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CC Combined Notes

Bachelor of Science in Medical Laboratory Science (Cagayan State University)

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CLINICAL CHEMISTRY 1 LECTURE NOTES RAISA M. MIRZA, RMT by God’s grace

AUGUST 2021 MEDICAL TECHNOLOGY LICENSURE EXAMINATION REVIEW (Klubsy Bear Online Tutorial)
Contents:
I. Basic Principles and Practices
II. Laboratory Safety
III. Quality Control
IV. Analytical Techniques
I. Basic Principles and Practices:
The primary purpose of a clinical chemistry laboratory is to facilitate the correct performance of analytic processes that yield
accurate and precise information, aiding patient diagnosis and treatment
UNIT OF MEASURE
Systeme International d’Unites (SI) (1960) – This system was devised to provide a global scientific community with a
uniform method to describing physical quantities. These SI system units are based on the metric system.
∑ Basic units- seven basic units having meter, kilogram, mole being the units most frequently encountered
∑ Derived units- derivative or a mathematical function describing one of the basic units
∑ Non-SI units-so widely used they have become acceptable but cannot be technically cathegorized under SI units

Standard prefixes are added to a given basic unit which can indicate decimal fractions or multiples of a unit (subunit)

REAGENTS: chemicals, standards, solutions, buffers and water

Spectroquality- designates the highest purity of a reagent
Grades of Purity of Analytical Chemicals:
1. Analytic Reagent (AR)- suitable Labels either state the actual impurities or the list of maximum allowable impurities
for use in most analytic laboratory Labels should reveal the percentage of impurities and either of the initials AR or
procedures ACS(American Chemical Society) for laboratory use
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2. Ultrapure Used in specific procedures such chromatography, atomic absorption, fluorometry,

immunoassays, molecular diagnostics, HPLC, standardization, or other techniques that
require pure chemicals
3. Chemically Pure (CP) ∑ Melting point analysis- used to ascertain the acceptable purity range
Impurity limitations not stated
Preparation not uniform; Not recommended for clinical laboratories
4. United States Pharmacopeia Chemicals may be pure enough for use in most chemical procedures but consider that
(USP) or National Formulary (NF) their purity standards are not based on the needs of the laboratory
∑ Used to manufacture drugs
5. Technical or Commercial grade Primarily in manufacturing and should never be used in the clinical laboratory
Reference Materials:
1. Primary Standard Highly purified chemical that can be measured directly to produce a substance of exact known
concentration and purity (ACS purity: 100 ± 0.02%)
Standard reference materials (SRMs)- substitute made by National Institute of Standards and
Technology (NIST)
2. Secondary Standard Substance of lower purity, with its concentration determined by comparison with a primary
standard or SRM
3. Control Represents a specimen that is in similar composition to the patient’s whole blood or plasma. The
value is known and measured the same way with the patient’s unknown sample
TWO levels of control: THREE levels of control:
-Used for majority of tests in Clinical Chemistry Used for:
∑ Normal -Blood Gases
∑ Pathologic -Therapeutic Drugs
-Hormones
∑ Low
∑ Normal
∑ High
Reasons for using a Standard/ Calibrator:
1. Change in the reagent lot number
2. Reagent control is out of range
3. When performing Method of Evaluation and Technical Validation/Verification
4. When establishing a Reference Range
Water Specifications:
Water is the most frequently used reagent in the laboratory (tapwater- unsuitable; purified water is used)
∑ Prefiltration- removes particulate matter from municipal water supplies before any additional treatments
Filtration cartridges are composed of glass, cotton, activated charcoal (removes organic materials and chlorine) and
submicron filters (≤0.2mm) (removes any substances larger than the filter’s pores including bacteria)
∑ Water Quality- Monitoring parameters are: resistance, pH, microbiological count, silicate, total dissolved solids
(chlorine, ammonia, nitrate or nitrite, iron, phosphate, sodium, metal detection, hardness) total oxidizable organic
carbon (carbon dioxide and chemical oxygen demand)
Classifications of Water according to their Preparation:
1. Distillation Distilled water has been purified to remove almost all organic materials
Water is boiled, vaporized, and condensed (can be done more than once)
∑ Double Distillation- used for tests requiring ammonia free water
2. Deionization Deionized water has been purified by ion exchange (either by an anion or cation exchange resins then
are replaced by hydroxyl or hydrogen ions)
∑ Two-bed system- uses both anion and cation resins; excellent in removing dissolved ionized
solids and dissolved gases
Deionization removes some or all ions although some organic materials may be present (neither pure
nor sterile); It is purified from a previously pre-filtered or distilled water
3. Reverse-Osmosis RO Water has been produced by pressure pumping water across a semipermeable membrane (filtered)
May be used as pretreatment of water
It does not remove dissolved gases
4. Ultrafiltration Excellent in removing particulate matter, microorganisms and any pyrogens or endotoxins
and nanofiltration ∑ UV oxidation (removes some trace organic material) or Sterilization (uses specific

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wavelengths), together with ozone treatment- can destroy bacteria but may leave residual
products (UV radiation at the biocidal UV of 245nm)

Classification of Water according to their Type (Conventional/Traditional):

Type I The most stringent requirements and generally suitable for routine laboratory use
Used in clinical chemistry (highest purity)
Used for preparation of reference buffer solutions and reconstitution of standard
Used for test methods requiring minimum interference such as trace metal, iron and enzyme analyses
Type II Acceptable for most analytic requirements including reagent quality control and standard preparations
Type III Autoclave wash water- acceptable for glassware washing but not for analysis or reagent preparation

Six Categories of Reagent Grade Water according to their use (Clinical and Laboratory Standards Institute):

1. Clinical Laboratory Reagent Water (CLRW)

2. Special Reagent Water (SRW)
3. Instrument Feed water
4. Water supplied by method manufacturer
5. Autoclave and wash water
6. Commercially bottled purified water
Solution Properties:
Solution: a mixture with a solute dissolved a solvent
Solute: substance dissolved in a liquid
Solvent: liquid in which the solute is dissolved
Any chemical or biologic solution is described by its basic properties, including concentration, saturation, colligative
properties, redox potential, conductivity, density, pH, and ionic strength
∑ Concentration
1. Percent Solution- expressed as equal parts per hundred or the amount of solute per 100 total units of the solution. Three
expressions are: weight/weight (%w/w), volume per volume (%v/v) and weight per volume (%w/v) –(most common; v/v-
g/dL)
2. Molarity (M)- expressed as the number of moles per 1 Liter of solution. One mole of a substance equals its gram molecular
weight (gmw). SI expressions are mol/L, mmol/L, µmol/L, and nmol/L (depends on volume, any significant physical changes
such as changes in temperature and pressure will influence)
3. Molality (m)- represents the amount of solute per 1 kg of solvent, Preferably expressed as weight/weight, mol/kg
4. Normality (N)- expressed as the number of gram equivalent weights per 1 Liter of solution. An equivalent weight is equal to
the gmw of a substance divided by its valence. The valence is the number of atoms/elements that can combine to a particular
compound.( gram combining weight of the material) Expressed as mEq/L but has been replaced as mmol/L. Often used in
chemical titrations and chemical reagent classification;
∑ Saturation- Temperature and presence of ions that influence the solubility constant for a solute in a given solution
and thus, affect the saturation
Dilute solution relatively little solute; lower solute concentration per volume of solvent when making a dilution
Concentrated solution large quantity of solute in a solution
Saturated solution excess of undissolved solute particles in a solution
Supersaturated solution greater concentration of undissolved solute particles than a saturated solution of the same
substance (thermodynamically unstable- addition of a crystal solute or by mechanical agitation can
result to crystallization of the excess material) –(Seen in Serum Osmolality by FPD)
∑ Colligative properties- behavior of particles or solutes in solution based only on the relative number of each kind of
molecules present
Freezing Point temperature at which the vapor pressure of the solid and liquid phases are the same
Vapor Pressure pressure at which the liquid solvent is in equilibrium with the water vapor
Boiling Point temperature at which the vapor pressure of the solvent reaches 1 atm
Osmotic Pressure pressure that allows solvent flow through a semipermeable membrane to establish equilibrium between
compartments of different concentrations
When a solute is dissolved in a solvent, these colligative properties change in a predictable manner. For each osmole of
substance present:
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ÿ Freezing point is lowered by 1.86C

ÿ Vapor pressure is lowered by 0.3 mmHg or torr
ÿ Boiling point is raised by 0.52C
ÿ Osmotic pressure is increased by a factor of 1.7 x 104 mmHg or torr
∑ Redox potential (Oxidation-Reduction potential) – measure of the ability of a solution to accept or donate electrons
Reducing agents substances that DONATE electrons LEO lose electrons, oxidized
Oxidizing agents substances that ACCEPT electrons GER gain electrons, reduced
∑ Conductivity – measure of how well electricity passes through a solution; depends on the number of respective
charges of the ions present; expressed as ohms-1 or mho
∑ Resistivity – reciprocal of conductivity; measure of a subtance’s resistance to a passage of electrical current; expressed
as ohms
∑ pH and Buffers:
Buffers- weak acids or bases and their related salts; as a result if their dissociation characteristics, they minimize changes in
the hydrogen ion concentration.
pH which is the negative logarithm or inverse log of hydrogen ion concentration; ranges from 0 to 14
= − log
Henderson-Hasselbach equation- mathematically describes the dissociation characteristics of weak acids (pKa) and bases
(pKb) on the effect on pH
= + log
ÿ Greatest buffering capacity- When the ratio of [A-] and [HA] is 1, pH equals the pK
ÿ Ionic strength- activity of ions in a solution or buffer; based on the charge and concentration of ions present;
important in separation techniques of buffers
Increasing the ionic strength increases the ionic cloud surrounding a compound and decreases the rate of particle migration
∑ Density and Specific Gravity
Density- expressed as pass per unit volume of a substance; expressed as g/mL
Specific gravity- ratio of the density of a material when compared to the density of a pure water at a given temperature; a
means of expressing density in terms of volume
CLINICAL LABORATORY SUPPLIES:
1. THERMOMETER DESCRIPTION
1. Liquid-in Glass Mercury (or red or other colored substance) encased in a plastic or glass material with a bulb at one
thermometer end and a graduated stem; usually measure between 20C and 400C
Visual inspection should reveal a continuous line of liquid free from separation or gas bubbles
Examples:
a. Partial immersion- heating blocks and water bath
b. Total immersion- refrigerator
c. Surface- incubators or heating oven
2. Electronic Advantage: Size and millisecond response time
thermometer Disadvantage: Initial cost
(Thermistor probe)
3. Digital thermometer
ÿ SRM thermometer and Gallium melting point cell – thermometer verification
ÿ Thermistor calibrated against gallium cell – reference for any type of thermometer
∑ Temperature:
Predominant practice for temperature measurement uses the Celcius C or centigrade scale
The SI designation for temperature is the Kelvin scale
Common Temperature Conversions:
ÿ Tc= ( ( − 32) or (Tf – 32) X 0.556
ÿ Tf= ( ( + 32) or (Tc + 32) X 1.8
ÿ K= Tc + 273.15
2. PLASTICWARES DESCRIPTION
1. Polystyrene Rigid clear; Should not be autoclaved
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Will crack and splinter

Used for test tubes and graduated cylinders
2. Polyethylene Relatively inert chemically; Should not be autoclaved
Resistant to most acids (exc. Conc’d H2SO4), alkali and salts
Used for test tubes, bottles, disposable transfer pipets, test tube racks
3. Polypropylene Relatively inert chemically; Can be autoclaved
Resistant to most acids, alkali and salts
Used for pipette tips and test tubes
4. Teflon Almost totally chemically inert; Resistant to a wide variety of temperature
5. Polycarbonate Very clear; Ideal for graduated cylinder
6. Polyvinylchloride Soft and flexible materials; Used for construct tubing
7. Polyolefins Noted for their strength and resistance to elevated temperature

3. GLASSWARES
1. High Thermal High resistance to thermal shock and chemical attack
Borosilicate Heavy walls to minimize breakage
(Kymax/Pyrex) Minimal contamination of liquids
Can be heated and autoclaved
Used for beakers, flasks, and pipets
2. High Silica (Vycor) Acid and alkali resistant, heat and electrical tolerance
Excellent optical pipettes
Used for high precision analytic work, optical reflectors and mirrors
3. Aluminosilicate (Corex) Six times stronger than borosilicate
Able to resist clouding due to alkali and scratching
4. Low Actinic Red or Amber- colored to prevent exposure of photosensitive substances to light
5. High Thermal Resist high temperature only
6. Boron Free Poor heat resistance; Alkali resistant
Used for highly alkaline solutions
7. Flint (Soda Lime) Glass Contains oxide of sodium, silicon and calcium
Least expensive; Used for some disposable glassware
Poor resistant to high temperature and fluctuations; Fair resistance to chemicals; Can release
alkali
∑ Glasswares are calibrated at 20C
∑ Two classes of Precision Tolerance:
1. Class A glasswares- Stamped with letter “A” in the glassware and are preferred in the laboratory applications
2. Class B glasswares- generally have twice the tolerance limits of Class A; often found in student laboratories where
durability is needed,
∑ Plasticware is beginning to replace glassware due to their high resistance to corrosion and breakage and their
flexibility as well. Some applications will also require plastic as glass absorbs metal ions
Cleaning Plasticwares and Glasswares:
∑ It is suggested that disposable glass and plastic be used whenever possible
∑ Immediately rinsing glass or plastic supplies after use, followed by washing with a powder or liquid detergent
designed for cleaning laboratory supplies and several distilled water rinses, may be sufficient.
∑ Presoaking glassware in soapy water is highly recommended whenever immediate cleaning is impractical.
∑ Successful cleaning solutions for glasswares are acid dichromate and nitric acid
∑ Brushes and abrasive cleaners should not be used in plasticwares
∑ Acid rinses or washes are not required for plasticwares
∑ Ultrasonic cleaners can help remove debris coating the surfaces of glasses and plastics
∑ Properly cleaned ware should be completely dried before using
1. Routine washing:
a. Soaking in dilute bleach followed by drying in an oven
b. Soaking in nitric acid solution for 12-24 hours
c. Soaking in acid dichromate solution (MCU)
2. For blood clots: Soak in 10% NaOH

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3. For New Pipets: Soak in 5% HCl or %% HNO3

4. For Metal Ion Determination: Soak in 20% HNO3
5. For Grease: Soak in any organic solvent or 50% KOH or Contrad 70 (Harleco)
6. For Permanganate Stains: Soak in 50% HCl or a mixture of 1%FeSO4 in 25% H2SO4
7. For Bacteriologic Glassware: Soak in 2-4% cresol followed by autoclave and thorough washing
8. For Iron Determination: Soak in HCL solution (conc’d HCl diluted 1:2) or HNO3 solution (conc’d HNO3 diluted 1:3) followed
by thorough washing
o LABORATORY VESSELS:
∑ Volumetric and Erlenmeyer flasks are two types of containers in general use in the clinical laboratory
1. Volumetric flask Has round flat lower portion and a long thin neck with the calibration line etched into the neck
-Calibrated to hold one exact volume
2. Erlenmeyer flask Has a wide bottom that gradually evolves into a smaller, short neck
-Calibrated to hold different volumes
3. Griffin Beaker Has a flat bottom, straight sides and an opening as wide as the flat base with a small spout in the lip
-Calibrated to hold different volumes
4. Graduated Cylinder Long cylindrical usually held upright by an octagonal or circular base
-Has calibration marks along its length and is used to measure volumes of liquids
o PIPETS: utensils made of glass or plastic that are used to transfer liquids; may be disposable or reusable
-Used for volumes 20 mL or less; with different tolerances depending on the volume (mL)
-Clean by soaking to soapy water with the pipet tips up.
Classifications and Types:
I. According to DESIGN:
a. To Contain (TC) Holds a particular volume but does not dispense that exact volume
-With an etched mark ring (near the mouth) Delivers the amount by washing it and emptying
-BLOW-OUT -The tips should be immersed in the liquid
b. To Deliver (TD) Dispenses the volume indicated; Transfers the exact volume
-Markings are absent Do not blow the last drop; Rate of flow is gravity
-SELF-DRAINING -The tip should not be in contain with the liquid
II. According to DRAINAGE CHARACTERISTICS:
Class: Used for: Calibrated by:
a. BLOW-OUT viscous solutions Mercury
b. SELF-DRAINING nonviscous solutions Distilled Water
III. According to TYPE:
A. MEASURING OR GRADUATED B. TRANSFER
Capable of dispensing several different volumes Designed to dispense one volume without further subdivisions
Examples: Examples:
Serologic – Blow-out, graduated up to the tip Pasteur – Do not have calibration marks; Transfer solutions
without considering of specific volume
Mohr – Self-draining, has dead space Ostwald-Folin – (bulb is nearer the mouthpiece) for accurate
measurement of viscous fluids such blood or serum; Blow-out
Ball, Kolmer or Khan – for volumes less than 1 mL Volumetric – (bulb is in the middle) for preparation of a standard;
∑ Sahli-Helige Self-draining; Used only once
∑ Lang-Levy -Pipet with greatest degree of accuracy and precision
∑ RBC or WBC pipet
Micropipet – total holding of less than 1 mL; may be mohr Micropipet/ Automatic pipet – for volumes less than 1 mL
or serologic - Draws up liquid and dispenses it automatically
IV. According to MECHANISM OF AUTOMATIC PIPETS:
Air Displacement Uses a suction aspirate and dispense sample through polypropylyne tip
Has one or two stops; Second stop (blow-out stroke) removes any remaining sample from the tip
Disposable tips
Seals require periodic lubrication
PISTON MECHANISM: air movement; relies on a piston for creating suction to draw sample into a
disposable tip that must be changed after use; The piston does not come in contact with the liquid
Positive Uses a glass capillary tip fitted with a Teflon-tipped plunger
Displacement Can be single channel (with one tip) or multichannel (with multitips)
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Reusable tips
Plunger setting must be checked and Teflon tip must be replaced periodically
PISTON MECHANISM: Hypodermic syringe-like; Operates by moving the piston in the pipet tip or
barrel; Piston comes in contact with the liquid; No air-interference
Direct contact enhances accuracy and precision for liquids which are too heavy or too viscous to be
displaced by air
Blow-out stroke is used during operation by the perfectly formed piston steel and prevent sample to
sample contamination (No carry-over)
Dispenser and Dispenser- obtain liquid from a common reservoir and dispense it repeatedly; can be:
Dilutor a. Bottle top
b. Motorized
c. Handheld
d. Attached to a dilutor
Dilutor- often combines sampling and dispensing functions
Precautions in Usage:
∑ Many automated pipets use a wash between samples to eliminate carryover problems.
∑ With manual or semiautomatic pipets, to minimize carryover contamination, careful wiping of the tip may remove
any liquid that adhered to the outside of the tip before dispensing any liquid. Care should be taken to ensure that the
orifice of the pipet tip is not blotted, drawing sample from the tip.
∑ In using manually operated semiautomatic pipets, move the plunger in a continuous and steady manner.
How to Use the Pipet:
1. Use the mechanical suction
2. Wipe off outside of the pipet with gauze
3. Adjust the meniscus (Read at the bottom of the curved liquid)
4. Drain into the receiving vessel

V. According to CALIBRATION:
Class A pipets do not need to be recalibrated by the laboratory. Automatic pipetting devices and non–Class A materials,
need recalibration.
Gravimetric Done by delivering and weighing a solution of known specific gravity such as water
Method Most desirable method
Photometric For automatic pipetting device
Method Spectrophotometer – molar extinction coefficient of a compound is obtained or comparison of different
dilution of potassium dichromate and its absorbance
∑ A method using a 0.1% solution of phenol red in distilled water has been used to compare the reproducibility of
different brands of pipet tips.
o BURETS: looks like a wide, long graduated pipet with a stopcock at one end; Used to dispense a particular volume of
liquid during titration; (Usual volume- 25 to 100 mL)
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o SYRINGES: sometimes used for transfer of small volumes (<500µL) in blood gas analysis or in separation techniques
such as chromatography and electrophoresis (disposable Teflon tips)
4. Desiccators and Desiccants: for prolonged storage
∑ Desiccants- excellent drying agents which are very hygroscopic and can remove moisture from air and other
materials; keeps other chemicals from being hydrated
∑ Desiccators- closed and sealed containers that contain desiccant material may be used to store more hygroscopic
substances
If these compounds absorb enough water from the atmosphere to cause dissolution, they are called deliquescent substances
5. Balances: A properly operating balance is essential in producing high-quality reagents and standards
Analytic and electronic balances are currently the most popular in the clinical laboratory.
∑ Mechanical Analytic balance (Substitution balance)- required for the preparation of the primary standard.
The weight range is 0.01mg to 60g
∑ Electronic balances- uses electromagnetic force to counterbalance the weighed sample’s mass. Advantage of a
fast response time (<10 seconds)
BASIC SEPARATION TECHNIQUES:
CENTRIFUGATION Process whereby centrifugal force is used to separate solid matter from a liquid suspension
Supernatant- liquid
Sediment- solid
Parts: Head/Rotor, Carriers, Shields
Rule of thumb: Placing the tube by exact positioning or by the exact opposite
Purpose of the hole in centrifuge machine: For speed verification
Revolution per minute (RPM) – actual speed
Relative Centrifugal Force (RCF) – force acting in sample; expressed in gravities (g)
= 1.118 10
Centrifugal force depends on three variables: mass, speed, and radius.
Types of Centrifuge:
1. Refrigerated Centrifuge Used for thermally labile test analytes; Temperature: 15-25C
Ex: Ultracentrifuge- up to 100,000 rpm; for lipoproteins
2. Fixed Angle/ Angle Head Cups are held in a rigid position at a fixed angle (52˚); Up to 10,000 to
Centrifuge 15,000 rpms
-Most Commonly used Used when rapid centrifugation of solutions containing small particles is
needed
Ex. Microhematocrit centrifuge
3. Swinging Bucket/ Not capable of higher speeds (up to 3,000 rpm)
Horizontal Centrifuge At rest, tubes are at a vertical position, during spinning, tubes attain a
horizontal position
4. Cytocentrifuge/ Cytospin Uses a very high torque and low inertia motor to speed monolayers of
cells rapidly across a special slide for critical morphologic studies
FILTRATION Filtrate- liquid that passes through the filter paper
Used to separate serum from a blood clot; Used in placed of centrifugation
Filter material is made of paper cellulose and its derivatives, polyester fibers, glass and a variety of resin
column material
Filter paper differs in terms of pore size and should be collected to separation needs. It should not be
used when using strong acids or bases
DIALYSIS Method of separating macromolecules (colloid) from a solvent or smaller substances (crystalloid)
LABORATORY MATHEMATICS:
Significant figures- are the minimum number of digits needed to express a particular value in scientific notation without the
loss of accuracy
Dilution- represents a ratio of concentrated or stock material to the final volume of a solution
Dilution factor- ratio of concentrated or stock solution to the total solution volume (inversely proportional to concentration)
Serial Dilution: multiple progressive dilutions ranging from more concentrated to less concentrated solutions; useful when
volume of concentrate or diluents is in short supply (minimize usage) or a number of dilutions are required (titer
determination)
DILUTION Expression of relative concentration of the solute in a solution
Solute:Solutions or Solute:Solute+Solvent
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RATIO Proportion of solute to the solvent

Solute:Solvent

1. Beer’s Law absorption and concentration

-concentration of unknown is α to the absorbance/ optical density and 1/α to the transmitted light
Absorbance= abc
∑ a=molar absorptivity
∑ b=length of light
∑ c=concentration of the solution
T=100
Abs= 2-log(%T)
Abs=0
2. Charle’s Law volume of gas and temperature
3. Dalton’s Law volume of gas to another
4. Boyle’s Law volume of gas and pressure
5. Avogrado’s Principle gram molecular weight and the number of atoms

