptors for
expression of hormonal activities
-evaluation of infertility
Adrenocorticotrophic Hormone (ACTH)
- Single-chain peptide without disulfide bonds
- It is produced in response to low serum cortisol
- Deficiency : atrophy of the zona glomerulosa and zona reticularis
- Higest level: 6:00 am to 8:00 am; Lowest level: 6:00 pm to 11pm
- Increased in Addison’s disease , ectopic tumors, after protein rich
meals
Adrenocorticotrophic Hormone (ACTH)
Spx Requirements:
-collected in a prechilled polysterene (plastic) EDTA
- not be allowed to have contact with glass
- Best time for collecting specimen: 8am to 10 am
Prolactin
• Pituitary lactogenic hormone; a stress hormone; a direct effector
hormone
• For initiation and maintenance of lactation
• Conjunction with estrogen and progesterone
• Major inhibitor: Dopamine
• Consequences of prolactin excess: hypogonadism
• Increased: pituitary adenoma, infertility, amenorrhea, galactorrhea,
acromegaly, renal failure, Polycystic ovary syndrome, cirrhosis, and
primary and secondary hypothyroidism
Prolactin
• Prolactin serum level > 200 mg/dL : pituitary tumor (prolactinoma can result in
anovulation)
• Specimen requirement: blood should be collected 3 to 4 hrs after the px is
awakened; fasting sample
• Highest serum level (during sleep): 4:00am - 8:00 am; 8:00pm - 10:00 pm
• Method: immunometric assay
• Physiologic stimuli: exercise, sleep, stress, postprandially, pain, coitus, pregnancy,
nipple stimulation or nursing
• Pharmacologic (increased): intake of verapamil, phenothiazines, olanzapine,
Prozac, cimetidine, and opiate
• Reference value:
• Male: 1-20 ng/mL (1-20ug/L)
• Female: 1-25 ng/mL (1-25 ug/L)
Posterior Pituitary
(Neurohypophysis)
Posterior Pituitary (Neurohypophysis)
Hormone Function

Vasopressin Regulate renal free

(antidiuretic H2O excretion
hormone)
Oxytocin Lactation, labor,
parturition
Oxytocin
• secreted in association with carrier protein
• stimulates contraction of the gravid uterus at term- “Fergusson
reflex”
• plays a role in homeostasis at the placental site following delivery
• Synthetic preparation: to increase weak uterine contraction during
labor and to aid in lactation
Anti-Diuretic Hormone
• Acts on the distal convoluted and collecting tubules of the kidneys
• Major Functions: to maintain osmotic homeostasis by regulating
water balance
• It increases blood
• affects blood clotting by promoting factor VII release from
hepatocytes and factor VIII (von Willebrands factor)release from the
endothelium
Anti-Diuretic Hormone
• Physiologic stimuli to ADH secretion: Nausea, cytokine, hypoglycemia,
hypercarbia, and nicotine
• Physiologic stimuli to ADH release: dehydration, physical and
emotional stress due to major surgery
• Potent physiologic stimuli to ADH release: emetic stimulus
• Inhibitor of ADH release: ethanol, cortisol, lithium, and
demeclocycline
• Reference value: 0.5-2 pg/uL
Anti-Diuretic Hormone: Clinical Disorder
- Diabetes insipidus is a deficiency of ADH
- Clinical pictures includes:
-Polyuria with low specific gravity
-Polydipsia
-Polyphagia
Major Types of Diabetes Insipidus
Nephrogenic Diabetes insipidus
- Characterized by having normal ADH but abnormal ADH receptor
- due to failure of the kidneys to respond to normal or elevated ADH
levels
- Nephrogenic DI is either congenital or acquired
Diagnostic test for Diabetes Insipidus: Overnight
water Deprivation test (Concentration test)
• Fasting : 10:00 pm onwards (8 -12 hrs)
• After 8-12 hrs without fluid intake, urine osmolality does not rise
above 300 mOsm/kg
• In neurogenic DI, ADH levels are low
• In nephrogenic DI, ADH levels are either normal or increase
Pituitary Tumors
Gigantism - ↑ GH
Acromegaly - ↑ GH
Dwarfism - ↓ GH
Kallmann’s Synd - ↓ GnRH
Diabetes insipidus - ↓ AVP
DEPARTMENT OF MEDICAL TECHNOLOGY
INSTITUTE OF ARTS AND SCIENCES
FAR EASTERN UNIVERSITY