Conversion- to convert one unit into another

Conversion of Traditional Units to SI Units for Common Chemistry Analytes:
Conventional/ Current SI Unit Conversion Factor
Albumin g/100 mL g/L 10
AST U/L (mU/mL) µkat/L 0.0167
Ammonia µg/dL µmol/L 0.587
Bicarbonate mEq/L mmol/L 1.0
Bilirubin mg/dL µmol/L 17.1
BUN mg/dL mmol/L 0.357
Calcium mg/dL mmol/L 0.25
Chloride mEq/L mmol/L 1.0
Cholesterol mg/dL mmol/L 0.026
Cortisol µg/dL µmol/L 0.0276
Creatinine mg/dL µmol/L 88.4
Crea Clearance mL/min mL/s 0.0167
Folic acid ng/mL nmol/L 2.27
Glucose mg/dL mmol/L 0.0555
Hemoglobin g/dL g/L 10
Iron mg/dL µmol/L 0.179
Lithium mEq/L µmol/L 1.0
Magnesium mEq/L mmol/L 0.5
Osmolality mOsm/kg mmol/kg 1.0
Phosphorus mg/dL mmol/L 0.323
Potassium mEq/L mmol/L 1.0
Sodium mEq/L mmol/L 1.0
Thyroxine µg/dL nmol/L 12.9
Total protein g/dL g/L 10
Triglyceride mg/dL mmol/L 0.0113
Uric acid mg/dL mmol/L 0.0595
Vit B.12 ng/mL pmol/L 0.0738
PCO2 mmHg kPa 0.133
PO2 mmHg kPa 0.133
SPECIMEN CONSIDERATIONS:
∑ Samples Analyzed in Clinical Chemistry
1. Blood
Types of Blood Specimen:
1. Whole Liquid portion of the blood (plasma) from arterial and for POCT and STAT
blood venous
2. Plasma Liquid portion of unclotted or anticoagulated blood with Specimen of choice for STAT requests
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fibrinogen
3. Serum Liquid portion of clotted blood without fibrinogen MCU specimen in CC
ÿ Heparin- least interference with analyses; Lithium heparin may be used for most chemistry test except litium and
folate levels
SITUATION: Comments:
IV Use the opposite arm or perform fingerstick if possible
Stop IV for 2 to 5 minutes
Fistula Draw from the opposite arm
Indwelling Line and Catheter, Heparin Lock, Cannulas Draw first 5 mL then discard
Draw blood below the IV line if nothing is being transfused
Sclerosed Veins Select another site
Hematoma Draw below
Streptokinase/Tissue Plasminogen Activator Hold pressure until bleeding has stopped
Edema, Scars, Burns, Tattoos Select another site
Mastectomy Draw from the opposite arm
Unidentified patient Ask the nurse to identify before drawing

Phlebotomy Sources of Error:

Misidentified patient Treatment errors
Drawing at incorrect time Treatment errors if samples for certain tests are not drawn at
appropriate time
Improper Skin Disinfection Contamination of blood culture and blood components
Isopropyl alcohol wipes can contaminate samples for blood
alcohol
Fist Pumping During Venipuncture ↑K+, Lactic acid, CA2+, Phosphorus, ↓pH
Prolonged Tourniquet (>1minute) Hemoconcentration: ↓ pO2 and pH
↑ K+, albumin, total protein, enzymes, lactate, cholesterol,
ammonia
IV Fluid Contamination ↑ Glucose, Electrolytes (For every 10% contamination with
Dextrose (D5W), the glucose will ↑ 500 mg/dL)
Expired Collection Tubes ↓ Vaccum, Failure to obtain specimen
Incorrect Anticoagulant/ Contamination from incorrect order K2EDTA before Serum/Heparin tube: ↑K+, ↓CA2+, Mg2+
of draw Sodium Heparin: ↑Na
Lithium Heparin: ↑Li
Gel separator: ↓TCAs and trace metals
Hemolysis from Alcohol contamination ↑K+, Mg2+, LDH, iron
Milking Site of Capillary puncture
Probing with needle
Vigorous shaking of tubes
Exposure of samples to extreme temperature

ANTICOAGULANT ACTION
Citrate Combines with Calcium in a non-ionized form
Oxalate Combines with Calcium to form an insoluble salt
Fluoride Forms weakly dissociated calcium compounds
EDTA Combines with calcium through chelation
Heparin Inhibits or neutralize thrombin

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2. Urine - the next most common fluid for determination; Most quantitative analyses of urine require a timed sample
(usually 24 hours)
ÿ Creatinine analysis is often used to assess the completeness of a 24-hour urine sample (Ave: excretes 1 to 2 g of
creatinine per 24 hours)
ÿ Urine volume differs widely among individuals; however, a 4 L container is adequate (Ave: output is ≈2 L)
ÿ Drug test urine volume: 60 mL
3. Cerebrospinal fluid
4. Paracentesis fluids (Pleural, Pericardial and Peritoneal)
ÿ The color and characteristics of the fluid before centrifugation should be noted for these samples
∑ Sample Processing:

ÿ It is important that serum samples be allowed to completely clot (≈20 minutes) before being centrifuged.
ÿ Plasma samples also require centrifugation but do not need to allow for clotting time and their use can decrease
turnaround time for reporting results.
ÿ Centrifugation of the sample accelerates the process of separating the plasma and cells. Specimens should be
centrifuged for approximately 10 minutes at an RCF of 1,000g to 2,000g but should avoid mechanical destruction of
red cells that can result in hemoglobin release
ÿ Samples should be analyzed within 4 hours
ÿ To minimize the effects of evaporation, samples should be properly capped and kept away from areas of rapid
airflow, light, and heat.
ÿ If testing is to occur after that time, samples should be appropriately stored.
o Refrigeration at 4°C for 8 hours
-Alkaline phosphatase (increases)
-Lactate dehydrogenase (decreases)
o Frozen at -20°C and stored for longer periods without deleterious effects on the results
Specimen Transport and Storage Requirements:
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Requirement Analyte/s
FASTING FBS, Lipid profile, Gastrin, Insulin
CHILLING ABG, ACTH, Acetone, Ammonia, Gastrin, Glucagon, Lactic acid,
Pyruvate, PTH, Renin, Cathecolamines
WARMING Cold Agglutinin, Cryoglobulins
PROTECTION FROM DIRECT LIGHT EXPOSURE Bilirubin, Beta-carotene, Folate, Porphyrins, Erythocyte
protoporphyrin, Vitamin A and Vitamin B12
CHAIN OF CUSTODY Blood Alcohol, Drug screens, DNA testing
FREEZING OF SAMPLE Affects LD, ALP, ACP, CK

∑ Sample Variables: include physiologic considerations, proper patient preparation, and problems in collection,
transportation, processing, and storage
Patient variables include physical activity, diet, age, sex, circadian variations, posture, stress, obesity, smoking, and
medication.
Minimize physiologic factors related to activities that might influence laboratory determinations. These include diurnal
variation, exercise, fasting, diet, ethanol consumption, tobacco smoking, drug ingestion, and posture
Physiologic variation refers to changes that occur within the body, such as cyclic changes (diurnal or circadian variation) or
those resulting from exercise, diet, stress, gender, age, underlying medical conditions (e.g., fever, asthma, and obesity), drugs,
or posture
Some frequently encountered influences are smoking, which causes an increase in glucose as a result of the action of nicotine;
growth hormone; cortisol; cholesterol; triglycerides; and urea. High amounts or chronic consumption of alcohol causes
hypoglycemia, increased triglycerides, and an increase in the enzyme gamma-glutamyltransferase and other liver function
tests.
ÿ Clerical errors are the most frequently encountered, followed by inadequate separation of cells from serum,
improper storage, and collection
ÿ Bar code labels on primary sample tubes are a popular means to detect errors and to minimize clerical errors

Lipemia TAG >400 mg/dL

Increased sample turbidity
False↑: HGB, Total Bilirubin
False↓: Na, Cl
Jaundice Bilirubin ≥ 2 mg/dL
Icteresia: Bilirubin >25 mg/dL
IM Injection ↑ CK-MM
Caffeine ↑fatty acids, hormone levels, glycerol, lipoproteins and serum gastrin
Short Term Exercise ↑Creatinine, Fatty acids, Lactate, AST, CK, LDH, Uric acid, Bilirubin, HDL, GH, Cortisol,
Aldosterone, Renin, Angiotensin and WBCs
↓pH and PCO2
Long Term Exercise ↑Aldolase, Creatinine, Sex hormones, AST, CK, LDH
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Fever ↑Protein, ALP, Estradiol, Free Fatty acids, Iron, RBCs

↓ ESR, Factors II, V, VII, IX, X, glucagon, cortisol
Pregnancy ↑Protein, ALP, Estradiol, Free Fatty acids, Iron, RBCs
↓ ESR, Factors II, V, VII, IX, X
Smoking ↑Glucose, BUN, TAG, Cholesterol, ALP, Cathecolamines, Cortisol, IgE, HGB,HCT, RBCs, WBCs
↓IgG, IgM, IgA
Alcohol ↑ TAG, GGT
↓Glucose

MEDICATIONS AFFECTED TEST/SYSTEMS

Acetaminophen and certain antibiotics ↑Liver Enzymes, Bilirubin
Cholesterol- lowering Drugs ↑ PT, APTT
Corticosteroid and estrogen ↑ AMY, LPS
Diuretics ↑ Calcium, Glucose, Uric acid
↓Sodium, Potassium
Chemotherapy ↓ RBCs, WBCs, PLTs
Aspirin, Salicylates and herbal supplements ↑ Bleeding time
Radiographic contrast media Routine urinalysis
Fluorescein Dye ↑ Creatinine, Cortisolm Digoxin
Oral Contraceptives ↓ Apoproteins, Transcortin, Cholesterol, HDL, Triglycerides, LH, FSH, Ferritin,
Iron

∑ Minimum Elements of Paper or Electronic Patient Reports:

Name and address of laboratory performing the analysis including any reference laboratories used
Patient name and identification number or unique identifier
Name of physician or person ordering the test
Date and time of specimen collection
Date and time of released results
Specimen source or type
Test results and units of measure if applicable
Reference ranges, when available
Comments relating to any sample or testing interferences that may alter interpretation
II. Laboratory Safety:

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MATERIAL SAFETY DATA SHEET Identification of Chemical

INFORMATION: Hazardous ingredients
Physical Data
Fire and Explosion Data
Health Hazard Information
Reactivity Data
Spill, Leak, and Disposal procedures
Personal Protective Information
Special Precaustions and Comments

ÿ Safety Awareness for Clinical Laboratory Personnel:

∑ Employer’s Responsibilities:
-Establish laboratory work methods
-Provide supervision and guidance to employees
-Provide safety information, training, PPE and medical surveillance to employees
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-Provide and maintain equipment and laboratory facilities that are free of recognized hazards and adequate for the tasks
required
-Maintain clean PPE used by more than one person
-Provision of the exposure control plan
∑ Employee’s Responsibilities:
-Know and comply with the established laboratory safe work practices
-Have a positive attitude towards supervisors, coworkers, facilities and safety training
-Be alert and give prompt notification of unsafe conditions or practices to the immediate supervisor and ensure that unsafe
conditions and practices are corrected
-Engage in the conduct of safe work practices and use of PPE
ÿ Appropriate Signs and Labeling of Reagents
1. Statement Hazard
2. Hazard Class
3. Safety Precautions
4. NFPA Code
5. Fire Extinguisher type
6. Safety Instructions
7. Formula Weight
8. Lot Number

Safety Equipments Safety showers

Eyewash stations
Fire extinguishers
Fire Blankets
Spill Kits
First Aid supplies
Mechanical pipetting devices
Fume Hood Required to contain and expel noxious and hazardous fumes
from chemical reagents
Should be visually inspected for blockages by putting a piece
of tissue paper at the good opening to indicate airflow
directions
Biosafety Cabinets Remove particles that may be harmful to employee working
with potentially infectious biologic specimens

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Personal Protective Equipment Safety glasses, goggles, visors, work shields, gloves,
rubberized sleeves, laboratory coats, proper footwear,
respirators (with HEPA filter) and hand washing

ÿ Storage of Chemicals
-According to compatibility

-Store room should be isolated in an area that is not used for routine work

ÿ Record keeping of Personnel Data working with Radioisotopes

-Records must be kept for the length of employment plus 30 years

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ÿ Nonionizing Radiation- equipments that emits a variety of wavelengths of electromagnetic radiation that must be
protected against through engineered shielding or use of PPE

∑ HAZARD CATEGORIES OF CHEMICALS:

Classification Effect Example Comments

1. Corrosives Visible GAA Chemicals with pH<2 or >12
destruction of HCL Concentrated acids and bases can generate large amounts
human tissue on H2SO4 of heat when mixed with water
contact; can cause HNO3 Separate inorganic and organic acids
injury on NAOH
inhalation or
contact
2. Toxic Interfere with Cyanides Threshold limit values (TLVs) – safe level of exposure
Substances metabolic Sulfides
processes, when
ingested, inhaled
or absorbed
through the skin
3. Carcinogens Capable of causing Benzidine OSHA requires monitoring of formaldehyde exposure
cancer Formaldehyde
4. Mutagens/ Mutagens- induce Benzene, Lead, Mercury, Special precaution during pregnancy
Teratogens genetic mutations Radioactive metal,
Teratogens- cause Toluene
defect in the
embryo
5. Ignitables May cause fire Acetone Flash point- lowest temperature that produce ignitable
Xylene vapor
Alcohols Flammables- <100˚F
Ether Combustibles- ≥100˚F
6. Reactives May cause Ether, Perchloric acid, Ether- forms explosive peroxides on exposure to air or light
explosion; Picric acid, Sodium azide Store in explosion proof refrigerator
Mixture of Perchloric acid- may react with organic compounds
oxidizing agents Separate with other acids
and reducing Picric acids- shock sensitive when dehydrated
agents generate Sodium azide solutions can react with explosive leads or
heat and may sopper azides in drains
explode

OTHER HAZARDS:
1. Electrical Have ground, polarity and leakage checks and other periodic preventive maintenance performed on
outlets and equipments
When accident occurs: the electrical source must be removed immediately:
a. Turning off the circuit breaker/ main switch
b. Unplugging the equipment

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C. Moving the equipment using a nonconductive glass or wood object

2. Compressed Danger of fire, explosion, asphyxiation or mechanical injuries
gases
3. Cryogenic Liquid Nitrogen- MCC cryogenic material used in the laboratory
Material Danger of fire or explosion, asphyxiation, pressure build-up, embrittlement of materials and tissue
damage like thermal burns
4. Mechanical Refers to equipments such as centrifuge, autoclaves and homogenizers, lavoratory glasswares, glass
pipets
5. Ergonomic Refers to the effect of repeated manipulation of instruments, containers, equipments for repetitive
stains such as tenpsynovitis, bursitis and ganglion cysts
Factors to consider: Position and posture/ Applied Force/ Frequency of Repetition/ Design of Hand
Tools
Ex. Twisting, Bending, Lifting, Static postures
Avoid:
Lifting heavy objects improperly
Keeping the load closely to the body using the muscles of the legs rather the back
Gradually increase force between pushing or pulling and avoid pounding actions with the extremeties
6. Others a. Chemical hazards- Chemical Spill: Best first aid is to flush the area with running water for 15 minutes
b. Physical hazards- Avoid running in the laboratory especially on wet floors, Seek help when lifting
heavy objects; Avoid wearing wrist watch or dangling jewelries; Tie back long hair
c. Biologic hazards- viruses, bacteria, parasites, fungi
d. Fire hazard- chemical reaction that involves the rapid oxidation of a combustible material or fuel with
the subsequent liberation of heat and light
Trigerring factors: fuel, heat or ignition source and oxygen (air)

ÿ Best type of All-purpose fire extinguisher: CO2 foam

∑ National Fire Protection Association (NFPA) Hazard Diamond:

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∑ Spills:

Basic First Aid Procedures:

Alkali or Acid burns on skin or in mouth Rinse thoroughly with large amounts of running tap water
Alkali or Acid burns in eye Wash out eye thoroughly for 15 minutes
Eye fountain is recommended
Heat burns Apply cold running water to relieve pain and stop further
tissue damage. Use a wet dressing of 2 tbsp of NAHCO3 in 1

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quart of warm water. Apply bandage securely but not tight. If

third degree burn occurs, do not use ointment or grease,
consult physician immediately
Minor cut Wash wound with soap and water the apply bandage
Serious cut Apply direct pressure to the wound area and call physician
immediately

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AUGUST 2021 MEDICAL TECHNOLOGY LICENSURE EXAMINATION REVIEW (Klubsy Bear Online Tutorial)
Contents:
I. Carbohydrates
II. Lipids and Lipoproteins
III. Amino Acids and Proteins
IV. Nonprotein Nitrogen Compounds
I. Carbohydrates:
-Composed of Carbon, Hydrogen and Oxygen
-Major source of energy in the body
CLASSIFICATION OF CARBOHYDRATES:
Monosaccharide Sugar that contains 3,4,5,6 carbon atoms
Sugar that cannot be hydrolyzed to a simpler form
Examples: Glucose, Galactose, Fructose, Ribose, Deoxyribose,
End product of CHO Metabolism: Glucose
Energy Mediator of Oxidation of Glucose is: ATP
Characteristic of Fructose: Levulose – rich in sperm
∑ The only carbohydrate directly used by the body for energy: Glucose
∑ Brain is completely dependent on blood glucose for energy. (2/3 of Glucose Utilization in
resting adults occurs in the CNS- Hypoglycemia-Neuroglucopenia)
Disaccharide Composed of 2 monosaccharides: (can be separated by hydrolysis)
Glucose + Glucose= Maltose(Maltase)
Glucose + Galactose = Lactose(Lactase)
Glucose + Fructose= Sucrose(Sucrase)
Most popular non-reducing sugar: Sucrose (w/o free aldehyde)
Oligosaccharide Composed of 2-10 monosaccharides
Polysaccharide Linkage of many monosaccharide units (glycosidic bond)
Examples: Starch (Amylase), Glycogen, Cellulose, Chitin
Storage form of sugar in the body: Glycogen

GLUCOSE METABOLISM:

GLUCOSE SUBSTRATE PRODUCT COMMENT

METABOLISM
PROCESS
Glycolysis Glucose Pyruvate/ Conversion of glucose into pyruvate or lactate for production of
Lactate + ATP energy (EMP)
Gluconeogenesis Amino Glucose Formation of glucose-6-phosphate from a non-carbohydrate source
acids when glycogen stores are already used up (48 hours-Starvation,
Glycerol Liver)
Lactate Primary played by cortisol

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Glycogenolysis Glycogen Glucose Breakdown of stored glycogen to glucose for energy

Primary played by glucagon
Glycogenesis Glucose Glycogen Conversion of excess glucose into glycogen for storage
Lipogenesis Glucose Glycerol Conversion of carbohydrates to fatty acids
Lipolysis - - Decomposition of fat
Trycyclic acid and Pyruvate ATP Energy production 2 ATP per molecule of Acetyl-CoA (Kreb’s)
Electron to Acetyl-
Transport System CoA
Hexose Glucose NADPH Energy source of many anabolic reactions and glycolysis in RBCs
Monophosphate since they lack mitochondria
Shunt

REGULATION OF GLUCOSE METABOLISM

1. Insulin only Primary hormone responsible for DECREASING blood glucose
hypoglycemic Regulation by: ↑Glycosis, Glycogenesis, Lipogenesis ↓Glycogenolysis
hormone in the Enhances glucose entry to the cells, storage of glucose to glycogen, synthesis of proteins and fatty acids,
body suppresses the breakdown of protein into amino acids and of adipose tissue into free fatty acids
STEP 1: Single chain pre-pro-insulin is cleaved into a two-chain pro-insulin and inactive C-peptide
STEP 2: Formation of Insulin- a two-chain polypeptide consisting of 51 amino acids
2. Glucagon Primary hormone responsible for INCREASING blood glucose
Regulation by: ↑Glycogenolysis, Gluconeogenesis, Some Lipolysis
Enhances release of glucose from glycogen and enhances synthesis of glucose from amino acids and
fatty acids
3. Epinephrine INCREASES BLOOD GLUCOSE
Regulation by: ↓ Insulin secretion ↑Glycogenolysis, Lipolysis
Enhances release of glucose from glycogen and release of fatty acids from adipose
4. Cortisol INCREASES BLOOD GLUCOSE
Regulation by: ↓ Entry of Glucose into the cell ↑GluconeogenesisGlycogenolysis, Lipolysis
Enhances synthesis of glucose from amino acids or fatty acids
5. Growth Hormone INCREASES BLOOD GLUCOSE
Regulation by: ↓ Entry of Glucose into the cell ↑Glycolysis, Glycogenolysis
Enhances release of glucose from glycogen
(antagonist of insulin)
6. Thyroxine INCREASES BLOOD GLUCOSE
Regulation by: ↑ Glycogenolysis, Gluconeogenesis and Glucose Intestinal Absorption
Enhances release of glucose from glycogen, synthesis of glucose from amino acids or fatty acids and
absorption of sugars from intestines
7. Somatostatin ACTS LIKE INSULIN but more of a hyperglycemic hormone
Produced by the Delta cells of the Islet of Langerhans in the Pancreas and Hypothalamus
Increases plasma glucose by the inhibition of insulin, glucagon, GH, etc.
8. ACTH ENHANCES RELEASE OF CORTISOL and ENHANCES RELEASE OF FATTY ACIDS FROM ADIPOSE TISSUE
(antagonist of insulin)

CLINICAL CONDITIONS:
1. Hyperglycemia – increase in plasma glucose
Complications: Retinopathy, Neuropathy, Nephropathy, Atherosclerosis
Diagnosis by glucose tolerance test and postprandial glucose testing
Optimal test for the glucose metabolism of the body: FBS and OGTT
Serum osmolality is high
Electrolyte imbalance:
-Metabolic acidosis
-Hyponatremia
-Hyperkalemia
Glucosuria (Hyperglycemia-associated) Renal threshold: 160-180 mg/dL

DIABETES MELLITUS
-Metabolic disease characterized by persisting hyperglycemia resulting from defects in insulin secretion, insulin action, or
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both
3P’s – Polyphagia, Polyuria, Polydypsia
Laboratory Findings:
-High Glucose in urine and serum
-High urine specific gravity
-High serum and urine osmolality
-Ketones in serum and urine
-Low blood and urine pH
-Electrolyte imbalance
Routine Test for DM:
1. FBS
2. HbA1c (not recommended for screening)
3. Microalbuminuria – renal complications
Best screening test: FBS
For monitoring: HbA1c
Other analytes commonly used in monitoring diabetic patients: Urea, Creatinine, Lipids Urinary albumin
Criteria by American Diabetes Association (ADA):
1. Symptoms and RBS≥200 mg/dL(11.1 mmol/L)
2. FBS ≥126 mg/dL (7 mmol/L)
3. 2-HR OGTT ≥200 mg/dL (11.1 mmol/L)
4. HbA1c ≥6.5%
TYPE 1 TYPE 2
Formerly Insulin Dependent Diabetes Mellitus (IDDM) Non- Insulin Dependent Diabetes Mellitus
known as Brittle DM Stable DM
Ketosis-prone DM Receptor deficient DM
Onset Juveline Onset Adult/ Maturity Onset
(<20 y/o) (>40 y/o)
Cause β-cell destruction Insulin resistance
Risk Genetic, Autoimmune Genetic, Obesity, Sedentary Lifestyle
Absolute Insulin Deficiency Progessive Insulin Deficiency
Antibodies: BMI >30 kg/m2
Anti-islet cell
Anti-tyrosine phosphatase IA-1 and IA-2B
Anti –glutamic acid decarboxylase (anti-GAD65)-
common in adults
Anti-insulin (IAA)- common in children
Incidence rate 5-10% (10-15%) 90-95%
C-peptide Undetectable Detectable
Symptom Abrupt Gradual
Ketosis Common Rare
Treatment Absolute Parenteral Insulin Oral Agents
TESTS:
x 0.0555 (to mmol/L) Normal Sugar Level Impaired FastingGlucose Diagnostic for DM
Fasting Blood Sugar 70-99 mg/dL 100-125 mg/dL ≥126 mg/dL
Random/Resting Blood Sugar <139 mg/dL 140-199 mg/dL ≥200 mg/dL
2-hr Post Prandial
2-hr OGTT
HbA1c <5.6% 5.7-6.4% ≥6.5%
3. GESTATIONAL DM- Glucose intolerance during pregnancy due to metabolic and hormonal changes
Disappears after delivery but prone to develop Type II DM in 5-10 years (10-40% of the cases)
Screening: Glucose Challenge Test – 50 g glucose load
Assessed after 1 hour
≥140 mg/dL –proceed to OGTT
Diagnostic: 3-hour Oral Glucose Tolerance Test – 100 g glucose load