Adrenal Gland
Prepared by: Charlene Princess Salvador Tolenada, RMT, MSMT
Adrenal Gland
• Pyramid like shape located above the kidney
• composed of Adrenal cortex and adrenal medulla
• Pathologic conditions are associated to blood pressure and
electrolyte balance
Adrenal Cortex •secretes steroid hormone
3 LAYERS OF THE ADRENAL CORTEX
• Zona glomerulosa : 10%
-mineralocorticoid

• Zona Fasciculata :75%

-Glucocorticoid and unsulfated DHEA

• Zona Reticularis :10%

- androstenedione and dehydroepiandrosterone
CORTISOL
• principal glucocorticoid
• synthesis is regulated by ACTH
• anti-insulin effect
• hormone that inhibit the secretion of ACTH
• high level (8:00-10:00 am); lowest at night (10:00 to 12:00 mn)
• Specimen: serum (red top), urine
-Blood sample should be drawn at 8am
• Urine free cortisol levels are sensitive indicators of adrenal hyperfunction
(endogenous corticolism) -24 hour urine collection
• Reference value: 5-25 ug/dL (140-690 nmol/L) at 8am to 10am
CORTISOL: Urinary metabolites
17-hydrocorticosteroid
• Method: Porter-Silber Method (yellow color)
• Reagent: Phenyl hydrazine in H2sO4 +alcohol

17-Ketogenic steroids
• Method: Zimmermann Reaction (reddish purple)
• Reagent: Meta-dinitrobenzene
• Oxidation procedure: Norymberski (Na+ bismuthate)
CORTISOL: Clinical Disorder
Hypercortisolism (Cushing’s Syndrome)
• adrenocortical hyperfunction
• excessive production of cortisol and ACTH but decreased aldosterone and renin
• Overuse of corticosteroids
• Sign and symptoms:
-obesity but with thin extremities (buffalo hump)
-hirsutism
-hyperglycemia
-thinning of the skin
-pour wound healing
-hypertension
-hypercholesterolemia
-Low WBC count (lymphocytes)
CORTISOL: Clinical Disorder
Hypercortisolism (Cushing’s Syndrome)
-SCREENING TEST: 24 hour urine free cortisol test
-overnight dexamethasone suppression test (blood level not
suppressed)
-midnight salivary cortisol test (high saliva cortisol)
-CONFIRMATORY TEST: Low dose dexamethasone suppression test
-midnight plasma cortisol (>7.5 ug/dL serum cortisol confirmatory)
-corticotrophin-releasing hormone (CRH) stimulation test

-HPLC-MS: reference method for measuring urinary free cortisol

-Saliva specimen is analyzed by immunoassay or by liquid chromatography-
mass spectrometry (LC-MS/MS)
CORTISOL: Clinical Disorder
HYPOCORTICOLISM
• Primary Hypocorticolism (Primary adrenal insufficiency)
-due to decreased cortisol production
-Disorder: Addison’s disease
-SCREENING TEST: ACTH Stimulation Test (low cortisol, aldosterone and
renin; high ACTH)
-Confirmatory Test: Insulin Tolerance Test
CORTISOL: Clinical Disorder
HYPOCORTICOLISM
• Secondary Hypercortisolism (Secondary Adrenal Insufficiency)
-due to hypothalamic-pituitary insufficiency with loss of ACTH
- No problem with mineralocorticoid secretion; absence of
hyperpigmentation
-Screening Test: ACTH stimulation Test (delayed response-low cortisol
and ACTH)
-Confirmatory Test: Insulin Tolerance Test
• ACTH Stimulation Test
-Known as corsyntropin stimulation test
-differentiate secondary adrenal insufficiency from tertiary adrenal
insufficiency
-Blood collection: Random
-Corsyntropin: synthetic cortisol and aldosterone stimulator

• Insulin Tolerance Test

-gold standard for secondary and tertiary hypocorticolism
-confirm borderline response to ACTH stimulation test
-Blood collection: Baseline, and then at 15, 30,45, 60, 90 and 120
minutes following insulin administration
• Overnight Metyrapone Test
-used as alternative test diagnostic or confirmatory test for secondary
or tertiary adrenal insufficiency
-Blood collection: blood is collected the following morning at 8am
CORTISOL: Clinical Disorder
HYPOCORTICOLISM
• Congenital Adrenal Hyperplasia
-result from deficiency of enzymes such as 21 hydroxylase, 11 beta
hydroxylase and 3 Beta hydroxysteroid dehydrogenase-isomerase
-Result to decreased plasma cortisol, and increased ACTH and androgen
levels
-Definitive test: 17-OHP measurement in amniotic fluid
-Genotyping cells from chorionic villous sampling: Most Preferred
CORTISOL: Clinical Disorder
Types of CAH
• 21-hydroxylase deficiency
-most common form of CAH
-leads to hirsutism in women and other symptoms
-Diagnosis: measuring 17-OHP in amniotic fluid (PCR) or by genotyping cells (Southern
blotting) obtained by chorionic villus sampling
-Definitive test: Genotyping
-Common method for detection: ACTH stimulation and 17 a-hydroxyprogesterone tests
-Lab findings: elevated plasma levels of 17 a-hydroxyprogesterone and pregnanetriol
-increased urinary pregnanetriol and 17 KS
-Defective Gene: CYP21
CORTISOL: Clinical Disorder
Types of CAH
• 11 Beta-Hydroxylase deficiency
-associated with virilization and hypertension
-Lab findings: increased serum deoxycortisol, and urinary 17-OHCS and
17 KS
-Defective gene: CYP11B1
CORTISOL: Clinical Disorder
Types of CAH
• 3 Beta-hydroxysteroid dehydrogenase-isomerase deficiency
-results to elevated ratio of 17 a-hydroxypregnenolone to 17 a-
hydroxyprogesterone and increased ratio of DHEA to androstenedione
-characterized by pseudohermaphroditism in female infants, and incomplete
masculinization in male infants
-Lab findings: elevated serum 17- hydroxypregnenolone, pregnenolone and
DHEA
-increased urinary 17-KS
-Defective gene: HSD3B2
CORTISOL: Clinical Disorder
Types of CAH
• 17-hydroxylase deficiency
-inability to convert 17-hydroxypregnenolone to DHEA and 17-
ahydroxyprogeterone to androstenedione
-result to decrease androgen, cortisol and estrogen synthesis; decrease
progesterone synthesis
-Indicators: amenorrhea, defective ovarian maturation,
pseudohermaphroditism in males, hypertension, hypokalemic alkalosis
-Lab findings: Increased serum deoxycortisol
-defective gene: CYP17
ALDOSTERONE
-most potent mineralocorticoid
-18-hydroxysteroid dehydrogenase is an enzyme needed for the
synthesis of aldosterone
-Method: RIA and Chromatography
ALDOSTERONE: Clinical Disorders
Primary hyperaldosteronism (Conn’s disease)
-Symptoms: hypertension, hypokalemia, mild hypernatremia and metabolic alkalosis
-Patient Preparation: Patient should be upright for atleast 2 hours prior to blood
collection
-Measurement: (PAC/ PRA) ratio, serum aldosterone and cortisol
-Screening test: Plasma aldosterone concentration/ Plasma renin activity ratio (PAC/
PRA)
(+) Result: >30 Ratio-suggestive of primary hyperaldosteronism
>50 Ratio-diagnostic of primary hyperaldosteronism
-Confirmatory Test:
-Saline suppression test
-Oral Sodium loading Test
-Fludrocortisone suppression test
-Catopril challenge test
Saline Supression Test:
-involves infusing 2 L of 0.9% saline over 4 hours, or by administering
10-12 mg NaCL Tablets daily for 3 days
-(+) Result: > 10 ng/dL
ALDOSTERONE: Clinical Disorders
Secondary Hyperaldosteronism
-Elevated plasma level of aldosterone and renin
-secondary hyperaldosteronism that result in hypokalemia: renal artery
stenosis, diuretic therapy, malignant hypertension, and congenital defects in
renal salt transport such as Bartter’s syndrome and Gitelman’s syndrome
 ALDOSTERONE: Clinical Disorders
Associated disorders