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Fasting: at least 10 hours and not longer than 16 hours

Diagnostic Criteria for Gestational Diabetes by ADA:
1. FBS >95 mg/dL (93-5.1)
2. 1hour≥185 mg/dL (180-10)
3. 2hour≥155 mg/dL (153-8.5)
4. 3hour≥140 mg/dL
GDM is diagnosed when 2 of the criteria are met
Guidelines for Administration of OGTT:
1. Unrestricted CHO diet: 150g/ day for 3 days
2. Patient is asked to fast overnight (8 hours) and avoid excessive physical activities
3. Measure fasting glucose level
If >140 mg/dL – terminate the test
If<140 mg/dL – administer glucose
Adult- 75 grams
Child-1.75g/kg of weight
4. Shout not smoke (false increase) and drink alcohol (false decrease) prior testing
Procedure for OGTT:
1. Collect fasting blood sample – 0 hour sample
Urine may also be collected
2. Instruct the patient to drink the glucose load for 5 minutes (Vomit-discontinue)
3. Collect sample after 1-hour, 2-hour, 3-hour respectively
FBS- Most important
2-hour- Confirmatory
3-hour- Optional
-It is recommended that any positive test be repeated on a subsequent day to confirm diagnosis
-OGTT is not recommended for routine clinical use
2. Hypoglycemia – decreased glucose in plasma
Diagnostic test: 72hr fasting then measure Glucose, Insulin, Proinsulin, Cpeptide at 6 hour intervals (Test is concluded if
glucose drops <45 mg/dL)
≤60 mg/dL- random blood glucose level which strongly suggest hypoglycemia
50- 55 mg/dL- observable symptoms of hypoglycemia
-Whipple’s triad – most common test
1. Low blood glucose
2. Symptoms consistent
3. Relief of symptoms after glucose administration
-Use of ethanol, propanolol and salicylate has been linked to the occurrence of hypoglycemia

TYPES:
Classification Glucose Level and Rate of Decrease Symptoms
Adrenergic Rapid Sweating, weakness, shakiness,
Activation of sympathetic nervous system Glucose level may or may not remain lightheadedness, rapid pulse, hunger,
which releases epinephrine within the reference range nausea
Neuroglycopenia Gradual Headache, Confusion, Seizures, Coma
Depriving the brain of glucose which Glucose level <20 to 30 mg/dL -May be asymptomatic until CNS is
cause CNS dysfunction impaired
-Repeated or Extended episodes results
in irreversible CNS damage

CAUSES OF ABNORMAL GLUCOSE LEVELS

HYPERGLYCEMIA HYPOGLYCEMIA
PERSISTENT TRANSIENT PERSISTENT TRANSIENT
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Diabetes Mellitus Pheochromocytoma Insulinoma Alcohol ingestion

Obesity Acute stress reaction (Physical Addison’s disease Drugs: Salicylates, Anti-TB
Cushing’s syndrome and emotional) Hypopituitarism Severe liver diasease
Hyperthyroidism Severe liver disease Galactosemia Several glycogen storage disease
Acromegaly Shock Ectopic insulin from tumors Functional hypoglycemia
Convulsions Hereditary fructose intolerance

CARBOHYDRATE INTOLERANCE
Disorder Comments
Lactose Intolerance Pathology: Lactose accumulates –GI irritation, diarrhea
Enzyme deficient: lactase
Test: Lactose Tolerance Test
1. Patients fast overnight; FBS determined
2. Oral lactose is administered
Load: 50 g lactose in 200-300mL of water
3. Blood samples are taken every 30 minutes for 2 hours
Results:
Normal Lactose tolerance- Rise in glucose 25 mg/dl or more
Presumptive Lactose Tolerance: Rise in glucose 20 mg/dL or less
LACTOSE TOLERANCE TEST DATA (mg/dL)
Patient Interval Normal Lactose Intolerance Ambiguous
Fasting 89 85 93
30 minutes 104 96 107
60 minutes 121 92 115
90 minutes - 90 104
120 minutes - 88 97
Causes: congenital or acquired
Galactosemia Pathology: Galactose accumulates – Cataracts, mental retardation
Enzyme Deficient: galactose-1-phosphate uridyl transferase, galactokinase, uridyl diphosphate
glucose-4-epimerase
Galactose-1-phosphate + UDP-glucose =(galactose-1-phosphate uridyl transferase)= UDP + galactose +
Glucose-1-phosphate
∑ High unphosphorylated galactose reacts with aldose reductase as a side reaction and produces
galactilol

URINE SERUM RBCS

+Reducing substances Elevated Bilirubin Greatly decreased galactose-1-
+Glactose Elevated ALP, AST, ALT phosphate uridyl transferase
-Glucose Normal glucose
+Protein Elevated galactose
Elevated galactilol
Newborn Screening Act (R.A 9288) of 2004
The disorders tested are:
-Congenital Hypothyroidism
-Congenital Adrenal Hyperplasia
-Galactosemia
-Phenylketonuria
-Glucose-6-phosphate dehydrogenase deficiency
-Maple Syrup Urine Disease ( DOH Memo 2012-0154)
The expanded screening panel tests 28 disorders

GLYCOGEN STORAGE DISEASE or Glycogenoses – defective glycogen metabolism (autosomal recessive inherited
disorders)
TYPE Comments Enzyme Deficiency Common Name
Type I Most common in the world Glucose-6-Phosphatase Ia- von Gierke
Diagnosed by IVGTT
Severe fasting, hypoglycemia and

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lactic acidosis
Type II Most common in the Philippines Acid α-1,4-Glucosidase Pompe
Increased glycogens in all organs Acid Maltase
Type III Hypoglycemia, Hepatomegaly and Amylo-1,6,-Glucosidase IIIa- Cari Forbes/ Limit
growth retardation (Debranching enzyme) Dextrinosis
Type IV Hepatomegaly, Cirrhosis and Branching Enzyme Andersen/ Amylopectinosis
muscle weakness
Type V - Muscle Phosphorylase McArdle
Type VI - Liver Phosphorylase or Hers
Phosphorylase kinase
Type VII - Muscle Phosphofructokinase Tarui
Type XI - Glucose transporter 2 Fanconi-Bickel

METHODS FOR THE DIAGNOSIS OF METABOLIC ALTERATIONS

1. Fasting Blood Sugar (Fasting 8 hours fasting
Plasma Glucose) NV- 70 to 110 mg/dL
Measures overall glucose homeostasis
2. Random / Resting Blood Sugar For emergency cases
Requested during insulin shock
For patients with diabetic coma (Hypo-<60; Hyper >400)
2. POCT Glucose Uses Whole Blood via Amperometric Method
Useful in monitoring Type I and Type II DM
∑ Strips are impregnated with glucose oxidase and peroxidase which tests color
change read by reflectance meter
∑ Hemocue: utilizes transmittance photometry and single test cartridges based on
glucose dehydrogenase method
3. 2-hour Post Prandial Blood Solution containing 75g for adults and 1.75g/kg in children of glucose is administered and
Sugar a specimen is drawn 2 hours later ; no fasting required
NV- <140 mg/dL
DM- >180 mg/dL
Measures how well the body metabolizes glucose
4. HbA1c (Glycated Hemoglobin) Index for long term plasma glucose control (Average plasma glucose in the last 2 to 4
months) indicating compliance and efficacy of therapy
Specimen: EDTA Whole blood
Principle: HPLC, Ion Exchange Chromatography, Isoelectric Focusing, Immunoassay
techniques
Cut off value: <6.5% (maintained at < 7%)
Compliant patient- twice a year (every 6 months)
Noncompliant patient- quarterly/ four times a year
Three Steps of Glycation Process:
1. Glucose reversibly binds to amino terminal end of hemoglobin to form a labile aldimine
2. The aldimine undergoes irreversible rearrangement (amadori) to form stable
ketoamine
3. Glucose undergoes a conformational change
-Largest fraction of glycated hemoglobin A1
-Depends on RBC lifespan:
FALSE HIGH- Older RBCs, Iron Deficiency Anemia, Hemolysis, Hemoglobin F and S
FALSE LOW- Shortened RBC life span, Hemolytic Anemia
5. Fructosamine (Glycated Monitors short term glucose control (Average plasma glucose in the last 3 to 6 weeks)
Albumin) NV- 205-285 umol/L
For Monitoring DM patients with:
1. Chronic Hemolytic anemia
2. HGB variants S or C
3. Shortened RBC life span
Method: HPLC, Affinity Chromatography
6. Ketones Product of fat decomposition by the liver
Determines Metabolic acidosis if present in urine
Three Ketone bodies:
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1. Acetone (2%)
2. Acetoacetic acid (20%)-most commonly measured
3. 3-β-hydroxybutyric acid (78%)-most abundant
Methods of Measurement:
1. Nitroprusside – NP- purple
2. Enzymatic-NADH+ β-Hydroxybutyrate Dehydrogenase (HBD) = HBA +NAD
(disappearance of absorbance at 340 nm)
3. Gerhard’s test – ferric chloride -red
7. Microalbuminuria Diagnose early stage of diabetic nephropathy and before development of protenuria
Persistent albuminuria in the range of 30-299 mg/day or Albumin:creatinine ratio of 30-
300 µg/mg
8. Oral Glucose Tolerance Test Patient’s fasting blood glucose is taken after a glucose load
Multiple blood sugar test
Performed to diagnose gestational DM
9. Glycomatic Blood Test Measures 1,5-anhydro-D-glucitol – a monosaccharide that is very close to glucose
structure
When Glucose>180mg/dL, kidney cannot reabsorde 1,5AG, thus when glucose level rises,
the 1,5-AG decreases (indicates poor glycemic control)
Monitors glycemic control for the last 1 to 2 weeks
Associated with hypertension, hyperglycemia, excess fat deposits around waist line, low
HDL and high TAG

Considerations for Specimen Processing:

1. Fasting glucose in whole blood is 12-15% lower than serum/plasma (10-15)
2. To convert whole blood glucose to serum or plasma level, use 1.15 as conversion factor
3. In fasting state, results of arterial, venous and capillary are comparable whereas after meal, venous glucose are lower than
arterial and capillary
4. Venous blood is 7 mg/dL lower than capillary and arterial blood (Fasting-5mg/dL)
5. Capillary blood glucose is the same with arterial blood glucose
6. Glycolysis rate: Room temp (20-25˚C) -7 mg/dL/hour decrease; Ref temp (4˚C) -2 mg/dL/hour decrease
7. Serum must be separated from cells within 30 minutes
8. Sodium Fluoride inhibits glycolysis- glucose preservative (inhibits enzyme and BUN determination)
9. In glucose meters, if hematocrit is low it yields false high results and vise versa. QC of glucose meters should be +/- 15% of
laboratory result
10. Other samples:
-CSF stored at 4 to -20˚C
-24-hour urine store at 4˚C
11. Blood sugar measured using Copper reduction is higher than enzymatic method by 5-15 mg/dL (due to reducing
substances such as urea)
12. Ten (10)% xontamination with 5% dexreose (D5W) will elevate glucose by 500mg/dL
NONENZYMATIC/ CHEMICAL METHODS
1. Alkaline Copper Nelson-Somogyi phosphomolydenum blue
Reduction Method -most commonly used chemical method for glucose
-uses BASO4 to Folin-Wu arsenomolybdenum blue
remove Neocuproine yellow or yellow orange cuprous-neocuproine complex
saccharoids Benedict’s Blue – Green – Yellow – Orange – Red
-Cupric ion to Uses citrate or tartrate as stabilizing agent (modified-more stable)
cuprous oxide
2. Alkaline Ferric Hagedorn Jensen (Inverse Colorimetry)
Reduction Method Yellow ferricyanide to Colorless ferrocyanide
3. Condensation Dubowski (Orthotoluidine)
Method Aromatic amines to Schiff’s bases (green)

ENZYMATIC METHODS
1. Glucose oxidase ∑ Saifer Gernstenfield – Coupled Enzymatic Reaction (Trinder’s reaction) -Colorimetric

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Measures β-D-glucose
Coupling enzyme: peroxidase
Mutarotase- converts other forms of glucose to β-D-glucose
Chromogen – o-dianisine (red)
Interferences: High Uric acid, Bilirubin, Ascorbic acid, Tetracycline, Hemoglobin, Glutathione (False
decrease)
∑ Oxygen (O2) Consumption
Principle: Polarography
Commonly used in glucose meter
Uses Oxygen (Clarke) electrode
Accurate and precise (no interferences)
2. Hexokinase- Based on the formation of NADPH and the increase in absorbance in 340 nm
reference method Interferences: Gross Hemolysis and Extremely elevated Bilirubin (False decrease)
and most specific Coupling enzyme: G6PD -derived from yeast
Uses Barium sulfate or Zinc sulfate to create a protein free filtrate (routine- x)
3. Glucose Based on the formation of NADH and the increase in absorbance in 340 nm
Dehydrogenase Mutarotase- shortens the time of test
Method for the diagnosis of metabolic alterations

D-xylose Absorption test

Distinguishing intestinal malabsortion vs maldigestion (pancreatic insufficiency)
-Orally administered (absorbed by passive diffusion in the small intestine)
-Not metabolized by the liver and is excreted unchanged in the kidneys
5 hour urine specimen
Malabsorption- Less than Normal
Maldigestion- Normal
II. Lipids and Lipoproteins:
LIPIDS:
-Composed mostly of Hydrogen and Carbon
-Insoluble to blood but soluble to organic solvents
-Used to assess risk of cardiovascular disease
-Transported by lipoproteins
∑ Functions as hormones precursors, hormones, cell membrane structure and function, fuel, energy source, aids in
nerve induction and digestion
∑ Regulates metabolism and clearance
∑ Increased levels: Hyperlipidemia
Major Lipids in the Body: Phospholipids, Fatty Acids, Triglycerides, Cholesterol
A. Phospholipids Composed of : 2 Fatty acids attached to 1 Glycerol (via ester bonds)
-Originated in the intestines and liver
-Most abundant lipid ( major constituents of cell membranes and outer shells of lipoprotein molecules
-Participate in cellular metabolism and blood coagulation
-Serves as a lung surfactant (alters surface tension)
L/S Ratio of ≥2 – Fetal Lung Maturity
L/S Ratio of <2 – Respiratory Distress Syndrome/ Infant Alveoli Collapse
Specimen: Amniotic fluid
Sphingolipids/Sphingomyelin – major lipids of the cell membranes of the central nervous system
(myelin sheath)
Reference Range: 150-380 mg/dL
Forms:
1. Lecithin/ Phosphatidy choline (70%)
2. Sphingomyelin (20%) – derived from sphingosine (Niemann- Pick disorder)
3. Cephalin (10%)
B. Fatty Acids Primary form of stored energy (leaves and enter the triglycerides from adipose tissue)
Derived from diet and from excess glucose in the liver
May exist as short, medium, and long chains
By-product of excessive mobilization of fatty acids is the formation of acetyl-CoA

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Classifications:
-Unesterified- bound to albumin
-Esterified- constituent of TAG and phospholipids
∑ Saturated (no double bonds), Monosaturated (one double bond), Polysaturated (≥ two
double bonds)
Types:
1. Transfat- started as liquid but changes to sloid fat by hydrogenation
-Prevents spoilage and increases the shell-life of processed foods
-It increases LDL and decreases LDL
-Ex. Pies, cookies, crackers, cakes
2. Saturated fats- comes from meat and dairy products; present in palm oil, coconut oil, or processed
foods
3. Monosaturated and Polysaturated fats- comes from fish, vegetable oils and nuts
C. Triglycerides Composed of: 3 Fatty Acids attached to 1 Glycerol
(Neutral Fat) Resynthesized in the intestinal epithelial cells after absorption then combines with cholesterol and Apo-
B48 to form chylomicrons
Contain unsaturated or saturated fatty acids
No charged groups- water insoluble, neutral lipids
-Acetyl-coA – intermediary in the metabolism of TAG through the process of oxidation (enters Kreb’s
cycle for energy conversion)
By-product: Ketone Bodies
-Main storage lipid in man (95% of adipose tissue)
-Energy source
Fasting requirement 10-12 hours (12-15 hours)
Peak after meal: 4-6 hours after meal
Carriers: Chylomicrons, VLDL
Clear Serum <200 mg/dL
Hazy or Turbid Serum >400 mg/dL
Opaque or Milky Serum >600 mg/dL
Nonfasting- resides in chylomicrons
Fasting- resides in VLDL
TRIGLYCERIDE LEVELS:
Normal <150 mg/dL
Borderline High 150-199 mg/dL
High 200-499 mg/dL
Very High ≥500 mg/dL
-Very high- acute pancreatitis
TRIGLYCERIDE MEASUREMENTS:
A. Non-enzymatic/Chemical Methods
1. TAG + Alcoholic KOH = Glycerol and Fatty acids
2. Glycerol + Periodic acid = Formaldehyde
a. Van Handel and Zilversmith
+Chromotropic acid = Blue solution
-Old reference method ( Modified Van Handel and Zilversmith) = Pink solution
b. Hantzch Condensation (Fluorometric Method)
+Diacetyl acetone + NH3= Yellow solution
B. Enzymatic Methods (Automated)
1. Conversion of TAG to Glycerol and Fatty Acids (Lipase)
2. Conversion of Glycerol +ATP to Glycel-3-phosphate + ADP (Glycerol kinase)
a. Glycerol Dehydrogenase
+NAD = Dihydroxyacetone +NADH =+ H+
NADH + H + Resazurin =Resorudin +NAD (Diaphorase)
b. Glycerol Phosphate Dehydrogenase (Formazan Colorimetric)
Glycerol-3-phosphate + NAD= Dihydroxyacetone phosphate +NADH + H+
NADH+ Oxidized tetrazoluim= Reduced Tetrazolium- Formazan (Diaphorase)
c. Pyruvate Kinase (NADH Consumption)
ADP + Phosphoenolpyruvate= ATP +Pyruvate
Pyruvate + NADH + H+ = Lactate + NAD (Lactate dehydrogenase)

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D. Cholesterol Biochemical structure: cyclopentanoperhydrophenanthrene

27-Carbon ringed structure; unsaturated steroid alcohol
2 forms:
Esterified- FA forms an ester bond at Carbon 3
Unsterified- No FA attached to the ring (free)
∑ Lecithin- cholesterol acyl transferase (LCAT) – catalyzes the esterification of cholesterol
∑ Apo A1 – activator of LCAT
UE to E – Esterification
E to UE - Saponification
- Majority exist esterified forms (70%)
-Integral component of cell membrane
-Not an Energy Source
-Precursor of steroids, bile acids, and Vit D
Converted to bile salt (Without it- no digestion of fat)
Considered as a secondary liver function test and secondary thyroid test
Carrier of cholesterol: HDL, LDL
Sources:
1. Exogenous (diet) – 15%
2. Endogenous (liver)- 85% - hypocholesterolemia
-Least affected by the fasting status of a patient ( fat ingestion- slight effect only)
∑ Transport and excretion is promoted by estrogen
CHOLESTEROL LEVEL:
DESIRABLE <200 mg/dL
BORDERLINE/ MODERATE 200-239 mg/dL
HIGH ≥240 mg/dL
NCEP GUIDELINE RECOMMENDATION FOR ADULTS IN TERMS OF RISK FOR CHD:
AGE MODERATE RISK HIGH RISK
2-19 >170 mg/dL >185 mg/dL
20-29 >200 mg/dL >220 mg/dL
30-39 >220 mg/dL >240 mg/dL
>40 >240 mg/dL >260 mg/dL
CHOLESTEROL MEASUREMENT:
-Use tube with gel separator to avoid exchange of cholesterol with RBC membranes, if not refrigerate at
4˚C
-Preferred: nonfasting sample for measurement of cholesterol: EDTA plasma
-Hemolysis- False Increase
Factores related to Quality Control that alters level: Fasting sample, Venous stasis, Posture
A. Non-Enzymatic/Chemical Methods:
Principle: Dehydration and Oxidation
GENERAL METHODS:
Colorimetry:
1. Liebermann Burchardt – Cholestadienyl Monosulfonic acid – green end color
-Most commonly used
2. Salkowski – Cholestadienyl Disulfonic acid – red end color
One step Colorimetry Pearson, Stern, Mac Gavack
Two steps C + Extraction Bloor
Three steps C + E + Saponification Abell-Kendall
Four steps C + E + S + Precipitation Schoenheimer-Sperry, Parekh-Jung
Extraction: Petroleum ether
Saponification: Alcoholic KOH
Precipitation: Acidic solution
∑ Abell Kendall:
Cholesteryl Esters are hydrolyzed with Alcoholic KOH
Unesterified Cholesterol is extracted with Hexane
Measured using Liebermann Burchardt Reaction
Cholesterol + Sulfuric Acid + Acetic Anhydride = green solution
-Old Reference Method: Modified Abell, Levy, Brodie (Extraction – zeolite)
B. Enzymatic Methods:
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1. Total cholesterol (cholesterol and cholesteryl ester) + H2o = free cholesterol + fatty acids (Cholesteryl
esterase/ Cholestery ester hydrolase) -Hydrolysis
2. Cholesterol + O2 = 4-cholesterone +H2O2 (Cholesterol oxidase)
a. CDC
H2O2 + 4-aminophenazone= oxidized dye + H2O2 (Peroxidase) – Max. Abs. @ 500 nm
b. Ciulla
H2O2 + phenol+ 4-aminoantipyrine= quinoemine dye + 2H2O2 (Peroxidase)
Enzymatic End-point – Most common method
Recommended Levels by National Cholesterol Education Program : Adult Treatment Panel
∑ Total Cholesterol <200 mg/dL
∑ LDL-c <100 mg/dL
<100 mg/dL with CHD or CHD risk equivalent
<130 mg/dL with two or more risk factors
<160 mg/dL with 0 or 1 risk factors
∑ HDL-c >40 mg/ dL (≥60 mg/dL –protective)
∑ Triglycerides <150 mg/dL
RISK FACTORS:
1. Cigarette smoking
2. Hypertension (BP >140/90) or on hypertensive medication
3. HDL <40 mg/dL
4. Family history of premature CHD
5. Age (Men >45 y/o; Women>55y/o)
6. CHD risk equivalent- Diabetes Mellitus
Summary:
ADULT VALUES DESIRABLE BORDERLINE/MODERATE RISK HIGH RISK
Cholesterol <199 mg/dL 200-239 mg/dL ≥240 mg/dL
Triglycerides <149 mg/ dL 150-199 mg/dL 200-249 mg/dL
HDL ≥40 mg/dL - ≤39 mg/dL
LDL ≤129 mg/dL 130-159 mg/dL ≥160 mg/dL
≤99 mg/dL – with
CHD
VLDL ≤30 mg/dL - -

CHILDREN AND DESIRABLE BORDERLINE/MODERATE RISK HIGH RISK

ADOSLESCENT (0-19 Y/O)
Cholesterol ≤169 mg/dL 170-199 mg/dL ≥200 mg/dL
LDL (measured) ≤109 mg/dL 110-129 mg/dL ≥130 mg/dL
LDL (direct)
0-19 y/o- 0-110 mg/dL
>20 y/o- 0-130 mg/dL
NCEP GUILINE FOR ACCETABLE MEASUREMENT ERROR
Analyte Total Error Bias CV
Cholesterol ≤9% ≤3% ≤3%
Triglyceride ≤15% ≤5% ≤5%
HDL-c ≤13% ≤5% ≤4%
LDL-c ≤12% ≤4% ≤4%

LIPOPROTEINS:
-Spherical lipid and protein complex
- Transports lipids throughout the body (TAG and cholesterol to sites of utilization and energy storage)
-Attached with apoproteins
∑ Increased levels: Hyperlipoproteinemia
Functions: maintain structural integrity, ligands of cellreceptor, activators anf inhibitors of enzymes, amphipathic

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LIPOPROTEIN METABOLISM:
1. Lipid Absorption
2. Exogenous / Dietary Pathway
1st- Absorption of TAG and Cholesterol in the intestine
2nd- Formation and release of chylomicrons into the lymph and subsequently in the blood by the way of thoracic duct
3rd- Chylomicrons release TAG in adipose and the ApoB48 in the surface of Chylomicrons activate lipoprotein lipase found in
vasculoendothelial cells
4th- LPL cleaves free fatty acids from the TAG
5th- Reduction of Chylomicrons into the remnants taken up by the liver
6th- Free fatty acids are liberated and taken up bym uscle and adipose cells
3. Endogenous Pathway
1st- Synthesis of TAG from fatty acids by the liver with the secretion of VLDL containing Apo B100 and ApoE
2nd- VLDL is also affected by LPL leading to the production of IDL (with the release of fatty acids) that can either be removed
by Apo E or lose the Apo E and bind to hepatocytes and be converted to LDL
3rd- Cholesterol-rich LDL taken by the livel (70%0 or by other tissues and become a factor in membrane synthesis, steroid
synthesis and deposits (ATHEROMAS)
4. Reverse Cholesterol Pathway
1st- Production of Nascent HDL particles containing phospholipids and Apo A1 by the liver
2md Nascent HDL particles pick up cholesterol from tissules to become HDL3
3rd- Esterification of cholesterol in HDL3 and converting it to HDL2 by LCAT
4th Some fraction of cholesterol is transferred to VLDL to participate in membrane or steroid synthesis or taken up by the liver
and excreted as bile
Enzymes involved:
1. Lipoprotein Lipase (LPL) – hydrolyzes TAG to glycerol, monoglycerol and fatty acids
2. Hepatic lipase- hydrolyzes HDL, VLDL, IDL
3. Lecithin Cholesterol Acyltransferase- converts free cholesterol to cholesterol esters
4. Endothelial lipase – hydrolyzes HDL
MAJOR LIPOPROTEINS: Chylomicron, LDL, HDL, VLDL
Minor/ Additional Lipoproteins: Chylomicron remnant, IDL, Lp (a)
Minor Apolipoproteins: ApoD, ApoJ, ApoH, ApoF, ApoG
∑ Chylomicron remnant- lipolytic product of CM metabolism; taken up by the liver
∑ Intermediate Density Lipoprotein (IDL)- transitional form; lipolytic product of VLDL catabolism (VLDL
remnant); taken up by the liver or converted to LDL (subclass of LDL) , migrates between beta and pre-beta
region
∑ Lp (A)- LDL-like molecule with Apo (a) linked to Apo B100 (sinking pre-beta lipoprotein)
- Density: similar to LDL
-Electrophoretic mobility: similar to VLDL
- High level of homology with plasminogen (competes with plasminogen and fibrin)
- Increased- risk for CHD
- Independent risk for atherosclerosis
-Increased- risk of premature CHD and stroke
Abnormal Lipoproteins: βVLDL, LpX
∑ βVLDL (floating beta-lipoprotein)
- Density: similar to VLDL
-Electrophoretic mobility: similar to LDL
-VLDL rich in cholesterol
-Found in dysbetalipoproteinemia or type III hyperlipoproteinemia
∑ LpX abnormal protein found in obstruction jaundice and LCAT deficiency
-Indicator of cholestasis
-Increased unsterified cholesterol
LIPOPROTEINS
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Chylomicrons Largest and least dense

Highest amount of fat
Formed in the intestines, enters blood via lymphatics
Transports dietary/exogenous TAG in plasma to hepatic and peripheral cells
(90%)TAG is the predominant component (Most TAG-rich lipoprotein)
Major Apolipoproteins: ApoA1, ApoA2, Apo C, ApoB48
Cleared 6-9 hours post prandial
-Produces creamy/opaque layer (after the standing plasma test)
-Imparts turbid appearance to serum (before standing)
Non-atherogenic
Very Low Pre-beta lipoprotein
Density Produced in the liver
Lipoprotein Most insoluble
Major Apolipoprotein: Apo C, Apo E, Apo B100
Transports endogenous TAG from the liver to peripheral tissues
-Imparts turbid appearance to serum (before and after standing)
Apo CII- activator of LPL (VLDL to IDL)
Intermediate Contains equal amounts of cholesterol, TAG and phospholipids
Density Major Apolipoproteins: Apo C, Apo E, ApoB100
Lipoprotein Further catabolized to LDL with the aid of Apo E
Can be converted to βVLDL
Low Density Beta lipoprotein
Lipoprotein BAD CHOLESTEROL
Most atherogenic
Primary marker for CHD
Most Cholesterol-rich lipoprotein
Most abundant lipoprotein (50%)
Major Apolipoprotein: Apo C, Apo E, Apo B100
Transports dietary/exogenous CHOLESTEROL from liver to peripheral tissues
-Basis from treatment of CHD:
Persons with multiple risk factors– LDL ≥100 mg/dL –Statin therapy is recommended
Target is <70 mg/dL for sersons with multiple risk factors
LEVEL RECOMMENDED USED BY PHYSICIAN FOR THERAPEUTIC CHANGES:
OPTIMAL <100 mg/dL
NEAR OPTIMAL 100-129 mg/dL
BORDERLINE HIGH 130-159 mg/dL
HIGH 160-189 mg/dL
VERY HIGH ≥190 mg/dL
High Density Alpha Lipoprotein
Lipoprotein GOOD CHOLESTEROL
Smallest but most dense
Smallest total amount of fat
Synthetized in the liver and intestines
Transports excess cholesterol from tissues and return it to the liver (Reverse Cholesterol Transport)
Facilitates the removal of cholesterol in the arterial wall (decreased risk for CHD-inverse relationship)
Major Apolipoprotein: Apo C, Apo E, Apo A2, Apo A1
NORMAL LOW RISK ≥40 mg/dL
PROTECTIVE ≥60 mg/dL
HIGH RISK <40 mg/dL
Reference Method: Ultracentrifugation, Precipitaion- Heparin Manganese, Abell- Kendall Assay

Lipoprotein Diameter Density Electrophoretic Major TAG Cholesterol Phospholipid Protein

(nm) Mobility Apolipoprotein
Chylomicron >70 <0.95 Origin Apo B48 90% 2-4% 3-6% 1-2%
VLDL 26-70 0.95- Pre-Beta Apo B100 65% 16-27% 15-20% 6-10%
1.006
LDL 19-23 1.019- Beta Apo B100 4-8% 50% 22% 18-22%
1.063

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HDL 4-10 1.063- Alpha Apo A1 2-7% 15-20% 30% 45-55%

1.21

HUMAN APOLIPOPROTEINS:
Apolipoprotein Function Major Source
Apo A1 Major structural protein in HDL- Ligand for HDL Binding Liver and intestine
Activates LCAT
Apo A2 Structural protein in HDL Liver
Activates LCAT
Enhances hepatic TAG lipase activity
Apo A4 Component of intestinal lipoproteins Intestine
Apo B100 Major structural protein in LDL and VLDL - Ligand for LDL receptor Liver
Apo B48 Primary structural protein in CM Intestine
Apo C1 Activates LPL Liver
Apo C2 Activates LPL Liver
Activates LCAT
Apo C3 Inhibits LPL Liver
Inhibits receptor recognition of ApoE
Apo E2,3,4 Binds to LDL receptor and remnant receptor Liver
Apo (a) Structural protein for Lp(a) Liver
May inhibit plasminogen binding
LIPID AND LIPOPROTEIN DISORDERS:
Type TAG Tc LDL VLDL CM Serum
Type 1 ↑ N or ↑ ↑ Creamy layer over clear
Familial LPL Deficiency ↑ serum
Exogenous Hypertriglyceridemia
Familial Fat Induced Lipemia
Hyperchylomicronemia
Type 2A N ↑ ↑ N N Clear serum
Familial Hypercholesterolemia
Type 2B ↑ ↑ ↑ ↑ N Clear or slightly turbid
Mixed Combined Hyperlipidemia serum
Type 3 ↑ ↑ - ↑ N or Creamy layer sometimes
Familial Dysbetalipoproteinemia ↑ over turbid serum
Type 4 ↑ N or N or ↑ N Turbid serum
Hypertriglyceridemia ↑ ↑
Endogenous Hypertriglyceridemia
Hyperprebetalipoproteinemia
Typoe 5 ↑ ↑ N or ↑ ↑ Creamy layer over turbid
Mixed Hypertriglyceridemia ↓ serum
Mixed Hyperlipidemia
Hyperprebetalipoproteinemia with Chylomicrons
LIPOPROTEIN MEASUREMENT:
Best sample for Lipoprotein analysis: EDTA plasma – preserves lipoproteins
A. HDL Methods:
1. Ultracentrifugation
-Sample adjusted to a density of 1.063 (Potassium bromide) – Expressed in Svedberg units
-Centrifuged at high speed for 24 hours
-Reference method for lipoprotein quantitation
2. Polyanion Precipitation
-Apo-B containing lipoproteins are precipitated using a polyanion divalent cation solutions: dextran sulfate, magnesium
sulfate, phosphotungstic acid, iron; no centrifugation needed
-HDL is quantified in the supernatant after precipitation
QUANTIFCIATION OF HDL:
Reagent: dextran sulfate-magnesium chloride or heparin sulfate-manganese chloride
-Precipitates Apo-B contanining compounds (VDL, IDL, LDL, CM)
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-Supernatant is mixed with cholesterol oxidase and chosterol esterase to quantitate HDL concentrations
3. Electrophoresis: separation based on size and charge (α1)
Cellulose Acetate or Agarose Gel

Polyacrylamide Gel

Lipoprotein Stains S
O
F
4. Homogenous Assay: uses an antibody to apo-B100 to bind LDL and VLDL then HDL is measure enzymatically
B. LDL Methods:
1. Computation:
1. Friedewald Formula
LDL-c= Total cholesterol – HDL-c + VLDL-c
VLDL-c = Plasma TAG/ 5 (mg/dL)
VLDL-c = Plasma TAG/ 2.175 (mmol/L)
-NOT RELIABLE WHEN TAG>400 mg/dL (TAG x0.16)
MOST COMMONLY USED
2. Delong Formula
LDL-c= Total cholesterol – HDL-c + VLDL-c
VLDL-c = Plasma TAG/ 6.5 (mg/dL)
VLDL-c = Plasma TAG/ 2.825 (mmol/L)
-MORE ACCURATE ESTIMATE OF VLDL-c
2. Beta-Quantification
Uses ultracentrifugation (to separate VLDL and CM) and precipitation (to remove HDL)
LDL is calculated as density >1.006
3. Homogenous (Direct)
-Uses detergent or other chemicals to block or solubilize lipoprotein classes to allow for quantification of LDL
C. VLDL Methods:
1. Computation:
1. Friedewald Formula
LDL-c= Total cholesterol – HDL-c + VLDL-c
VLDL-c = Plasma TAG/ 5 (mg/dL)
VLDL-c = Plasma TAG/ 2.175 (mmol/L)
-NOT RELIABLE WHEN TAG>400 mg/dL (TAG x0.16)
-MOST COMMONLY USED
2. Delong Formula
LDL-c= Total cholesterol – HDL-c + VLDL-c
VLDL-c = Plasma TAG/ 6.5 (mg/dL)
VLDL-c = Plasma TAG/ 2.825 (mmol/L)
-MORE ACCURATE ESTIMATE OF VLDL-c

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AUGUST 2021 MEDICAL TECHNOLOGY LICENSURE EXAMINATION REVIEW (Klubsy Bear Online Tutorial)
Contents:
III. Proteins
IV. Nonprotein Nitrogen Compounds
V. Liver Function
VI. Renal Function
III. Proteins
Composed of Carbon, Hydrogen, Oxygen and Nitrogen (CHON); These are large molecules referred to as macromolecules
Element that distinguishes protein from CHO and Lipids is Nitrogen; accounts for approx. 16% of the protein molecule
Fundamental building blocks of protein: Amino Acids
Catabolism and Nitrogen Balance:
Anabolism: protein synthesis; All are synthesized in the liver except immunoglobulins (plasma cells)
∑ TPAG – most significant liver function test
∑ Central Dogma of Molecular Biology:

Catabolism: protein breakdown

∑ Positive Balance – more nitrogen is incorporated in the body than excreted
Ex. Pregnant women, Growing children and Adults recovering from major illness
∑ Negative Balance – more nitrogen is excreted than is incorporated in the body
Ex. Burns, Wasting disease, Continual high fever and Starvation
Central reaction that remove amino acid nitrogen from the body: Deamination/ Transamination
Two Main Routes for Converting Intracellular proteins to Free Amino Acids:
∑ Lysosomal Pathway – degrades extracellular proteins
∑ Cytosolic Pathway – degrades important intracellular proteins
Essential Amino Acids: Valine, Methionine, Arginine, Phenylalanine, Histidine, Isoleucine, Leucine, Lysine, Taurine,
Threonine, Tryptophan
Protein Charges:
Form of amino acid with two differing charges and with a net charge of zero: Zwitter ion
The pH value at which the sum of a molecule’s charges equals zero: Isoelectric point
The point at which the number of positive and negative charged group equals each other in a protein: pI
pH>pI = protein charge is NEGATIVE
pH<pI= protein charge is POSITIVE
Enzymes for Protein Digestion:
1. Pepsin
2. Trypsin
3. Chymotrypsin
4. Elastase
5. Carboxypeptidase
Functions of Proteins:
1. Biocatalysts (enzymes)
2. Repair body tissue (structural proteins)
3. Important in blood coagulation and immunologic function (C, Ig)
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4. For transport of metabolic substances (protein transporters)

5. Maintenance of osmotic pressure (albumin)
6. Maintenance of blood pH (buffers)
7. Production of peptide hormones (insulin-glucose metab, endorphins-pain perception)
PROTEIN STRUCTURE:
PRIMARY Amino acids arranged in specific sequence Peptide bond
SECONDARY Twisted shape of primary structure due to rotation Hydrogen bond
of bonds
Shapes may be α-helix, plated sheet or random coil
TERTIARY 3-D structure that forms when AA chain folds back Disulfide linkages and noncovalent attraction like
on itself hydrogen bonds, electrostatic, hydrophobic and
Vander Waals forces
QUATERNARY Interaction ir combination of more than one Noncovalent attrations
protein molecule or subunits
∑ The loss of the naturally occurring folded structure of proteins: Denaturation (Mechanical, Heat or Chemical –
alteration of the tertiary structure)
CLASSIFICATION
SIMPLE Composed of AA inked by peptide bonds
Globular- albumins and immunoglobulins
Fibrous- connective tissues, tendons, bone and muscle
CONJUGATED Composed of a prosthetic group and a protein
∑ Metalloproteins – Metal ions attached: Ferritin, Ceruloplasmin; Complex metals attached:
Hemoglobin, Flavoproteins
∑ Lipoproteins – lipid attached: HDL, LDL, VLDL
∑ Mucoproteins – mucin attached Pproteoglycans)
∑ Glycoproteins: CHO attached: Haptoglobin, α1-antitrypsin
∑ Nucleoproteins: nucleic acids attached: Chromatin
PLASMA PROTEINS: Protein Electrophoretic Fractions

Plasma Protein Usage Increased Decreased

PREALBUMIN Indicator of nutrition Steroids, Hepatic damage, Acute Phase
-↓ MARKER OF POOR NUTRITIONAL STATUS Alcoholism, Inflammatory response, Tissue
Transthyretin: Binds with Thyroid hormones Chronic Renal Necrosis
and retinol (Vit.A) Failure
Confirms if a specimen is really CSF (crosses
more easily than other proteins)
ALBUMIN -Most anodal protein (fast migrating) Dehydration, Liver disorders, GIT associated
-Major contributor to oncotic colloidal pressure excessive malabsorption, Muscle wasting,
-Present in highest concentration in plasma albumin Severe burns, Renal disease,
Binds bilirubin, steroids and fatty acids, ions infusion Starvation and Malnutrition
such as calcium and magnesium, and drugs -Sudden – Nephrosis (lowest plasma
albumin levels)
-Gradual - Cirrhosis
∑ Analbuminemia – abscense of albumin
∑ Bisalbuminemia – presence of unusual molecular characteristics (two albumin bands) -hereditary; acquired
Malnutrition:

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∑ Kwashiorkor- diet severely deficient of protein (high starch foods)

↓ serum albumin, Immune deficiency, Edema, Ascites, Growth failure, Apathy, etc.
∑ Marasmus- protein and energy sources are deficient
Protein-calorie starvation which leads to general wasting of muscles and subcutaneous tissues and lesser edema
ALPHA-1 GLOBULINS
1. α1-antitrypsin Major protease inhibitor Inflammation, Genetic: Pulmonary Emphysema
Most abundant protein in α1globulin Pregnancy, Juvenile Cirrhosis
fraction and APR Contraceptive Liver disease, Neonatal cholestasis,
Neutralize trypsin and other proteolytic use Hepatitis, Cirrhosis, HEpatocellular
enzymes used to combat infections but can carcinoma
destroy alveoli
2. α1-fetoprotein Principal fetal protein (fetal yolk Neural tube defects- Spina Bifida, Trisomy 21 – Down
sac/liver) Anenchephaly, abdominal wall defects, Syndrome
Used to measure risk for general fetal distress Trisomy 18
chromosomal abnormalities in fetus Twins
-Tumor marker: ↑ in hepatic and
gonadal cancer
3. α1- acid Major plasma glycoprotein Stress, Inflammation, Tissue damage, -
glycoprotein - Only protein still negatively Acute Myocardial Infarction,
Orosomucoid charged even in acidic solution Nephrotic syndrome, trauma,
Greatest affinity to progesterone pregnancy, cancer, pneumonia,
Binds with quinidine rheumatoid arthritis, surgery
4. α1- Inhibits serine proteinases Inflammation, Alzheimers disease Liver disease
antichymotrypsin Major transporter of PSA
5. α1- lipoprotein transports lipids (HDL) Risk of CHD Tangier’s disease
6. Group-specific Transports cholecalciferol (Major carrier protein of Third trimester of Severe liver disease
Component Vit.D) pregnancy, Oral and protein-losing
(Gc)-globulin Immunonephelometry – method of choice for contraceptives syndromes
measurement
Migrates between α1 and α2 globulin
ALPHA-2 GLOBULINS
1. Haptoglobulin Transport, salvage and conserve hemoglobin Inflammatory diseases Hemolytic anemia
Marker for intravascular hemolysis (HDN and HTR) (Rheumatic diseases), (detect and evaluate)
Independent risk factor for cardiovascular disease Burns, Nephrotic
Used in paternity testing syndrome
2. Ceruloplasmin Binds to copper (>90%) Inflammation, Infection, Wilson’s disease (marker) –
The enzyme present in highest Cancer, Tissue damage, Kayser-Fleischer rings in
circulating concentration Pregnancy, Oral cornea
- Ferroxidase- has oxidase activity; contraceptive, Menke’s syndrome – kinky
Oxidizes ferrous to ferric ion to allow Medications such as hair
transferring binding carbamazepine, Malnutrition, Malabsorption,
-Indirectly associated with Iron Deficiency phenobarbital, valproic Severe liver diasesa, Nephrotic
Anemia acid syndrome
3. α2- Inhibits proteases Nephrosis(↑10x), Genetic conditions,
macroglobulin -Largest major nonimmunoglobulin Diabetes, Liver disease, Pancreatitis, Prostatic
protein present in plasma Pregnancy and Oral Carcinoma
-Major component of α2 globulin fraction contraceptives (↑20%)
BETA GLOBULINS
1. Transferrin - -Major component of β globulin fraction Hemochromatosis, Liver disease, Malnutrition,
Siderophilin -Transport iron into its storage sites Iron Deficiency Nephrotic syndrome, Infection
-Prevents loss of Fe in urine Anemia and Malignancy
2. Hemopexin Binds heme from degradation Inflammation, Diabetes, Duchenne Hemolytic anemia (diagnostic)
of hemoglobin muscular dystrophy, Malignancies – early hemolysis
(melanoma)
3. Complement Natural defense mechanism Inflammatory states Malnutrition, Hemolytic anemia, Autoimmune
against infections disease, Neonatal distress syndrome, Bacteremia,
Tissue injury, Chronic hepatitis
4. Fibrinogen Precursor of fibrin clot Inflammation, Pregnancy, Oral Extensive coagulation
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Most abundant coagulation factor (F1) contraceptives

Plasma Electrophoresis – distinct band
between beta and gamma globulins
5. β2- Single polypeptide chain that is the light chain of the human Renal failure, -
microglobulin leukocyte antigen (HLA) found in the surface of nucleated cells and Lymphocytosis, SLE, RA
present in lymphocytes
( MHC Class 1)
6. C-reactive Binds to C-polysaccharide of the Screen organic diseases; Assess activity of inflammatory
protein pneumococcus; motivates phagocytosis disease (RA, SLE, leukemia, surgery, rejection in renal
No.1 Acute Phase Reactant allograft recipients and neonatal septicemia and
Normal to have undetected CRP meningitis
<1.0 mg/L – Low CVD risk
1-3 mg/L – Average CVD risk
>3 mg/L – High CVD risk
>10 mg/L - Acute Inflammatory condition
GAMMA GLOBULINS
1. Immunoglobulins Antibodies- IgG, IgA, IgM, IgD, IgE IgG- most abundant
IgM- first antibody to appear
IgD- surface of B cells
IgA- secretions
IgE- allergic and anaphylactic reactions
Other Proteins:
Amyloid Amyloidosis - Fibrils may infiltrate organs including heart, blood vessels, brain and peripheral
nerves, kidneys, liver, spleen and intestines causing localized or widespread organ failure
Amyloid vs. TAU protein – used to differentiatae Amyloidosis and Alzheimer’s disease
↓Amyloid, ↑ TAU protein – Alzheimer’s Disease
Abnormal Amyloid and TAU protein: Dementia
β-trace protein Accurate marker of CSF leakage
potential marker in detecting renal function impairment
Cross-linked C-telopeptide Biochemical marker of bone resorption (serum and urine)
(CTX)
Cystatin C new marker for early assessment of GFR (not affected by muscle mass, gender, age, race; affected
by drugs, infections, diet, inflammation)
Fibronectin Glycoprotein composed of two nearly identical subunits
Roles in cell adhesion, Tissue differentiation, Growth, Wound healing (cellular interactions)
Plasma fibronectin has been used as a nutritional marker
Myoglobulin Primary oxygen carrying protein in striated skeletal and cardiac muscle (approx. 2 % of total
muscle protein)
General Clinical Significance:
TOTAL PROTEIN ALBUMIN GLOBULIN DISEASE
Dehydration
Immunodeficiency Syndrome
Salt retention Syndrome
Hepatic damage
Cirrhosis
Hepatitis
Obstructive jaundice
Burns, trauma, infections
Malabsorption, Inadequate diet
Nephrotic Syndrome
Multiple Myeloma, monoclonal and polyclonal gammopathies
Reference Values:
∑ Albumin – 3.5-5.0 g/dL (53-65%)
∑ α1-globulin – 0.1-0.3 g/dL (2.5-5%)
∑ α2-globulin – 0.6-1.0 g/dL (7-13%)
∑ β-globulin – 0.7-1.1 g/dL (8-14%)
∑ γ-globulin – 0,8-1.6 g/dL (12-22%)
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Methods for Total Protein Measurement:

1. Kjeldahl – Acid digestion of protein based on the Reagents: Sulfuric Acid

measurement of the nitrogen content (assumes there is 16% Endproduct: Ammonia
nitrogen in protein)
- Reference method (Gold standard) but not routinely used
(Nitrogen is difficult to measure)
2. Biuret – In an alkaline medium, cupric ions complex with Reagents:
proteins forming a violet-colored chelate based on the Potassium iodide- stabilizes cupric ions
measurement of peptide bonds (requires at least 2 peptide Tartrate salts- keeps copper in the solution
bonds) Cupric ions- break the peptide bonds
- Most common method (routinely used) -Absorbance at 540nm is proportional to total protein
concentration
3. Dye binding – proteins bind to dye and causes a special Proteins absorbed light at 210 nm and 280 nm
shift in the maximum absorbance of the dye -Coomasie Brilliant Blue- preferred dye of choice
4. Serum Protein Electrophoresis – migration of charged Normal spikes: 5 spikes
particles in an electric field Albumin- 1 spike
-Most significant use: identification of monoclonal spike of Globulin- 4 spikes
immunoglobulins
ELECTROPHORETIC PATTERNS:
Multiple Myeloma M-spike/ Gamma spike; hyperglobulinemia
Dysproteinemia Sharply elevated spike in gamma globulin (Monoclonal Gammopathy)
Chronic Liver Disease Diffuse but large elevation in gamma globulin(Cirrhosis- Polyclonal
Gammopathy with beta-gamma binding; Increased IgA)
Chronic Inflammatory Disease Diffuse but small elevation in gamma globulin (Polyclonal Gammopathy)
Hypoalbuminemia Low spike in albumin
Hypogammaglobulinemia Flat curve in gamma globulin
Nephrotic syndrome Low albumin spike, Increased α2 globulin, Very high beta globulin
Protein Losing Enteropathy Low albumin spike, Moderate α2 globulin elevation
Malabsorption syndrome Low albumin and gamma globulin; High α2 globulin
α1- Antitrypsin Deficiency Flat curve in α1 globulin
Acute Inflammation Increased α1 and α2 (APRs)
5. Folin-Lowry – highest analytical sensitivity
Methods for Albumin Measurement: can be quantified by Simple Colorimetric Assay

1. Salt Precipitation / Fractionization – Globulins are Reagents: Sodium sulfate, sodium sulfite, Ammonium sulfate
precipitated in high salt concentration and Albumins in and methanol
supernatant is quantified by Biuret rxn
2. Dye-Binding HABA vs BCG:
a. Methyl orange – nonspecific dye for albumin HABA- 500 nm Interferences BCG- 500 nm & 630 nm
b. Hydroxyazobenzene benzoic acid (HABA) ↑ Hemolysis sl. ↑
c. Bromcresol green (BCG) – most commonly used dye for ↓ Bilirubin ↓
albumin; overestimates low albumin levels (more sensitive) sl. ↓ Salicylate No apparent effect
d. Bromcresol purple (BCP) – most specific, sensitive and ↑ Heparin ↓ (add detergent)
precise
3. Electrophoresis
IV. Nonprotein Nitrogen Compounds:
Measuring the concentration of nitrogen-containing compound in a protein-free filtrate by converting nitrogen to ammonia
Present in concentration of mg/dL or less because of ready clearance into the urine
∑ Principal excretory form of nitrogen: Urea
∑ Calculation of Nitrogen Balance: Urinary Urea
CLINICALLY SIGNIFICANT NONPROTEIN NITROGEN COMPOUNDS
Compound Concentration in Plasma Concentration in Urine
1. Urea 45-50 86.0
2. Amino Acids 25 absent
3. Uric Acids 10 1.7
4. Creatinine 5 4.5
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5. Creatine 1-2 absent

6. Ammonia 0.2 2.8
∑ UREA: major excretory product of protein metabolism in the liver form amino groups and free ammonia (urea cycle)
-Filtered from the plasma by the glomerulus and reabsorbed by passive diffusion in the renal tubules (affected by urine flow
and extent of hydration)
-45% of total NPNs
-90% are excreted by the kidneys
Concentration of Urea in the blood is determined by:
1. Protein diet
2. Rate of protein catabolism
3. Renal function/ perfusion
Clinical applications: Aid in diagnosis of renal disease; Verify adequacy of dialysis
Increased concentration: Uremia and Azotemia
Decreased concentration: Low Protein intake, Severe vomiting and diarrhea, Liver disease and Pregnancy
METHODOLOGY:
Specimen Consideration:
∑ Acceptable specimens: plasma, serum, 24-hour urine
∑ Avoid sodium citrate and fluoride
∑ Fasting is not required
∑ Urine samples should be refrigerated
ENZYMATIC (INDIRECT) METHOD Step 1: Urease hydrolyzes urea
-High specificity; more expensive Step 2: Quantify ammonium ions
General principle: Ammonia production General reaction: Urea +2H2O (UREASE)= 2NH4 +CO3
1. GLDH Coupled Enzymatic Reaction NH4 + 2-oxoglutarate/ α-ketoglutaric acid + NADH + H+ (GLUTAMATE
-Most frequently used DEHYDROGENASE) = Glutamate/ Glutamic acid + NAD+ + H2O
-Best Done kinetically -UV enzymatic method for BUN
2. Indicator Dye NH4 + pH indicator = color change
-Very common in dry chemistry ∑ NESSLER’s Reaction
NH4 + Nessler’s salt (KI/HgI2) – gum ghatti = YELLOW-ORANGE
∑ BERTHELOT Reaction
NH4 + Phenol + Alkaline hypochlorite – sodium nitroprusside = BLUE
3. Conductimetric Uses electrode selective for ammonia
Conversion results in increased conductivity
4. Isotope Dilution Mass Spectrometry Detection of characteristic fragments following ionization and
-Reference Method quantification using isotopically labeled compound
COLORIMETRIC (DIRECT) METHOD Urea + Diacetyl monoxime (DAM) = yellow solution (Diazine derivative)
-No interference by ammonia -Diacetyl condenses with urea forming a colored complex
-Sulfa drugs causes positive interference ∑ Iron and thiosemicarbizide – stabilizes the color of the solution
Reference Range:
Plasma/ Serum UREA: 5-38 mg/dL BUN:
Adult: 7-18 mg/dL
Infant/ Child: 5-18 mg/dL
>60 years old: 8-21 mg/dL
24-hour Urine 12-20g/ day 428-714 mmol urea/ 24 hour
∑ URIC ACID: major end product of endogenous purine catabolism and degradation of dietary purines primarily in the
liver (catalyzed by xanthine oxidase)
- Produced even in the absence of dietary purine intake (Organ meats, legumes and yeast)
-Daily excretion: Low purine diet= 0.5 g; Normal purine diet= 1g)
-Present in plasma as monosodium urates (>6.8 mg/dL = urate crystals may precipitate in tissues)
- 100% Filtered from the plasma by the glomerulus and reabsorbed in the proximal convoluted tubule and secreted in the
distal convoluted tubule by 6-12%
Clinical applications:
1. Assess inherited disorders of purine metabolism
2. Confirms diagnosis and monitor gout
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3. Diagnosis of renal calculi

4. Prevent uric acid nephropathy during chemotherapy
5. Detect kidney dysfunction
Increased concentration:
Increased production, Raised serum levels Idiopathic mechanisms associated with primary gout
Lesch-Nyhan syndrome
Excessive dietary purines
Cytolytic treatment of malignancies (lymphomas and leukemias)
Polycythemia
Myeloid Metaplasia
Psoriasis
Sickle cell anemia
Decreased excretion, Raised serum levels Alcohol ingestion
Thiazide diuretics
Lactic acidosis
Aspirin doses <2g/day
Ketoacidosis, esp. in children
Renal Failure
Increased excretion, Lowered serum levels Probenecid, Sulfinpyrazone, Aspirin doses above 4g/day
Corticosteroids and ACTH
Coumarin Anticoagulants
Estrogen

Decreased concentration:
1. Liver disease
2. Defective tubular reabsorption (Fanconi’s syndrome)
3. Chemotherapy with azathioprine or 6-mercaptopurine
4. Overtreatment of allopurinol
Treatment:
1. Allopurinol – inhibits xanthine oxidase activity; lowers serum levels without increased secretory load in kidney
2. Uricosuric drugs (Probenecid, Sulfinoyrazone) – lowers serum levels by increasing secretory of uric acid in the urine (risk
of stone formation if urine is not maintaine at an alkaline ph)
3. Colchicine – alters the phagocytic response of leukocytes to urate crystals in tisuue
METHODOLOGY:
Specimen Consideration:
∑ Acceptable specimens: heparinized plasma, serum, urine
∑ Serum or plasma samples can be refrigerated up to 3-5 days
∑ EDTA and Sodium fluoride should be avoided
∑ Lipemic, icteric and hemolyzed samples causes falsely decreased values
∑ Salicylates and thiazides causes falsely increased values
∑ Allopurinol, probenecid, corticosteroid and aspirin (large doses) increases urate excretion
ENZYMATIC METHOD:
∑ Enzymatic –H2O2 production – interference by I. Coupling enzyme: Catalase
reducing substances H2O2 + reagent = colored compound
-Commonly used and increased specificity
First Step: Uric acid + O2 + 2H2O (URICASE)= allantoin + CO2 + II. Coupling enzyme: Peroxidase
H2O2 H2O2 + indicator dye = colored compound
∑ Enzymatic –UV – needs special instrumentation and Decrease in absorbance at 293 nm is measured (allantoin)
optical cells
-Spectrophotometric (Blauch and Koch)
CHEMICAL METHOD:
Caraway Method (PTA) Interferences: proteins and lipids cause turbidity
Uric Acid + Phosphotungstic acid + O2 = tungsten blue +allantoin + CO2 and quench absorbance (false decrease) ; glucose,
∑ Allantoin- oxidation product of uric acid in an alkaline solution ascorbic acid, acetaminophen, caffeine, theophylline,

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∑ Phosphotungstic acid – reducing agent glutathione, ergothionine and cysteine released

∑ Tungsten blue is measured from RBC (false increase)

Reference Range: Men 4-8.5 mg/dL

Women 2.7-7.3 mg/dL

∑ CREATININE: chief product of muscle metabolism; end product of creatine metabolism (muscle’s energy storage –
creatine phosphate)
-Dependent on muscle mass (proportional to skeletal muscle)
-Not affected of protein diet and not metabolized by the liver
∑ Creatine phosphate + ADP (CREATINE KINASE) = ATP + creatine
-Some creatinine are irreversibly converted to creatinine which is excreted in the kidneys. Daily excretion is constant except in
crushing injuries and degenerative disease (massive muscle damage)
Clinical applications:
1. Determine sufficiency of kidney function
2. Determine severity of kidney damage
3. Monitor the progression of kidney disease
4. Measures completeness of 24-hour urine
∑ Blood level becomes elevated when renal function declines
∑ In aging patients, blood level is stable but 24-hour urine shows a decreased creatinine clearance
METHODOLOGY:
Specimen Considerations:
∑ Accepted specimens: Plasma, serum and urine
∑ Hemolyzed, icteric and lipemic samples should be avoided
∑ Fasting is not required
∑ Urine should be refrigerated or frozen after collection if longer storage than 4 days is anticipated
FALSE INCREASE Glucose, α-ketoacids, ascorbate (enzymatic that uses peroxidase), uric acid, cephalosphorins, dopamine;
Lidocaine (enzymatic)
FLASE DECREASE Bilirubin (both jaffe and enzymatic), Hemoglobin, Lipemic samples

ENZYMATIC METHOD:
Creatininase-CK Creatinine + H2O (CREATININASE) = Creatine
-Requires large sample Creatine + ATP (CREATININE KINASE) = Creatine phosphate + ADP
-Not widely used Phosphoenolpyruvate + ADP (PYRUVATE KINASE) = Pyruvate + ATP
Pyruvate + NADH + H+ (LACTATE DEHYDROGENASE)= Lactate + NAD
Cretininase-H2O2 Creatinine + H2O2 (CREATININASE) = Creatine
-Adapted for dry chemistry Creatine + H2O (CREATINASE) = Sarcosine + ADP
-No interferences from Sarcosine + O2 + H2O + ADP (SARCOSINE OXIDASE) = Glycine + CH2O + H2O2
acetoacetate/cephalosphorin H2O2 + Colorless substrate (PEROXIDASE) = Colored product + H2O
-Some positive bias to lidocaine -Potential to replace Jaffe
Isotope Dilution Mass Spectrometry Detection of characteristic fragments following ionization and
-Highly specific quantification using isotopically labeled compound
-Accepted Reference Method
CHEMICAL METHOD: Colorimetric: End point – simple and nonspecific
Colorimetric: Kinetic – rapid and increased specificity
Direct Jaffe Reaction: Creatinine + Alkaline picrate = red-orange complex
Jaffe-Kinetic Rate of change of abcorbance is detected to avoid interference with
-Requires automated equipment noncreatinine chromogens
-Positive bias from αketoacids and cephalosphorins
Jaffe with Absorbent Creatinine in protein-free filtrate is absorbed onto an absorbent then
-Absorbent improves specificity eluted and reacted with alkaline picrate
-Previously considered as reference method Fuller’s Earth Lloyd’s Reagent
Aluminum Magnesium Silicate Sodium Aluminum Silicate
Jaffe without Absorbent Creatinine in protein-free filtrate is reacted with alkaline
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-Positive bias from ascorbic acid, glucose, gluthathione, αketoacids, picrate to form colored complex
uric acids and cephalosphorin
Reference Range:
Sex Jaffe Enzymatic
Men 0.9-1.3 mg/dL 0.6-1.1 mg/dL
Women 0.6-1.1 mg/dL 0.5-0.8 mg/dL

∑ AMMONIA: product of amino acid deamination

-Source: skeletal muscle and gut; Converted to urea in the liver
-Neurotoxic; associated with encephalopathy

Clinical Applications:

1. Diagnosis of hepatic failure and coma (If in children, suspect inborn error of metabolism- urea cycle disorders)

2. Reye’s syndrome (acute metabolic disorder of the liver)

3. Inherited deficiencies of the urea cycle

Increased Levels:
1. Significant hepatocyte dysfunction
2. Too much collateral circulation
3. Excess protein in the gut
4. Reye’s syndrome
5. Renal failure
6. Chronic liver failure
METHODOLOGY:

Specimen Considerations:

∑ Accepted specimen: arterial blood w/o tourniquet

∑ False Elevations in all methods:

1. Smoking of patient

2. Laboratory atmosphere

3. Poor venipuncture (Vein probing, Use of Heparin lock, Transfer of syringe to evacuated tube, Partially filled tube allowing
air to enter)

4. Metabolism of nitrogen constituents (Prevented by: Keeping on ice, Separation of serum from cells)

5. Hemolysis

6. Tourniquet Application

1. Two-stage: requires separation of ammonia and ammonium

2. One-stage: direct measurement of ammonia
ENZYMATIC METHOD:
Glutamate Dehydrogenase (GLDH) NH4 + 2-oxoglutarate + NADPH + H+ (Glutamate Dehydrogenase) = Glutamate +
-Most common on automated instrument NADP+ + H2O
Accurate and precise
CHEMICAL METHODS:
Ion-selective electrode Diffusion of NH3 through selective membrane into NH4Cl causing pH change
-Good accuracy and precision which is measured potentiometrically
-Membrane stability may be a problem

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Spectrophotometric NH3 + bromphenol blue = blue dye

Reference Range: 19-60 µg/dL

V. Liver Function Tests:

Liver is the chief metabolic organ in the body
-To abolish liver tissue function, more than 80% of the liver must be destroyed
Functions of the Liver:
1. Synthetic Function- last function to be affected
-Synthesis of proteins- enzymes, clotting factors, lipoproteins
-Metabolism of cholesterol into bile acids
-Normal liver produces 12 grams of albumin daily
2. Conjugation Function
-Conjugation of bilirubin
3. Detoxification
-Drug metabolism
-Conversion of Ammonia to Urea
4. Excretory and secretory function
-Excretion of Bile
5. Storage
-Storage site for all fat soluble and water soluble vitamins
-Storage site for glycogen
A. Tests Measuring Hepatic Synthetic Function:
-Useful in quantitating the severity of hepatic dysfunction
-Serum albumin and Vit, K dependent coagulation factors provide the most useful indices for assessing the severity of liver
disease
1. Total Protein – performed on serum which has no fibrinogen and no anticoagulant that may slightly dilute proteins in
plasma; hemolysis may falsely elevate the total protein
Methodology:
a. Kjeldahl Method
b. Biuret Method
c. Serum Protein Electrophoresis
2. Albumin
-Concentration is inversely proportional to the severity of liver disease
-Levels decline when severe hepatocellular disease last more than 3 weeks
Gradual loss: Cirrhosis
Abrupt loss: Nephrosis
-Negative APR
Dyes used for measurement:
Albumin can be measured based in its dye-binding property
1. BCG
2. MO
3. HABA
4. BCP
-BCG and HABA are widely used in manual and automated procedures
Hyperalbuminemia
1. Severe Dehydration
2. Artifactual Hyperalbuminemia (Prolonged tourniquet application)
Hypoalbuminemia
1. Reduced synthesis- Cirrhosis
2. Increases loss- Nephrosis
3. Increased catabolism- Severe burns, Malignancy, Thyrotoxicosis

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3. Albumin/Globulin Ratio
-If globulin is higher than albumin, it is known to be INVERTED A/G ratio seend in
Hepatic Cirrhosis – IgA
Multiple Myeloma – IgG
Waldenstrom’s macroglobulinemia – IgM
Reference Range:
Total Protein: 6.5-8.3 g/dL
Albumin: 3.5 -5.0 g/dL
Globulin: 2.3-3.5 g/dL
A/G Ration: 1.3-3: 1 (2:1)
B. Tests Measuring Conjugation and Excretion Function
1. Bilirubin: end product of hemoglobin metabolism and the principal pigment in bile
BILIRUBIN METABOLISM:

Reference Range:
Total Bilirubin: 0.2-1.0 mg/dL
Conjugated Bilirubin: 0-0.2 mg/dL
Unconjugated Bilirubin: 0.2-0.8 mg/dL
2. Delta Bilirubin – conjugated bilirubin tightly bound to albumin
-Formed due to prolonged elevation of conjugated bilirubin in biliary obstruction
-Reacts directly with the color reagent and contributes to the direct or conjugated value
3. Jaundice (Icterus/ Hyperbilirubinemia)
-Yellow discoloration of the skin, sclera and mucous membranes
-Indirect or unconjugated bilirubin can cross the myelin rich structures of the brain causing kernicterus
Classification of Jaundice:
Causes Description B1 B2 Urobilinogen Urine
Bilirubin
Pre-Hepatic Jaundice Too much destruction of RBC ↑ N N
Elevated Indirect Bilirubin
Hepatic Jaundice Hepatocyte injury caused by virus, alcohol ↑ ↑ ↓ +
and parasites
Inherited Defects of Bilirubin Metabolism:
a. Gilbert’s – Bilirubin Transport Deficit
-Ligandin deficiency; impaired cellular uptake of bilirubin
↑B1
b. Crigler-Najar – Conjugation Deficit
-Glucoronyl transferase (UDPGT) deficiency
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↑B1
-Physiologic Neonatal Jaundice Infants are treated with phototherapy
Type 1- Complete deficiency
-Total B2 absence + Kernicterus
-Bile is colorless
Type 2- Partial deficiency
-Small amount of B2 produced
c. Dubin-Johnson and Rotor – Bilirubin Excretion Deficit
- blockage of the excretion of bilirubin
-Intense dark pigmentation of the liver due to accumulation of lipofuscin pigment
↑B2 and Total Bilirubin
d. Lucey-Driscoll- Conjugation Inhibitor
-Circulationg inhibitor of bilirubin conjugation
↑B1
Post-Hepatic Jaundice Failure of bile to flow to the intestine/ N ↑ ↓ +
impaired bilirubin excretion due to presence
of gall stone/ cholelithiasis
Bile fluid: Cholesterol, Bile salt, B2
Elevated Direct bilirubin
Methodology:
Principle: Van den Berg Reaction
-Bilirubin + Sulfanilic acid= Azobilirubin (Diazotization)
Assay Evelyn-Malloy Jendrassik-Grof
pH Acid Alkaline
Dissociating agent Methanol Caffeine-Sodium Benzoate
(Coupling accelerator)
Diazo product Red or Reddish-purple Blue or Blue-purple
Maximum Absorbance 560 nm 600 nm
∑ Jendrassik-Grof
-Candidate reference method
-Most sensitive and widely used
-Preferred by automated analyzers (popular for discrete)
-Alkaline Tartrate- provides alkalinity
-Ascorbic acid- terminates the reaction
C. Tests for the Detoxification Function
1. Ammonia- marker for detoxification; least NPN
-the only NPN that is not a kidney function test
-Liver disease- ↑NH3, ↓Urea
-Lowers GABA, a neurotransmitter
Methodology:
a. Nesslerization
b. Berthelot
VI. Kidney Function Tests:
A. Test for Glomerular Filtration Rate
-Best overall indicator of level of kidney function
-Measure clearance of normal molecules that are not bound to protein and are freely filtered by the glomeruli and neither
reabsorbed nor secreted by the tubules
1. Clearance tests:
-removal of the substance from plasma into urine
-Plasma concentration and Clearance are inversely proportional
Formula:
mL 1.73
= ( )
min min
U-conc. of analyte in urine

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P-conc. of analyte in plasma

V- volume of urine in ml/24 hours (÷ 1440) =ml/min
BSA- body surface are of the patient
1.73- constants value (Average body surface area) ; if infant use 0.17
Estimated Glomerular Filtration Rate (eGFR)
-does not require the collection of timed (24-hour) urine specimen
Cockroft –Gault
Modified Diet in Renal Disease (MDRD)
Chronic Kidney Disease-Epidemiology collaboration (CKP-EPI)
Modified Schwartz- pediatric population (<18 years old)
a. Inulin Clearance
-Gold standard; Reference method
-Extremely stable substance; Most reliable
-Not reabsorbed nor secreted by the tubules
-Not routinely done due to continuous IV infusion of Inulin
b. Creatinine Clearance
-Best Alternative method
-Excellent measure of renal function since creatinine is freely filtered by the glomeruli and not reabsorbed
-Production and excretion is directly related to muscle mass
Reference Range: Men 85-125 mL/min
Women 75-112 mL/min
c. Urea Clearance
-The earliest GRF
-Variably reabsorbed by the tubules (40% of the filtered urea is reabsorbed)
d. Cystatin C
-freely filtered in the glomerulus but completely reabsorbed and broken down by the renal tubular cells
-Presence in urine denotes damage of the Proximal Convoluted tubule
-Not affected by muscle mass, age, gender
Specimen: Serum or Plasma
-The only kidney function test measured in serum
-Increased in acute and chronic renal failure; for early diagnosis
Reference Range: 0.5-1 mg/dL
B. Tests for Renal Blood Flow
-BUN, BUA, CREA
-Routine: Enzymatic
-Reference: Isotope Dilution Mass Spectrometry (IDMS)
1. Blood Urea Nitrogen
-First metabolite to elevate in kidney disease
-Easily removed by dialysis
Reference Range: 8-23 mg/dL
BUN: Creatinine Ratio: 10-20:1
-Kidney disease- ↓BUN in urine, ↑BUN in blood
-Severe Hepatocellular disease- ↓BUN in blood
Methods: Comments:
Colorimetric: DAM Inexpensive, lacks specificity
Enzymatic: Ammonia formation Greater specificity, more expensive

2. Creatinine
-Not easily removed by dialysis
-An index of renal function
-Most commonly used test
Methods: Comments:
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Colorimetric: End point Simple, nonspecific

Colorimetric: Kinetic Rapid, increased specificity
Enzymatic Measures ammonia colorimetry or with ion selective
electrode