Liddle’s syndrome (pseudohyperaldosteronism)

-increased ENaC (Epithelial Sodium Channel)
-resembles primary aldosteronism clinically, but aldosterone and renin are low, with absence
of hypertension

Bartter’s syndrome (Bumetanide-sensitive chloride channel mutation)

-rare potassium-losing autosomal recessive disorder
-elevated concentration of plasma aldosterone and renin

Gitelman’s syndrome (Thiazide-sensitive transporter mutation)

-defect in NaCl reabsorption
-accompanied by elevated aldosterone
 ALDOSTERONE: Clinical Disorders
Hypoaldosteronism
-destruction of the adrenal glands and deficiency of glucocorticoid
-associated with 21-hydroxylase deficiency
-Symptoms: hyperkalemia and metabolic acidosis
-Test:
-Furosemide stimulation test or upright posture- (+) result: Low
aldosterone levels
-Saline Suppression test (+) Result: High Aldosterone Level
• Px Prep:
-Blood samples should be drawn in the morning before the px has
gotten out of bed
-Blood sample for renin should be drawn into an iced EDTA tube
WEAK ANDROGEN/ ADRENAL ANDROGENS
- by products of cortisol synthesis that are regulated by ACTH
-Circulate bound to steroid hormone binding globulin (SHBG)
-Ex: Dehydroepiandrosterone (DHEA) and androstenedione
Adrenal Medulla Synthesis of Medullary Hormones

-composed primarily of
chromaffin cells that
secretes cathecolamines
-L-tyrosine is the precursor
of cathecolamines

Dopamine ->
Methoxytyramine
Norepinephrine (Primary amine)
-produced by sympathetic ganglia
-highest conc found in CNS

-Major metabolite:
-3 methoxy-4-hydroxyphenolglycol (MHPG) -CSF and Urine
-Vanillylmandelic acid (VMA)
Epinephrine (adrenaline / secondary amine)
-Flight or Fight Hormone
-Inc glucose conc

-Major metabolite: VMA

-Minor metabolite: metanephrines, normetanephrines homovanillic
acid
Dopamine (Primary Amine)
-catecholamine produced in the body by the decarboxylation of 3,4
dihydroxyphenylalanine (DOPA)
-the major intact catecholamines present in urine

-Major metabolite: HVA

Clinical Disorder:
• Pheochromocytoma
-due to over production of cathecolamines
-Screening test: Plasma metanephrines and normetanephrines by HPLC
-Diagnostic test: 24 hr urinary excretion of metanephrines and
noremetanephrines (Inc levels)
-Px Prep: avoid caffeine, nicotine, alcoholand acetaminophen,
monoamine oxidase inhibitor, and tricyclic antidepressants for at least
5 days before testing
Pharmacologic test
• Clonidine Test: differentiate pheochromocytoma (not suppressed) to
neurogenic hypertension (50% decreased in catecholamines level)

• Glucagon Stimulation Test: highly suggestive of pheochromocytoma

• Neuroblastoma: fatal malignant condition in children resulting to

excessive production of norephinephrine
-(+) high urinary excretion of HVA or VMA or both, and dopamine
Methods
• Specimen: 24 hr urine and blood (Plasma)
Px Prep:
-overnight fasting
-px in placed in a reclining position in a quiet environment and heparin lock
is inserted intravenously. After 20-30 minutes blood is collected in a
prechilled EDTA tube

• Chromatography: HPLC or GC-MS (VMA and metanephrines)

• Radioimmunoassay: sensitive screening test for total plasma
catecholamines
-> 2000 pg/mL of plasma catecholamines- diagnostic for
pheochromocytoma