BUN: Creatinine Ratio Conditions Associated

Low <10:1 Low protein diet
Acute tubular necrosis
Repeated dialysis
Hepatic Disease
Vomiting
Starvation
High >20:1 with Normal High protein diet
Creatinine Prerenal Azotemia
Dehydration
Muscle wasting
Tissue breakdown
Catabolic states
GI hemorrhage
Cortisol treatment
High >20:1 with Increased Postrenal azotemia
Creatinine Prerenal azotemia with renal disease
Prostatitis
Severe infection
Normal 10-20:1 with End Stage Renal Disease (ESRD)
Increased Creatinine and Urea Acute Renal failure
Tubular disease (acute pyelonephritis)
Glomerular disease (nephrosis/ glomerulonephritis)
Disease Correlation:
a. Azotemia- elevated concentrations of nitrogenous substances such as urea and creatinine in blood
Pre-renal Azotemia CHF, hemorrhage, dehydration, increased protein catabolism,
-Reduced blood flow, poor perfusion of high-protein diet, shock, blood loss, crush injuries, burns, fever,
the kidney resulting to decrease GFR hemorrhage into soft tissues of the body cavites and hemolysis
Renal Azotemia Acute or chronic glomerulonephritis, polycystic kidney disease
-Damaged filtering structures of the and nephrosclerosis, acute or chronic renal failure and renal
kidney due to acute or chronic renal disease (tubular necrosis, pyelonephritis, DM, arteriosclerosis,
diseases amyloidosis, collagen-vascular disease), malignant hypertension,
nephrotoxic drugs or metals
Post-renal Azotemia Renal calculi, tumors of the bladder or prostate, enlarged prostate
-Urinary Tract Obstruction; where urea gland, inflammation, surgical misadventure, obstruction of
is reabsorbed into the circulation bladder neck or urethra by prostate, tumor, inflammation
b. Uremia- marked elevation of plasma urea and other NPNs accompanied by acidemia and hyperkalemia
-Very high plasma urea with renal failure
- Anemia, Uremic frost (dirty skin), Foul breath, Sweat is urine –like
-Burr Cell (Echinocyte) – signas the development of renal dysfunction
3. Blood Uric Acid
-Formed from xanthine by xanthine oxidase
-Uric acids in joints- Tophi
Methods: Comments:
Colorimetric Problems with turbidity and several common drugs
Enzymatic-UV Needs special instrument and optical cells
Enzymatic-H2O2 Interferences by reducing substances

C. Tests Measuring Tubular Function

1. Concentration tests – commonly requested test
a. Specific Gravity (SG)
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-Simplest renal concentrating ability test

-Perform 3-5 days
Fixed SG at 1.010 – severe loss of concentrating ability
Reference Range: 1.002-1.030
b. Osmolality
-More reliable; Under tubular function test
-Affected by the number of solutes present
-Urine osmolality is primarily due to Urea
-Serum osmolality is primarily due to Sodium and Chloride
-Sample: Serum or Urine (24-hr urine)
METHODOLOGY:
a. Direct Method:
-Measurement of Colligative properties
-An increase in osmolality increases the osmotic pressure and boiling point and decreases vapor pressure and freezing point
Freezing point depression- most popular
b. Indirect Method:
-Measurement by computation
∑ mg/dL
1.86 + + + 9
18 2.8
2 + +
20 3
∑ mmol/L
1.86Na + Glu + BUN
2Na + Glu + BUN
Reference Range:
Serum: 275-295 mOsm/kg
24-hour Urine: 300-900 mOsm/kg
Normal Urine:Serum Osmolality Ratio: 1-3:1
Urine Osmolality of ≤290 = Chronic Renal Disease

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AUGUST 2021 MEDICAL TECHNOLOGY LICENSURE EXAMINATION REVIEW (Klubsy Bear Online Tutorial)
Contents:
VII. Enzymology
A. Characteristics of Enzymology:
-Proteins produced by living cells that hastens chemical reactions in organic matter (biochemical catalysts)
-Lowers the activation energy needed for the reaction to proceed
-Majority is of intracellular origin; Normally secreted in minute concentration; Increased serum levels are used diagnostically
to assess tissue damage
-Only analyte in blood measured not based on absolute concentration but in their activity in catalyzing reaction
-Highly specific; based on
1. Nature of substrate
2. Active site composition
3. Active site spatial arrangement
Tissue Specificity of Enzymes:
ENZYME PRINCIPAL TISSUE/S
High Specificity Acid Phosphate RBCs. Prostate
Alanine Aminotransferase Liver
Amylase Pancreas, Salivary gland
Lipase Pancreas
Moderate Specificity Aspartate Aminotransferase Liver, heart, skeletal muscles
Creatinine Kinase Brain, heart, skeletal muscles
Low Specificity Alkaline Phosphatase Liver, bone, kidney
Lactate Dehydrogenase All tissues
∑ There is no truly “organ specific enzyme”; The most tissue specific enzyme: Alcohol dehydrogenase (liver)
-Requires substrates, activators and metallic ions
∑ Michaelis-Menten Hypothesis:
-Rate of substrate conversion to product is determined by substrate concentration and the rate of dissociation of the enzyme-
substrate complex
-Rate of enzyme activity varies linearly with substrate concentration up until enzymes are fully saturated with substrates
ÿ Vmax (Saturation point) – maximum velocity ; the point at which all substrates are bound to an enzyme
ÿ Kmax (Michaelis constant) substrate concentration where the reaction is half maximum (1/2 Vmax)
Components:
1. Active site- site where substrate attach to undergo chemical reaction
2. Allosteric site- site opposite to the active site where regulatory molecules attach
∑ Isoenzyme- enzyme with the same activity but different in characteristics or structure
No isoenzyme: ALT, GGT, 5’NT, LPS
∑ Apoenzyme- protein nature of the enzyme subject to denaturation and loss of activity
-affected by pH and Temperature (Optimal pH: 7.0-8.0; Optimal Temperature: 37˚C)
Temperature is directly proportional to reaction rate
Temperature coefficient: For every 10˚C increase, there will be 2x increase in enzyme activity
∑ Denaturation: 40-50˚C; Repeated freezing and thawing
∑ Inactivation: 60-65˚C
Ideal Storage Temp: 2-8˚C (Refrigerated-4˚C)
For longer period of storage: -20˚C
B. Classification of Enzymes:
Classification Function Example
Oxidoreductase Catalyzes oxidation-reduction reaction (removal/ addition of LDH, HBD, GLDH, MDH
electrons)
Transferase Catalyzes the transfer of a chemical or a functional group (intact AST, ALT, CK
group of atoms) from one substance or molecule to another
Hydrolase Catalyzes the hydrolysis of compound with introduction of water ACP, ALP, PCHE, CHY, E1, AMS, LPS
into the structure (cleavage of bonds with water)

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Lyase Catalyzes the removal of one group without hydrolysis, leaving Aldolase
double bonds in the molecular structure of the product
-Cleavage of C-C, C-O, and C-N and other types of bonds
(without water)
Isomerase Catalyzes intramolecular rearrangement of the substrate Glucose phosphate isomerase
compound (Convert one isomer to another)
Ligase Catalyzes the joining together of two substrate molecules to form Glutathione synthase
a larger molecule with simultaneous breakdown of the
pyrophosphate bond with ATP as energy source

C. Factors that Affect Enzyme Reaction:

1. Substrate- molecules or substances attached with enzyme
2. Enzymes- as enzyme concentration increases, reaction rate also increases
First-order kinetics Reaction rate is directly proportional to substrate concentration; enzyme in excess
Zero-order kinetics Reaction rate is dependent only on the enzyme concentration; substrate in excess
3. pH and temperature
4. Buffer- to regulate pH
5. Cofactors
Cofactors are nonprotein entities that must bind to particular enzymes to enhance activity
Coenzyme (Organic cofactor) Coenzymes serve as a second substrate for enzymatic reactions
-Ex. Nucleotide phosphatases (NAD), Protein S (for Protein C) and vitamins (Vit B6)
Prosthetic group- when it is tightly bound to an enzyme
Activators (Inorganic cofactor) Metallic –Ca, Fe, Mg, Mn, Zn, K
Nonmetallic – Br, Cl
6. Inhibitors
Types of Inhibition:
a. Competitive Inhibition Inhibitor and substrate compete for the active site
-Resolved by increasing substrate concentration
-Line weaver burke plot: 1/Vmax unchanged and Km decreases
b. Noncompetitive Inhibitor binds to the allosteric site
Inhibition -Cannot be resolved by increasing substrate concentration
-Line weaver burke plot: 1/Vmax changed and Km unchanged
c. Uncompetetive Inhibitor binds to the E-S complex and stabilizes it
inhibition -Line weaver burke plot: 1/Vmax changed and Km changed
D. Measurement of Catalytic Activity:
1. Increase in product concentration
2. Decrease in substrate concentration
3. Decrease in coenzyme concentration
4. Increase in altered enzyme concentration
Types of Enzyme Assay Principle
End point/ Fixed time -Simplest and most widely used technique
Analysis -Substrate is added and allowed to proceed for a set time then reaction is stopped; the amount of
product formed during the time period is measured
-Underestimation of enzyme activity when substrate depletes due to a sample with high activity
-There is no way to assess linearity with a one-point system
Multipoint Analysis -Measures change in concentration at several intervals during the course of assay (change in
absorbance related to change in concentration)
-Allows the measurement whether the reaction is linear throughout the entire time of the assay
-Practical or Automated systems only
Kinetic Analysis Involves continuous measurement of change in concentration as a function of time
-Multiple measurements at a specific time interval
-Coupled Enzyme Assay: the product of second reaction is quantitated or monitored

ÿ Measurement of Enzyme Antigen Concentration: quantity of enzyme is proportional to enzyme activity

-Usually by Immunoassays

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-If values do not agree, it may be due to:

1. Presence of serum enzyme inhibitors
2. Deficiency of a necessary cofactor
3. Macroenzymes
4. Defective enzymes
5. Proteolytically inactivated enzymes
∑ Macroenzymes- an ordinary enzyme bound to an antibody ( IgA, IgG); not capable of any function and not
cleared in the circulation
∑ Defective enzymes- antigenically active nut enzymatically inactive
∑ Units for Expressing Enzymatic Activity:
1. International unit (IU) – 1 micromole of substrate per minute (µmol/min)
-Amount of enzyme that catalyzes 1µmol of substrate per minute
2. Katal unit (KU) – 1 mole of substrate per second (mol/s)
-Amount of enzyme that catalyzes q mol of substrate per second
-The unit recognized by the International System of Units (SI Unit)
∑ 1 IU=16.7 nanokatals
∑ Enzyme concentration is then expressed as katals per Liter (kat/L)
∑ Interpretation of Enzyme Data:
1. There is no true tissue-specific organ
2. Serial measurements provide most useful data; single ones can be misleading
3. Negative (Normal) results are useful
4. Must be integrated with other laboratory information
D. Clinically Significant Enzymes:
1. Aspartate Amino Transferase/ Serum Glutamic-Oxaloacetic Transferase (AST/SGOT)- 2.6.1.1
Reaction Catalyzed Transfer of an amino group in aspartic acid for an α-keto group in α-ketoglutaric acid forming
oxaloacetate and glutamate
Cofactor: Vit. B6 (Pyridoxal)
Tissue Source/s Cardiac muscle>Liver> Skeletal muscle> Kidney>Brain> Lung> Pancreas
Isoenzymes -Mitochondrial (80%)
-Cytoplasmic (20%)
Laboratory Methods: 1. KARMEN Method – uses Malate dehydrogenase as coupling enzyme and monitors the decrease in
absorbance at 340 nm
Aspartate + α-ketoglutaric acid (AST)= Oxaloacetate +Glutamate
Oxaloacetate +NADH (MD)= Malate + NAD
2. REITMAN-FRANKEL Method – uses 2,4 DNPH which produces an intense blue color measured at
505 nm
Ketoacid + 2,4 DNPH + NaOH= Ketoacidhydrazone
-lacks specificity
3. Coupling with Diazonium Salt
Ketoacid + Diazo compound= Diazonium derivative
4. BABSON Method- violet colored product
Specimen Serum or Plasma (Avoid H,B,L)
Reference Range <55 U/L
Clinical significance Increased: AMI, Hepatocellular disorders, skeletal muscle involvements, muscular dystrophy, acute
pancreatitis
Decreased: Uremia
3. Alanine Amino Transferase/ Serum Glutamic-Pyruvic Transferase (ALT/SGPT)- 2.6.1.2
Reaction Catalyzed Transfer of an amino group in alanine for an α-keto group in α-ketoglutaric acid forming pyruvate
and glutamate
Cofactor: Vit. B6 (Pyridoxal)
Tissue Source/s Liver
Laboratory Methods: 1. KARMEN Method – uses Lactate dehydrogenase as coupling enzyme and monitors the decrease in
absorbance at 340 nm
L-alanine + α-ketoglutaric acid (ALT)= Pyruvate +Glutamate

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Pyruvate +NADH (LD)= Lactate + NAD

2. REITMAN-FRANKEL Method – uses 2,4 DNPH which produces an intense blue color measured at
505 nm
Ketoacid + 2,4 DNPH + NaOH= Ketoacidhydrazone
-lacks specificity
3. Coupling with Diazonium Salt
Ketoacid + Diazo compound= Diazonium derivative
4. BABSON Method- violet colored product
Specimen Serum or Plasma (Avoid H,B,L)
Reference Range 5-35 U/L
Clinical significance Increased: Hepatic parenchymal disease, Viral hepatitis, Obstructive jaundice, Reye’s syndrome, Heart
failure or AMI, IM, Muscular dystrophy
Decreased: Not clinically significant
∑ De Ritis Ratio: AST/ALT
-In children: AST>ALT
-In adults: ALT>AST
<1 Viral in origin (Viral Hepatitis)
>1 Nonviral in origin hepatic disease
1-2 Hepatic cirrhosis
>2 Alcoholism; Hepatocellular carcinoma
3. Lactate Dehydrogenase (LD/ LDH)- 1.1.1.27
Reaction Catalyzed Oxidation of L-lactate to pyruvate with the mediation of NAD or its hydrogen receptor (reversible
reaction)
Cofactor: Zinc (Zn2+)
Tissue Source/s Liver, heart, skeletal muscle, kidney, erythrocytes
Isoenzymes LD1 (HHHH)- Heart, RBC, Kidneys
LD2 (HHHM)- Heart, RBC, Kidneys
LD3 (HHMM)- Lungs, Pancreas, Spleen, WBC
LD4 (HMMM)- Skeletal muscles, Liver, intestine
LD5 (MMMM)- Skeletal muscles, Liver, intestine
LD6- Alcohol dehydrogenase
Normal Serum: LD2>1>3>4>5
AMI: LD1>2>3>4>5
Normal CSF: LD1>2>3>4>5
Bacterial Meningitis: LD5>4>3>2>1
+ 1,2,3,4,5 –
Laboratory Methods: 1. WACKER Method- Forward reaction ; pH 8.8-9.8
Lactate + NADH=Pyruvate +NAD (decrease in absorbance at 340 nm)
-LD1 prefers this reaction
-Most commonly used
-Lactate is more specific
2. WROBLEWSKI LADUE Method- Reverse reaction ; pH 7.4-7.8
Pyruvate + NAD=Lactate +NADH (increase in absorbance at 340 nm)
-LD5 prefers this reaction
-2x Faster
-Preferred in Dry chemistry
-Smaller specimen volume
3. Heat denaturation LD1 is stable; the rest is denatured
Specimen Serum (Avoid anticoagulants; Freezing destroys sample)
Reference Range Forward: 35-90 U/L
Reverse: 95-200 U/L
Clinical significance Increased:
Cardiac muscle diseases: AMI, CHF, Myocarditis
Skeletal muscle disease: Muscular dystrophy, Muscle trauma
Hepatic parenchymal diseases: Viral hepatitis, Cirrhosis, Obstructive jaundice, IM
Others: Megaloblastic and Pernicious anemia
Decreased: Not clinically significant

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∑ The only hematologic marker; erythrocytic enzyme

∑ LD1 and LD2 – most abundant, most anodal, heat stable
∑ LD4 and LD5 – least conc, least anodal, heat and cold labile
∑ Can be stored at room temperature without appreciable loss of activity
∑ Highest elevation: Pernicious anemia, Megaloblastic anemia, Hemolytic disorders
∑ LDH: HBD Normal Ratio: 1.2-1.6; AMI: 0.8-1.2

4. Cretinine Kinase (CK/CPK)- 2.7.3.2

Reaction Catalyzed Reversible phosphorylation of creatine by ATP
Cofactor: Magnesium (Mg2+)
Tissue Source/s Brain, heart (cardiac muscle/myocardium). Skeletal muscle
ISoenzymes CK-BB (CK-1) – Brain
CK-MB (CK-2) – Hybrid (Heart- cardiac muscle)
CK-MM (CK-3) – Skeletal and cardiac muscle, lung, thyroid, liver, spleen, placenta
Laboratory Methods: 1. TANZER-GILVARG Method- Forward reaction
Creatine +ATP=Creatine Phosphate +ADP
Coupling Enzyme: G6PD, Hexokinase ( Decrease in absorbance at 340nm)
N-acetyl cysteine- reagent added to activate
2. OLIVER-ROSALKI Method- Reverse reaction
Creatine Phosphate +ADP= Creatine +ATP
Coupling Enzyme: G6PD, Hexokinase ( Increase in absorbance at 340nm)
N-acetyl cysteine- reagent added to activate
3. Isoenzyme Analysis by Electrophoresis
-Ratio of CK-MB to total-CK
+CK-BB, CK-MB, Macro-CK,CK-MM, Mito-CK –
Increases ability to distinguish myocardial infarction; Immunoinhibition test alone are every prone to
false increase due to hemolysis
Specimen Serum
Sensitive to light
CK-MB and CK-BB – heat sensitive
Reference Range Male: up to 160 U/L
Female: up to 130 U/L
Clinical significance Increased:
Cardiac muscle disease: AMI
Non-ischemic cause of cardiac injury: Cardiac contusion and Myocarditis
Skeletal muscle disease: Muscular Dystrophy
Brain and CNS disease: Stroke
Decreased: Not clinically significant
∑ CK-BB – rare in adults; only increased in cerebrovascular diseases and severe head trauma
∑ CK-MB >6% is considered the most specific indicator for AMI
∑ CK-MM- major isoenzyme (94-100%) both abundantly present in cardiac and skeletal muscle
∑ Most abundant in skeletal muscle: Highest elevation of CK: Duchenne disorder
ÿ Macro-CK- common among elderly women
ÿ Mito-CK- seen in advanced malignancies; indicator of poor prognosis
AMI/ Cardiac Profile
Cardiac Marker Elevation Peak Normal
Myoglobin 4-10 12-49 7-10
Troponin T, I 4-10 - -
CK-Total 4-6 12-24 1-2 days
CK-MB 4-8 24 -
AST 6-8 48 4-5
LD-Total (1-2) 8-10 72 5-7

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∑ Cardiac Proteins:
1. Myoglobin (MGB)
-Screening test for AMI
-Small heme portion found in skeletal and cardiac muscle
-Higher affinity for oxygen than HGB
-Nephrotoxin
-Earliest but not specific cardiac marker
2. Troponin T (TnT)
-Assessment of Early and Late AMI
-Sensitive marker for the diagnosis of unstable angina
-Elvated in skeletal and renal diseases
3. Troponin I (TnI)
-Only found in myocardium
-Greater cardiac specificity than TnT
-Highly specific for AMI
-Not a marker for late AMI
4. B-type Natriuretic Peptide (BNP)
-Marker for Congestive Heart failure
-Specimen: Heparinized plasma
5. Alkaline Phosphatase (ALP)- 3.1.3.1
Reaction Catalyzed Hydrolysis of organic phosphate esters with formation of alcohol and phosphate ion at alkaline pH
Component: Zinc (Zn2+); Cofactor: Magnesium (Mg2+), Manganese (Mn2+)
Tissue Source/s Bone, Bile ducts, Liver, Intestines, Kidney, Placenta, Carcinoplacental ALP (TUMOR MARKERS:
Regan- Lung CA breast, ovarian, colon, Nagao- Adenocarcinoma, pleural metastasis, Kasahara-
gastrointestinal cancer and hepatoma)
Major Isoenzymes Liver
Bone
Placental
Intestinal
Laboratory Methods: a. Enzyme Measurement
Reaction Name Substrate Comments
Shinowara- Jones-Reinhart Beta-glycerophosphate Long incubation time; High blank values
King-Armstrong Phenylphosphate End point; requires protein removal
Bessey-Lowry-Brock p-nitrophenylphosphate Rapid endpoint or kinetic
Bowers-McComb p-nitrophenylphosphate Uses phosphate-accepting buffer;
reference method
-Szasz modification – most specific
b. Isoenzyme Differentiation:
1. Electrophoresis
- I, P,B, L+
-Can distinguish fractions but not quantitate
Liver and Bone- most anodal
Intestinal- least anodal
2. Heat Fractionation/Stability
S-P,I,L,B-L
-Performed at 56˚C for 10-15 minutes
Placental- most heat stable
Bone- most heat labile
3. Chemical Inhibition test
∑ L-phenylalanine – inhibits Placenta and Intestinal
∑ Urea (2M/3M) inhibition – useful when used with electrophoresis; inhibits Bone
∑ Levamisole – inhibits Liver and Bone
Specimen Serum or heparinized plasma
∑ ALP is sensitive if stored at low temperature or room temperature (false high) –due to loss of
CO2
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Reference Range Male: 90-190 U/L

Female: 85-165 U/L
Clinical significance Increased: Biliary/Obstructive jaundice, Bone disease, Cholestasis, viral hepatitis, alcoholic cirrhosis,
hepatic malignancy, hyperparathyroidism, bone tumors, rickets, osteomalacia, osteitis deformans
(Paget’s Disease), 3rd trimester pregnancy, bone growth, CHF< hyperthyroidism, Certain drugs
(Ibuprofen)
Decreased: Hypophosphatasia (Inborn deficiency), Malnutrition
∑ Increased ALP activity between 16-20 weeks of gestation; bone growing (osteoblast)
∑ B or O blood group increases intestinal ALP after a fatty meal intake (complexed with Lp (X)
∑ Greatest elevation: Obstructive Jaundice
∑ Bone and Liver Marker
To differentiate:
Obstructive Jaundice Pagets disease
ALP ↑ ↑
GGT ↑ N
ÿ Carcinoplacental ALP: indicates malignancy
1. Regan- found in lung, breast, ovarian and gynecological cancers
-Most heat stable ALP (65˚C for 30 minutes)
-Inhibited by phenylalanine
2. Nagao- found in adenocarcinoma of the pancreas and bile duct, pleural cancer
-Inhibited by L-leucine and phenylalanine
6. Acid Phosphatase (ALP)- 3.1.3.1
Reaction Catalyzed Hydrolysis of organic phosphate esters with formation of alcohol and phosphate ion at acid pH
Component: Zinc (Zn2+); Cofactor: Magnesium (Mg2+), Manganese (Mn2+)
Tissue Source/s Prostate (major source), RBCs, PLTs, Liver, Bone marrow, Spleen, kidney, Seminal fluid
Isoenzymes Prostatic ACP
Non-Prostatic ACP
Laboratory Methods: a. Enzyme Measurement
Reaction Name Substrate Comments
Bodansky Beta-glycerophosphate Long incubation time; Nonspecific
Gutman, King-Armstrong Phenylphosphate Nonspecific
Babson and Reed Alpha-naphthylphosphate Complicated, less sensitive
Hudson p-nitrophenylphosphate Rapid, nonspecific
Roy Thymolphthalein More specific for prostatic form
monophosphate
Rietz and Guilbalt 4- Fluorescent; some improved
methylumbelliferonephosphate specificity
ÿ Alpha-naphthylphosphate- preferred for continuous monitoring methods
ÿ Thymolphthalein monophosphate- specific substrate; substrate of choice for quantitative
endpoint reaction
b. Isoenzyme Differentiation:
Technique Comments
Chemical inhibition Cumbersome, not specific
Electrophoresis Not easily reproduced, too complicated for routine use
Immunoassay Best approach for prostatic isoenzyme
Isoenzyme Other Name Inhibitors
Prostatic Specific ACP L-tartrate
Non-Prostatic Nonspecific/ Erythrocytic ACP 2% Formaldehyde, Cupric ions
Specimen Anticoagulant of choice: Citrate buffered to pH 6.2-6.6
Enzyme is labile when not stored in proper pH and temperature
∑ ACP decreases within 1-2 hours if left at room temperature (false low) –due to loss of CO2
∑ If not assayed immediately, serum should be frozen (-20˚C) or acidified to a pH lower than 6.5
∑ With acidification, ACP is stable for 2 days at room temperature
Reference Range 0.5-1.9 U/L
Clinical significance Increased: Metastatic carcinoma of the prostate, Gaucher’s diseases

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Useful in investigation of rape cases (Medico-legal forensic studies) –SPX: Vaginal washings/
Secretion – seminalfluid-ACP activity can persist up to 4 days
Bone disease: Osteoporosis, Multiple Myeloma, Paget’s disease
Decreased: not clinically significant
ÿ Hepatic Diseases:

1. Acute injury (Hepatitis) and Necrosis

Increased ALT>AST>ALP>Bilirubin (B1&B2)

Normal: TPAG

2nd Markers: Increased LD4 & 5

2. Biliary Tract Obsjuction (Obstructive Jaundice-OJ)

Increased ALP, B2

Slightly Increased ALT, AST

2nd Markers: Increased GGT, LAP, 5’NT, OCT

3. Cirrhosis

Increased Bilirubin (B1&B2); Globulin (Inverted A/G ratio- 1:2)

Decreased Total protein and albumin

Slightly Increased ALT, AST, ALP

2nd Markers: Increased Ammonia, LD4 and 5

Hepatocellular Damage ALT and AST

Hepatobiliary Damage ALP and GGT

7. Amylase (AMS)- 3.2.1.1

Reaction Catalyzed Hydrolysis of polysaccharide such as amylase, amylopectin, glycogen (responsible for starch
digestion= maltose is the end product)
Cofactors: Calcium and Chloride (Ca2+ and Cl-)
Tissue Source/s Pancreas, salivary gland
Isoenzymes Salivary AMS- Mumps (Anodal)
Pancreatic AMS- Acute Pancreatitis (Cathodal)
Laboratory Methods: Substrate: Starch
1. Amyloclastic- degradation of starch; time required for the disappearance of colored substrate
2. Saccharogenic- expressed in somogyi units; amount of reducing sugars produced after hydrolysis
3. Chromogenic- Uses chromogen to measure increase in color intensity
4. Coupled enzymatic- Uses continuoud monitoring technique
5. Electrophoresis (First 3-salivary; Slowest 3- pancreatic)
6. Inhibition test- sensitivity to wheat germ lectin (triticumuilgaris) –Interfering substance- TAG ( no
lipemic samples should be used
7. Monoclonal Antibodies- directed against specific isoenzymes
8. Amylase:Creatinine Ratio:
%= 100
Reference Range: 1-5%
Increased: Acute pancreatitis, Diabetic acidosis, Severe burns, recovery from thoracic surgery
9. Specificity of Amylase

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-Fractional excretion= ratio of renal clearance of X divided by GFR (clearance of X/ clearance of crea)
Specimen Serum or plasma (Avoid citrate, oxalate, EDTA)
Reference Range Method dependent; 15% higher value in women; Increased during 25th week of gestation
Clinical significance Increased: Acute non-hemorrhagic pancreatitis, bacterial parotitis,
Nonpancretic cause: Diabetic ketoacidosis, Peptic ulcer, Acute cholecystitis, ectopic pregnancy,
salphingitis, bowel ischemia, intestinal obstruction, macroamylasemia (high serum level and low
urine level, renal insufficiency
Drugs: codein, morphine, glucocorticoids, dexamethasone, oral contraceptives, radiographic contrast
dye
Decreased: pancreatic insufficiency
∑ Samllest enzyme in size
∑ Noramlly filtered in the glomerulus and appears in the urine
∑ Earliest pancreatic marker (2-12 hours)
∑ STAT and commonly used
7. Lipase (LPS) / Triacylglycerol Acylhydrolase- 3.1.1.3
Reaction Catalyzed Hydrolysis of fats (TAG) into fatty acids and monoglycerides
Cofactors:
Tissue Source/s Pancreas, GI mucosa, RBC, WBC
Laboratory Methods: 1. Turbidimetric Enzyme Reaction
Substrate: 50% Olive oil/ Triolein (pure form of TAG)
Indicator Reaction: Decrease in turbidity
2. Titration/ Classic Method – Cherry-Crandall Method
Substrate: 50% Olive oil/ Triolein (pure form of TAG)
Reagent: Sodium hydroxide
-Measure liberated fatty acids by titration after 24 hour incubation
-Reference Method
3. Colorimetric method- utilize copper and bind the generated fatty acid;
∑ Colipase- prevents inactivation of lipase
4. Peroxidase Coupling – most commonly used method
-Does not used 50% olive oil
5. Tietz and Fiereck
Specimen Serum- specimen of choice
Bacterial contamination- falsely increased
Clinical significance Increased: Acute pancreatitis, Pancretic cyst or pseudocyst, obstructive jaundice, peritonitis, intestinal
obstruction
Decreased: pancreatic insufficiency
∑ Most specific pancreatic marker (levels rise 6 hours after onset)
ÿ Pancreatic Function Markers: AMS, LPS, Trypsin, Chymotrypsin, Elastase 1 (E1)
8. Gamma-Glutamyl Transferase (GGT)- 2.3.2.1
Reaction Catalyzed Transfer gamma glutamyl group from a gamma glutamyl peptide to another peptide or amino acid
Tissue Source/s Kidney, Liver, Pancreas, Prostate, Brain
Isoenzymes No clinically significant isoenzymes
Laboratory Methods: 1. Szasz Assay – Photometric Enzyme Reaction
Substrate: L-gamma-p-nitroanilide or L-gamma-3-carboxy-nitroanilide
-Production of p-nitroaniline or 5-amino-2-nitrobenzoate, monitored at 405 nm or 410 nm
Specimen Serum
Avoid hemolysis and anticoagulant
Samples can be frozen and remain stable for weeks
Reference Range Male- up to 40U/L
Female- up to 25 U/L
Clinical significance Increased: Obstructive jaundice, cirrhosis, tumors, IM, hepatotoxicity (acetaminophen), Alcohol abuse
(Chronic alcoholism), antiepileptic/anticonvulsant drus administration, renal disease
Decreased: not clinically significant
∑ Highest elevation: Chronic Cholestasis due to Primary Biliary Cirrhosis or Sclerosing Cholangitis (>10x UL of Normal
Value)
∑ Secondary marker for OJ

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∑ Marker used with ALP to differentiate liver from bone disorder

∑ Test for occult alcoholism
8. Cholinesterase (CHe)
Reaction Catalyzed Hydrolyzes a number of esters with acetylcholine to choline and acetic acid
Tissue Source/s True- RBCs, Lungs, Kidneys, Liver, Pancreas, Prostate, Brain (nerve tissue/cell)
Pseudo- Serum, Heart, Liver, Pancreas, Brain (white matter of CNS)
Isoenzymes True Cholinesterase (Acylcholine Acylhydrolase) – 3.1.1.7
Pseudocholinesterase (Acetylcholine Acetylhydrolase) – 3.1.1.8
Laboratory Methods: 1. Szasz Assay – Photometric Enzyme Reaction
Substrate: L-gamma-p-nitroanilide or L-gamma-3-carboxy-nitroanilide
-Production of p-nitroaniline or 5-amino-2-nitrobenzoate, monitored at 405 nm or 410 nm
Specimen Serum or heparinized plasma
Reference Range 6000-12000 U/L
Clinical significance Increased: not clinically significant
Decreased: Insecticide/Pesticide poisoning (Organophosphorus
Metastatic carcinoma, Parenchymatous disease including viral hepatitis and cirrhosis
9. Aldolase- 4.1.2.13
Reaction Catalyzed Catalyzes the splitting of fructose-1,6-diphosphate to glyceraldehydes-3-phosphate and
dihydroxyacetone phosphate which is one of the reactions in the glycolytic breakdown of glucose to
lactic acid
Tissue Source/s Widely distributed in the body specially prominent in skeletal muscles, cardiac muscle, liver and RBC
Isoenzymes A-Skeletal muscle
B-WBC, liver, kidney
C-Brain tissue
Laboratory Methods: 1. Rate of Triose Formation- Measuring colored dinitrophenyl hydrazine (DNPH)
Clinical significance Increased: Progressive muscular dystrophy, Inflammatory muscle disease, MI, Liver disease or
necrosis, pulmonary infarction, Some cases of malignancy and megaloblastic anemia
Decreased: not clinically significant
∑ Significant in skeletal muscle disease; High in muscle wasting disease
ÿ 5’Nucleotidase (5’NT) – Secondary Heapatobiliary Marker for OJ
ÿ Ornithine Carbamoyl Transferase (OCT) - Hepatobiliary disease
ÿ Leucine Aminopeptidase (LAP)– Hepatobiliary disease

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AUGUST 2021 MEDICAL TECHNOLOGY LICENSURE EXAMINATION REVIEW (Klubsy Bear Online Tutorial)
Contents:
I. ENDOCRINOLOGY
1. Major Endocrine Glands and their Hormones
2. Hypothalamus and Endocrine glands
II. THERAPEUTIC DRUG MONITORING
1. Pharmacology
2. Drugs in the Human Body
3. Laboratory Assessment of Therapeutic Drugs
4. Classifications of Therapeutically Monitored Drugs
III. TOXICOLOGY
1. Toxic Agents
2. Drugs of Abuse
3. Laboratory Assessment of Toxic Drugs
I. ENDOCRINOLOGY:
Endrocine system- a network of endocrine glands, which are ductless specialized organs capable of producing hormones.
(the regulatory system of the body)
∑ Hormones- chemical signals secreted in the bloodstream that targets specific tissues to elicit a physiological response
ÿ Major function: To maintain the constancy of chemical composition of ECF and ICF
To control metabolism, growth, fertility, and responses to stress
ÿ Multiple hormones can affect one physiological function or a single hormone can affect several
organs to produce different physiological effects
∑ Characteristics of the Endocrine System:
a. Cyclicity- characteristic pattern in the production of hormones
b. Pulsatility- characteristic interval in the production of hormones
c. Feedback mechanism- response of the endocrine glands for stimulation or inhibition in synthesis of hormones
ÿ Positive Feedback System- An decreased in the product results to the elevation of the activity of the
system and production rate (e.g., gonadal, thyroidal and adrenocortical hormones)
ÿ Negative Feedback- An increased in the product results to the decreased activity of the system and
production rate (e.g., leutenizing hormones)
∑ Classification of Hormones according to their actions:
1. Endocrine- secreted in one location and release in blood circulation (binds with specific receptor to elicit a
physiological response)
2. Autocrine- secreted in endocrine cells and sometimes released in interstitial space (binds with specific receptor of
itself for self-regulation)
3. Paracrine- secreted in endocrine cells and released in interstitial space (binds with specific receptor and targets
adjacent cells)
4. Intracrine- secreted in endocrine cells and remains inside the synthesis of origin to affect its own function
5. Juxtacrine- secreted in endocrine cells and remains in the plasma membrane (acts immediately on the adjacent
cell by direct cell-to-cell contact)
6. Exocrine- secreted in endocrine cells and released into the lumen of gut and interacts with receptors of cell at a
distant site
7. Neurocrine- secreted in neurons and released into extracellular space (binds with receptor of nearby cell and
affect their function)
8. Neuroendocrine- secreted in neurons and released from nerve endings (binds with receptor of cells at a distant
site and affect their function)
∑ Classification of Hormones according to their structure:
Proteins (Peptides) Glycopeptide FSH
-water soluble TSH
-not bound to carrier proteins LH
-synthesized ans stored within the hCG

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cell as secretory granules EPO

-cannot cross cell membrane due to Polypeptide GHRH,CRH, TRH, GnRH, PRF, GH,
their large molecular size ACTH, MSH, ADH, Oxytocin,
-most hormones are in this type Calcitonin, Cholecystokinin, Gastrin,
(2/3) Vasointestinal peptide (VIP),
Secretin, Insulin, Glucagon, PRL, hPL,
PTH, Somatostatin, Angiotensin
Amines (Amino acid derivatives) Tryptophan derivatives Melatonin
-intermediary between steroid and Serotonin
protein hormones Tyrosine derivatives Epinephrine
Norepinephrine
Thyroid hormones (T3 and T4)
Dopamine (PIF)
Steroids Aldosterone
-water insoluble Cortisol
-bound to carrier proteins Estrogen
-lipid molecules with cholesterol as a Progesterone
common precursor Testosterone
-primarily involved in the regulation Activated Vit. D3
of sexual development and
characteristics
Fatty acids Prostaglandins (from arachidonic acid)

I.1. Major Endocrine Glands and their Hormones:

Endocrine Gland Hormone Function Hyposecretion Hypersecretion
Anterior Pituitary 1. Growth hormone Major effect- Growth of skeletal Pituitary Gigantism (child)
Gland (GH) or Somatotropin muscles and long bones; IGF-1 dwarfism Acromegaly
(Adenohypophysis) production (adult)
2. Prolactin (PRL) Breast milk production; Proliferation Hypogonadism
of mammary gland; antagonist of
insulin
3. Adrenocorticotropic Stimulates adrenocortical steroid
hormone (ACTH) or formation and secretion
Corticotropin
4. Thyrotropic Stimuates thyroid gland to release
hormone (TH) or thyroid hormones
Thyroid-stimulating
hormone (TSH) or
Thyrotropin
5. Follicle-stimulating Female- Male- Sterility in both
hormone (FSH) ovulation, follicle spermatogenesis; male and female
development semineferous
with LH, tubule
estrogen development
production
6. Luteinizing hormone Female- Male-testosterone Sterility in both
(LH) ovulation, (androgen) male and female
corpora lutea production;
formation and stimulates
progesterone interstitial tissue
production
7. Beta-endorphin Endogenous opiates; raises pain
threshold and influence
extrapyramidal motor activity
8. Alpha-melanocyte- Dispersion of pigment granules,
stimulating hormone darkening of skin
9. Leu-enkephalin and Same as beta-endorphin
met-enkephalin
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Posterior Pituitary 1. Oxytocin Uterine contraction and milk ejection

Gland in nursing woman; action in
(Neurohypophysis) parturition and sperm transport
2. Antidiuretic Reabsorb body water and Constricting DI SIADH
hormone (ADH) or arterioles to increase blood pressure
Vasopressin
Thyroid Gland 1. Thyroxine or Body’s metabolic hormone; Increase Cretinism or Grave’s disease
Tetraiodothyronine glucose oxidation of cells Hashimoto’s and
(T4) and disease thyrotoxicosis
Triiodothyronine (T3)
2. Calcitonin or Calcium deposition in long bones
Thyrocalcitonin
Parathyroid Gland 1. Parathyroid Bone calcium liberation in blood Tetany Extreme bone
hormone (PTH) wasting and
fractures
Adrenal Gland: 1. Mineralocorticoids Regulate sodium and potassium General Hyperaldosteroni
Cortex (Aldosterone) reabsorption; Salt and water balance hypoactivity of sm
2. Glucocorticoids Resist long-term stress by increasing the cortex- Cushing’s disease
(Cortisone and blood glucose and decreasing the Addison’s
cortisol) inflammatory response; (CHO, fat and disease
protein metabolism)
3. Converted to estrogen and Masculinization
Dehydroepiandrostero testosterone (androstenedione)
ne (DHEA) or
Dehydroepiandrostene
dione sulfate (DHEAS)
4. 17- Converted to cortisol
hydroxyprogesterone
Adrenal Gland: 1. Catecholamines Symptoms of
Medulla typical
sympathetic
nervous activity
Pancreas (Islet of 1. Insulin Increased glucose uptake and DM
Langerhans) metabolism of cells
2. Glucagon Stimulates liver to release glucose,
increasing its blood level
3. Somatostatin (SS) Inhibits secretion of insulin and
glucagon
4. Amylin Inhibits secretion of insulin and
glucagon
5. Pancreatic Increased gut motility and gastric
polypeptide emptying; inhibits gall bladder
contraction
GI Tract 1. Gastrin Secretion of gastric acid; gastric
mucosal growth
2. Ghrelin Secretion of GH; for appetite
3. Secretin Secretion of pancreatic bicarbonate
and digestive enzymes
4. Cholecystokinin- Gall bladder contraction and secretion
pancreozymin (CCK- of pancreatic enzymes
PZ)
5. Motilin Gastrointestinal motility
6. VIP Neurotransmitter; relaxation of gut
smooth muscles and circulation;
Increased release of hormones from
pancreas
7. Gatruc Inhibitory Inhibition of gastric secretion and
Peptide (GIP) motility; Increase in insulin secretion
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Ovaries 1. Estrogen Maturation of female repro. organs Hampers the

and secondary sex characteristics ability of a
Causes menstrual cycle with woman to
progesterone conceive and
bear children
2. Progesterobe Causes menstrual cycle with estrogen Hampers the
ability of a
woman to
conceive and
bear children
Testes 1. Testosterone Maturation of female repro. organs Man becomes
and secondary sex characteristics sterile
Spermatogenesis
Pineal Gland 1. Melatonin Affects biological rhythms and
reproductive behavior; Sleep
induction; (Inhibits Gonadotropin and
GH secretion)
2. Serotonin or 5- Neurotransmitter; stimulation or
hydroxytryptamine inhibition of smooth muscles and
neurons
Kidneys 1. 1,25- (OH)2 Absorption of calcium and
cholecalciferol phosphorus; increase bone resorption
2. Erythropoietin Red cell formation
(EPO)
3. Renin (RAAS) Converts angiotensinogen to
angiotensin I
Liver 1. IGF-1 Cellular and linear growth
(Somatomedin)
2. IGF-2 Insulin-like activity
Heart 1. Atrial natriuretic Blood volume and pressure regulation
peptide
2. B-type natriuretic Blood volume and pressure regulation
peptide
Adipose tissue 1. Adiponectin Increased fatty acid oxidation;
Suppresses glucose formation
2. Leptin Inhibition of appetite, stimulation of
metabolism
3. Resistin Insulin resistance
Thymus gland 1. Thymosin or Maturation of T lymphocytes
Thymopoietin
Monocyte, 1. Cytokines Stimulation and inhibition of cellular
Lymphocyte, growth and others
MAcropahages
I.2. Hypothalamus and the Endocrine glands:
A. HYPOTHALAMUS- the link between the nervous and endocrine systems, which is located in the walls and floor of the
third ventricle just above the pituitary gland (connected to the posterior pituitary by the infundibulum or pituitary stalk)
The neurons in the anterior portion are capable of producing both releasing hormones and inhibiting hormones
(Hypophyseal hormones) which then, affects the anterior pituitary gland. The hormones produced and secreted are all
peptides except for dopamine.
∑ Releasing hormones: which stimulates for the secretion of the following hormones
Corticotropin-releasing hormone (CRH) Adrenocorticotropic hormone (ACTH) and
Lipotropic hormone (LPH)
Gonadotropin-releasing hormone (GnRH) Follicle-stimulating hormone (FSH) and
Luteinizing hormone (LH)
Growth hormone-releasing hormone (GHRH) Growth hormone (GH)
Thyrotropin-releasing hormone (TRH) Thyroid-stimulating hormone (TSH) and
Prolactin (PRL)
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∑ Inhibiting hormones: which inhibits the for the secretion of the following hormones
Dopamine or Prolactin-inhibiting factor (PIF) Prolactin (PRL)
Somatostatin or Growth hormone-inhibiting Thyroid-stimulating hormone (TSH), Growth
hormone (GHIH) hormone (GH), Gastrin, Vasoactive Intestinal
Polypeptide (VIP), Gastric Inhibitory Polypeptide
(GIP), Secritin, Motilin, Glucagon and Insulin
∑ Hypothalamus also produces antidiuretic hormone and oxytocin via the supraoptic and paraventricular nuclei
which are then transported to the posterior pituitary gland for storage.
B. Endocrine glands:
1. PITUITARY GLAND (HYPOPHYSIS)- master gland; Pea shaped organ located in the small cavity of the sphenoid bone of
the skull (Sella Turcica/ Turkish saddle). All the hormones it produces have circadian rhythms. It is divided into two lobes:
-Anterior Pituitary (Adenohypophysis)- true endocrine gland; The hormones produced and secreted are either peptides
and glycoproteins
∑ Five types of cells: by immunochemical test
1. Somatotrophs- secretes GH
2. Lactotrophs or mammotrophs- secretes PRL
3. Thyrotrophs- secretes TSH
4. Gonadotrophs- secretes LH and FSH
5. Corticotrophs- secretes proopiomelanocortin (POMC) which is cleaved to produce ACTH, beta-endorphin, and beta-
lipotropin
The failure of either the pituitary or hypothalamus results in the loss of anterior pituitary function.
Complete loss of function- Panhypopituitarism Loss of only a single pituitary hormone- Monotropic Hormone Deficiency
Tropic Hormone - Hormones that ∑ The tropic hormones of the anterior pituitary are mediated by negative
have other endocrine glands as feedback, which involves interaction of the effector hormones with the
their target hypothalamus and the anterior pituitary gland.
∑ The loss of a tropic hormone (ACTH, TSH, LH, and FSH) is reflected in
function cessation of the affected endocrine gland
∑ Hypophysiotropic hormones/ hypothalamic neurohormones (releasing and
release-inhibiting hormones) are also tropic hormones which are released
to the hypothalamo-hypophyseal portal system and act on the anterior
pituitary gland.
Direct Effector/ Non-tropic ∑ Diffuse target tissue and lacks a single endocrine end organ
Hormone- Hormone that acts directly ∑ Loss of the direct effectors (GH and Prolactin) may not be readily apparent
on peripheral tissues (directly ∑ Other examples include: Vasopressin (ADH), Oxytocin, Glucocorticoids such
stimulate target cells to induce as cortisol and corticosterone, Estrogen, Testosterone, Epinephrine and
effects) Norepinephrine

Trophic hormones - Hormones of ∑ These hormones affect growth, function, or nutrition of other endocrine
the anterior lobe of the pituitary cells.
(adenohypophysis) ∑ Trophic hormones can be found in body systems including the endocrine,
gastrointestinal, urinary, and nervous systems.
∑ Anterior Pituary Gland Hormones:
1. Somatotropin (GH)- controlled by GHRH and GHIH; structurally similar to PRL and hPL
Overall metabolic effect: Metabolize fat stores while conserving glucose
Major stimuli: Deep sleep (Others: Physiologic- stress, fasting, high protein diet; Pharmacologic- sex steroids, amorphine,
levodopa)
Major inhibitor: Somatostatin (Others: glucocorticoids, elevated fatty acids)
Increased: chronic malnutrition, renal disease, cirrhosis and sepsis
Decreased: hyperglycemia, hypothyroidism and obesity
Disorders:
Excess- gigantism or acromegaly (Gigantism- high GH prior to epiphyseal plate closure; occurs in children)
Deficiency- idiopathic GH deficiency or pituitary adenoma (drawfism or subnormal growth)
∑ Laron syndrome- primary GH resistance or insensitivity)

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∑ Severe growth failure with normal or elevated GH caused by a GH receptor gene mutation (anorexia nervosa/
uncontrolled DM)
Method: Chemiluminescent assay
Reference Range: <1 ng/mL (fasting)
Diagnostic Tests of Major Disorders:
1. GH Deficiency
Screening – Physical Activity or Exercise test
Negative: elevated serum GH
Positive: GH fails to increase
Confirmatory – Insulin Tolerance test (Gold standard)
2nd Confirmatory – Arginine stimulation test
Procedure: 24-hr/ nighttime GH monitoring
Positive: Failure of GH to rise >5 ng/mL (adults) and >10 ng/mL (child)
2. Acromegaly
Screening – Somatomedin C or Insulin-like Growth Factor (IGF-1) – produce in the liver
Negative: low IGF-1 – GH deficiency
Positive: increased IGF-1 – Acromegaly
Confirmatory –Oral Glucose Suppression Test (OGTT) – 75g glucose load
Procedure: 4 blood specimens every after 30 minutes (for 2 hours) and fasting specimen is required
Negative: Suppresion of GH less than 1 ng/mL (<1g/L)
Positive: GH fails to decline less than 1 ng/mL (<1g/L)
∑ Suppression of GH less than 0.3 µg/L and Normal IGF-1- excludes acromegaly
∑ Suppression of GH less than 0.3 µg/L and Increased IGF-1- follow-up and monitoring
∑ Failure of GH to be suppressed below 0.3 µg/L and Increased IGF-1- diagnostic of acromegaly
Specimen requirement: preferably fasting serum; complete rest for 30 minutes prior blood collection
2. Gonadotropins (FSH and LH)- present in blood of both male and female at all ages; important markers in diagnosing
fertility and menstrual cycle disorders
∑ LH acts on thecal cells to cause synthesis of androgen, estrogens and progesterone
∑ FSH elevation is a clue in the diagnosis of premature menopause
3. Thyrotropin (TSH)- main stimulus for the uptake of iodide by the thyroid gland; acts to increase the number and size of
follicular cells
∑ Composed of 2 monocovalently α and β subunits (α has the same AA sequences with LH, FSH and hCG; β carries the
specific information to the binding receptors for the expression of hormonal activity)
∑ Blood levels may contribute in the evaluation of infertility
4. Corticotropin (ACTH)- single-chain peptide without disulfide bonds; regulator of adrenal androgen synthesis
Stimuli- low serum cortisol
Increased- Addison’s disease, Ectopic tumors, Protein-rich meals
∑ Deficiency will lead to atrophy of zona glomerulosa and reticularis
∑ Highest level (6-8AM); Lowest level (6-11PM); Best time to collect specimen (8-10AM)
Specimen requirement: Prechilled plastic EDTA tube (To prevent degradation; ACTH adheres to glass surfaces)
5. Prolactin- pituitary lactogenic hormone; stress hormone; direct effector hormone
∑ AA structure similar to GH
∑ Most common pituitary tumor is prolactinoma (can result to anovulation ; >200 mg/dL)
∑ Highest level (4-8AM) and (8-10PM) – during sleep
Specimen requirement: fasting sample, 3-4 hours upon waking up
Function: Initiation and maintenance of lactation; Breast tissue development (with estrogen and progesterone)
Physiologic stimuli: exercise, sleep, stress, postprandial, pain, coitus, pregnancy, nipple stimulation, nursing
Pharmacologic stimuli: verapamil, phenothiazines, olanzapine, prozac, cimetidine, opiate
Major inhibitor: Dopamine
Three forms of circulating prolactin:
1. Nonglycosylated monomer- major form
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2. Big prolactin- dimeric and trimeric glycosylated forms

3. Macro-prolactin- less physiologically active form
Method: Immunometric assay
Reference range: Male- 1-20 ng/mL; Female- 1-25 ng/mL
∑ Excess levels will cause hypogonadism
Increased: pituitary adenoma, infertility, amenorrhea, galactorrhea, acromegaly, renal failure, PCOS, cirrhosis and primary
and secondary hypothyroidism
-Posterior Pituitary (Neurohypophysis)- does not produce hormones; storage site only of hormones produced by the
hypothalamus; controlled by the CNS
∑ Posterior Pituary Gland Hormones:
1. Oxytocin- stimulates the gravid uterus at term (Fergusson reflex) and prostaglandin secretion by the desidua
Half-life: 3-5 minutes
Main effect: smooth muscle contraction during delivery and lactation (burst of secretion with anticipation of nursing and
hearing a baby cry)
Stimuli: neural stimulation of receptors in the birth canal and uterus; touch receptors in the breast
∑ Plays a role in hemostasis at the placental site following delivery
Significance: Useful in predicting premature onset of labor; Can be found in tumors that produce ectopic oxytocin (oat
cell carcinoma of the lung and adenocarcinoma of the pancreas); Synthetic preparations can aid weak uterine contrations
during labor
2. Arginine Vasopressin (AVP) or Antidiuretic hormone (ADH)
Major function: maintain osmotic homeostasis by regulating of water balance (promotes water reabsoption in DCT and
CD)
Stimuli: increased plasma osmolality and thirst (shrinks the hypothalamic osmoreceptor cells); nausea, cytokine,
hypoglycemia, hypercarbia and nicotine, physical and emotional stress, emetic stimulus-potent
Inhibitors: ethanol, cortisol, lithium, demeclocycline
∑ Potent pressor agent that affects blood clotting by promoting factor VII and VIII release
∑ Increases blood volume, pressure and tonicity (5-1% drop triggers baroreceptors)
Differential Diagnosis of Water homoestasis Disorders:
a. Excess-Syndrome of Inappropriate ADH Secretion (SIADH)
b. Deficiency-Diabetes Insipidus (DI)
c. Psychogenic polydypsia
Reference Range: 0.5-2 pg/µL
Diagnostic Tests of Major Disorders:
1. Diabetes Insipidus
Clinical picture:
∑ Normoglycemia
∑ Polydypsia (secondary)
∑ Polyphagia (occasional)
∑ Polyuria with low SG
∑ Without ADH, urine osmolality is about 50 mOsm/kg, while serum osmolality increases
Treatment: Desmopressin (DDAVP)
Types: (distinguished by the response to either endogenous or exogenous vasopressin)
a. True DI (Hypothalamic/ Neurogenic/ Cranial/ Central/ Pituitary related DI)- deficiency of ADH with normal ADH receptor;
failure of pituitary to secrete ADH
∑ Large volume of urine secreted (3 to 20L/ day)
b. Nephrogenic DI (Kidney-related DI)- normal ADH level but abnormal ADH receptor (renal ADH resistance)
Can be congenital or acquired

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Diagnostic Test: Water Deprivation/ Overnight WD/Concentration Test

8 HOUR FLUID DESMOPRESSIN INTERPRETATION
DEPRIVATION
<300 >750 Central DI
<300 <300 Nephrogenic DI
>750 >750 Primary polydypsia
300-750 <750 Non-diagnostic; Proceed to Saline Infusion Set
No increase in ADH- CDI
Normal increase in ADH- NDI (if low osmolality despite high ADH)
-PP (if low osmolality and low ADH)

2. SIADH- sustained ADH production in the absence of a known stimuli

∑ Characterized with decreased urine volume, low plasma osmolality (low serum electrolytes) and normal or elevated
urine sodium levels
Diagnostic Test: Water Load Test
2. THYROID GLAND- butterfly shaped organ in which lobes are connected by a ligament called isthmus; located just below the
larynx, in lower part of the neck
∑ Compose of two cell types:
1. Follicular cells- produce T3 and T4 (metabolic function)
2. Parafollicular/ Perifollicular/ C cells- produce calcitonin (calcium and phosphate regulator)
Regulation: Hypothalamic-Pituitary-Thyroid Axis (HPTA)

Biosynthesis and Metabolism of Thyroid hormones

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Iodine is the most important element in the biosynthesis of thyroid hormones

∑ Iodine intake below 50µg/day is an indication of deficiency of hormone secretion
∑ When iodine sources are diminished, MIT is produced in greater quantities, leading to increased T3 formation and
release

Thyroid peroxidase (TPO)- catalyze the binding of iodine and tyrosine in the follicular colloidal thyroglobulin
∑ In tissues, peripheral deiodonation of T4 occurs using the enzyme thyroid deiodinase
Thyroglobulin(Tg)- glycopeptides that acts as a preformed matrix containing tyrosyl groups; synthesized and secreted by the
follicles
∑ Reflects thyroid mass, thyroid injury and TSH receptor stimulation
∑ Useful in monitoring the course of thyroid disease or response to treatment (not recommended in pre-operative
identification of thyroid malignancy)
∑ Conditions: Grave’s disease, Thyroiditis, Nodular goiter
∑ Reference range: up to 30 ng/mL (45 pmol/L)

Protein-bound thyroid hormones- biologically inert; cannot enter cells

Free hormones (FT3 and FT4) – physiologically active (mediated by T3 receptors located in the nucleus of the cell
Thyroid Hormone Binding Proteins:
1. Thyroxine-binding globulin (TBG)- main serum carrier fir T3 and T4 (70-75% of total T4)
-Transports majority of T3 (less affinity for T3 than T4)
Increased TBG Decreased TBG
Liver disease Liver failure
Genetic Malnutrition
Pregnancy Nephrotic syndrome
Newborns Genetic (complete or partial deficiency)
Drugs Drug administration by androgen and glucocorticoids

2. Tyroxine-binding prealbumin (TBPA or Transthyretin)- (15-20% of total T4)

-has no affinity for T3
3. Thyroxine-binding albumin (TBA)- (10% of total T4)
-transports T3 also
Hormone Description
T4 Principal secretory product – 100% secreted (amount is a good indicator of the thyroid secretory rate)
Major fraction of organic iodine in the circulation (most abundant thyroid hormone)
Prohormone for T3 production
Elevated levels imhibits TSH secretion, vice versa
T3 Most active thyroid hormonal activity- 4-5x (less tightly bound to serum proteins than T4)
75-80% is produced from tissue deiodination of T4 (liver and kidneys)
Principal application- Diagnosing T3 thyrotoxicosis
Helpful in confirming the diagnosis of hyperthyroidism
Better indicator of recovery from hyperthyroidism (recognition of recurrence)
rT3 Major metabolite of thyronine (inner ring enzymatic monodeiodination of T4)
40% of secreted T4 undergoes this biologically inactive form
TRH TRH test is performed when:
∑ Borderline cases of hyperthyroidism
∑ Classifying hyperthyroidism
∑ Evaluating I-Thyroxine suppression
∑ Evaluating Euthyroid Grave’s disease
TSH Most important test in determining thyroid dysfunction (best screening test for thyroid disease) – Most
clinically sensitive for primary thyroid disorders; Early detection of hypothyroidism
FT4 Most sensitive marker for thyroid disease (esp. in subclinical contiditions) – differentiate drug induced
TSH elevation and hypothyrodism
FT3 Valuable in confirming hyperthyroidism

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Functions of Thyroid hormones:

1. Tissue growth
2. Mental and CNS development
3. Elevated heat production
4. Oxygen consumption control
5. Carbohydrate, Fat and Protein Metabolism
6. Energy conservation
Disorders: TSH T3 Total T4 rT3 T3RU Serum Creatinine
cholesterol Kinase
Hyperthyroidism ↓ ↑ ↑ N ↑ ↓ N/↑
Hypothyroidism ↑ ↓ ↓ N ↓ ↑ N/↑
Euthyroid Sick N ↓ N ↑ N N N/↑
Syndrome
Excess TBG N ↑ ↑ N ↓ N N/↑
HYPOTHYROIDISM HYPERTHYROIDISM
Forms: TSH T3 and T4 Forms: TSH T3 and T4
1° ↑ ↓ 1° ↓ ↑
2° ↓ ↓ 2° -pituitary lesion ↑ ↑
3° ↓ ↓ There is no 3° hyperthyroidism
Subclinical ↑ N Subclinical ↓ N
Increased metabolic rate Decreased metabolic rate
Increased sympathetic response Decreased sympathetic response
Weight gain Weight loss
Cold intolerance (Decreased sweating) Heat intolerance (Increased sweating)
Constipation (Decreased appetite) Diarrhea (Increased appetite)
Decreased cardiac output (bradycardia) Increased cardiac output (tachycardia)
Hypoventilation Dyspnea
Myxedema Exophthalmos
Deep voice Decreased blinking
Impaired growth (in children) Enlarged thyroid
Mental retardation (in children) Restlessness Wakefulness
Mental dullness Irritability Tremors
Mental and physical sluggishness Anxiety Menstrual changes
Somnolence Hyperkinesis
Increased serum cholesterol and triglycerides Decreased serum cholesterol and triglycerides; altered
glucose insulin relationship
Increased serum levels of muscle enzymes: CK, AST,LD Increased serum levels of AST, ALT and ALP
Increased serum prolactin, serum carotene (YELLOW Increased retention of Sulphobromophthalein (BSP) ;
COARSENED SKIN) and spinal fluid protein Increased urinary excretion of calcium
Normochromic anemia, (HGB 10g/dL); increased capillary Increased proportion of lymphocytes in the WBC diff ct
fragility
Decreased urinary excretion of 17-KS, 17-OHCS Increased skin temp, pulse rate and pulse pressure
Primary cause: deficiency of elemental iodine, destruction 1. Thyroxicosis- high free thyroid hormones
or ablation of thyroid gland, surgical removal of gland, a. T3 thyroxicosis (Plummer’s disease) ↑FT3, NFT4, ↓TSH
radioactive iodine treatment for hyperthyroidism, b. T4 thyroxicosis N/↓FT3, ↑FT4, ↓TSH
radiation exposure, drugs such as lithium 2. Grave’s disease- (diffuse toxic goiter)
1. Hashimoto’s disease- (chronic autoimmune thyroiditis) -6x more common in women
-associated with enlargement of the thyroid gland (goiter); Features- exophthalmos, pritibial myxedema
thyroid replaced by a nest of lymphoid tissue Dx test- TSH receptor antibody test
-Positive TPO antibody 3. Riedel’s thyroiditis- woody/stony hard thyroid mass
2. Myxedema- peculiar nonpitting swelling of the skin 4. Quervain’s thyroiditis- Subacute granulomatous/ Subacute
(mucopolysaccharides infiltration) nonsuppurative thyroiditis (painful thyroiditis)
Features: puffy face, slow speech, thinned eyebrows, dry -↑ESR,Tg; associated with neck pain, low-grade fever and
yellow skin, andanemia swings in thyroid fxn test

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3. Congenital/ Neonatal hypothyroidism ( can lead to Cretinism)- inadequate thyroid hormone production (hyposecretion) in
newborn infants
∑ It can occur because of an anatomic defect in the gland. inborn error of thyroid metabolism or iodine deficiency
Symptoms: mental and physical development retardation
Screening Program for Detection of Neonatal Hypothyroidism: FT4, TSH- (↑, most sensitive test for neonatal screen)
∑ TSH<10 mIU/L- no further test
∑ TSH 10-20 mIU/L- repeat test in 2-6 weeks
∑ TSH >20 mIU/L- for endocrinologic evaluation to diagnose hypothyroidism
*Very low birth weight infants should have additional screening at 2 and 4-6 weeks to detect late-onset transient
hypothyroidism
4. Euthyroid Sick Syndrome (Non-thyroidal illness)- normal/ non dysfunctional thyroid, but unusually low T3/T4 (critically-ill
hospitalized patients) – high rT3; normal/increased TSH
∑ Secondary VS Tertiary Hypothyroidism:
Hypothyroidism TRH TRH Stimulation/ Administration Test
2°- pituitary adenoma/ destruction N/↑ TSH before administration ↓
TSH after administration ↓
3°- hypothalamic disease ↓ TSH before administration ↓
TSH after administration ↑
∑ Thyroid Autoantibodies:
1. Thyroperoxidase (TPO) or anti microsomal antibodies- Hashimoto’s disease
2. Thyroglobulin (Tg)
3. TSH receptor (TR) or thyroid stimulating immunoglobulin - Grave’s disease
∑ Myxedema coma- most severe form of primary hypothyroidism
∑ Hashimoto’s- most common cause of hypothyroidism
∑ Grave’s- most common cause of hyperthyroidism (thyrotoxicosis)
THYROID FUNCTION TEST:
1. Thyrotropin-releasing hormone (TRH) stimulation test- measures relationship between TRH and TSH secretions
-Differentiate euthyroid and hyperthyroid patients with undetectable TSH
-Detection of thyroid hormones resistance syndromes
-Confirm borderline cases and euthyroid’s grave’s disease
Dose needed: 500 µg TRH by IV
Increase- primary hypothyroidism
Decrease- hyperthyroidism
2. Thyroid Stimulating Hormone (TSH) test- differentiate primary and secondary hypothyroidism
Increased TSH Decreased TSH
Primary hypothyroidisim Primary hyperthyroidism
Hashimoto’s thyroiditis Secondary and tertiary hypothyroidism
Thyrotoxicosis due to pituitary tumor Treated Grave’s disease
TSH antibodies Euthyroid Sick Disease
Thyroid hormone resistance Over replacement of thyroid hormone in hypothyroidism

3. Radioactive Iodine Uptake (RAIU)- measure ability of the gland to trap iodine (cause of hyperthyroidism)
High Uptake- metabolically active gland (with TSH deficiency- autonomous thyroid activity)
4. Thyroglobulin (Tg) assay- postoperative marker of thyroid cancer
Increased: untreated and metastatic differentiated thyroid cancer, hyperthyroidism, nodular goiter
Decrease: infants with goitorous hypothyroidism and thyrotoxicosis factitia (↓; differentiates it with subacute thyroiditis-↑)
Reference Range: 0.5-5 µU/mL
5. Reverse Ts (rT3) – end product of T4 metabolism, 3rd major circulating thyroid hormone
-used to assess borderline or conflicting laboratory results (identifies euthyroid sickness)
Reference Range: 38-44 ng/dL
6. Free Thyroxine Index (FTI or T7)- assesses level of FT4 in blood (equilibrium of bound T4 and FT4)
↑-Hyperthyroidism, ↓-Hypothyroidism

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-Important in correcting euthyroid individuals

Reference Method: equilibrium dialysis
Reference Range: 4-12%
7. T3 Uptake Test- measure TBG
Increased: hyperthyroidism, euthyroid patients, chronic liver disease
Decreased: Hypothyroidism, Oral contraceptives, pregnancy, acute hepatitis
Reference Range: 25-35%
8. Thyroxine binding globulin (TBG) test- confirm FT3 or FT$ abnormalities or abnormalities in the TT4 and THBR
-Total serum (TT3 and TT4) levels are dependent on the amount of TBG
-Distinguish between hyperthyroidism causing high thyroxine and euthyroidism with increased binding by TBG
Increased: hypothyroidism, pregnancy, estrogen
Decreased: anabolic steroids, nephrosis
9. Fine needle aspiration- most accurate tool in the evaluation of thyroid nodules
10. Recombinant human TSH- test for thyroid cancers for residual and recurrent disease
11. Tanned erythrocyte hemagglutination method- for antithyroglobulin antibodies
12. Serum calcitonin test-tumor marker for detecting residual thyroid metastasis in medullary thyroid carcinoma (MTC)
(before and six months after surgery
13. Pentagastrin (Pg) stimulation test-diagnosis of MTC
Summary of Thyroid Diseases and Laboaratory Tests:
Disorders T3 T4 TSH FT4 rT3 Tg TBG
1. Grave’s disease ↑ ↑ ↓ ↑ ↑ ↑ N
2. Primary hypothyroidism N/↓ ↓ ↑ ↓ ↓ N/↓ N
3. Hashimotos’s thyroiditis N/↓ N/↓ ↑ N/↓ ↓ N/↓ N
4. Nonthyroidal illness ↓ N/↓ V V N/↑ N N
5. Thyroidal hormone resistance ↑ ↑ N/↑ ↑ ↑ ↑ N
6. Neonatal hypothyroidism ↓ ↓ ↑ ↓ ↓ N/↓ N

3. ADRENAL GLANDS- shaped like pyramids located above the kidney (arise from neural crest cells)
Hypofunction- treated with exogenous hormone replacement
Hyperfunction- treasted with suppressive drugs or surgery
∑ Composed of two distinct gland:
1. Outer adrenal cortex
(10%)Zona glomerulosa (G cell)- Aldosterone
(75%)Zona fasciculata (F cell)- Cortisol
(10%)Zona reticularis (R cell) - DHEAS
2. Inner adrenal medulla- Cathecolamines
21-C steroid- progesterone, deoxycortisone, aldosterone and cortisol
19-C steroid- dehydroepiandosterone, androstenedione
18-C steroid- estrogen
-Adrenocortical hormones:
1. Cortisol- oscillates with a 24-hr periodicity or circadian rhythm
-Highest in the morning and lowest in afternoon
Regulation: Negative feedback of cortisol in ACTH and CRH production
Urinary metabolites:
1. 17-hydroxycorticosteroid (OHCS)- Porter-Silber
2. 17-ketogenic steroids (KS)- Zimmerman
Disorders:
1. Congenital Adrenal Hyperplasia- inherited family of enzyme disorders affecting cortisol, aldosterone and sex steroid
production
a. 21-hydroxylase deficiency- Increased levels of 17A- hydroxyprogesterone and unrinary excretion of estriol, ACTH, urine
17-KS; Decreased cortisol and aldosterone
b. 11B- hydroxylase deficienciency- Increased 11-deoxycortisol
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c. 3B- hydroxysteroid dehydrogenase-isomerase deficiency- increased ratio of 17A-hydroxypregnenolone to 17A-

hydroxyprogesterone and increased ratio of DHEA to androstenedione
d. 17A-hydroxylase deficiency- decreased androgen, cortisol, estrogen, progesterone synthesis; increased aldosterone and
ACTH
2. Hypercortisolism- due to excessive production of CRH and ACTH, glucocorticoids or androgens; can be caused by
corticosteroids overuse
A. CUSHING’S SYNDROME- caused by ACTH secreting pituitary adenoma (Cushing’s disease), autonomous cortisol
production from adrenal tumor and ectopic ACTH/CRH production
Signs and symptoms: weight gain but with thin extremities (Buffalo hump), central obesity hyperglycemia, thinning of the
skin, poor wound healing, hypertension, hypercholesterolemia, decreased WBC (lymphocytes)
Diagnosis: Cortisol excess, Loss of diurnal rhythm, Suppression resistance
Lab Findings: Persistent elevated levels of serum and urinary 17-OHCS, Loss of circadian rhythm, Loss of dexamethasone
suppression
Screening tests:
ÿ 24-hour Urinary Free Cortisol (four-fold increase)
ÿ Overnight Dexamethasone Suppression Test (blood level not suppressed)
ÿ Midnight Salivary Cortisol Test (high saliva cortisol)
Confirmatory tests:
ÿ Low Dose Dexamethasone Suppression Test
ÿ Midnight Plasma Cortisol (>7.5µg/dL serum cortisol-confirmatory)
ÿ Corticotrophin-releasing Hormone (CRH) Stimulation Test
∑ Dexamethasone suppression test- based on the ability of dexamethasone to inhibit cortisol
Low Dose Dexamethasone-inhibits ACTH and cortisol secretion
High Dose Dexamethasone- inhibits ACTH secreting tumors
-Adrenal cortical tumors cannot be inhibited by both of these
TEST Pituitary ACTH Secretion Ectopic ACTH Secretion
ACTH High Higher
Urinary free cortisol High Higher
Hypokalemia No YES
Dexamethasone Relatively resistant Completely resistant
3. Hypocortisolism- due to decreased cortisol production, aldosterone deficiency, excess ACTH release
A. ADDISON’S DISEASE- hypotension, hyponatremia, hyperkalemia, weight loss, hyperpigmentation
Screening test: ACTH stimulation test
B. SECONDARY HYPOCORTISOLISM (Secondary Adrenal insufficiency)
-abnormality of the hypothalamic-pituitary level
C. GLUCORTICOID deficiency- most common cause but abnormality in mineralocorticoid is not observed and no pigementation
involved. (Normal response to Cosyntropin test but Abnormal Metyrapone test)
Symptoms: Failure to thrive, weakness, fatigue, anorexia, nausea, diarrhea, abdominal pain
Lab findings: Low cortisol, Elevated ACTH (>200 pg/mL), Hyponatremia, Hyperkalemia, Hypercalcemia, Prerenal azotemia,
Mild metabolic acidosis
Diagnosis:
1. Cosyntropin synthetic stimulation of Cortisol and ACTH- not for secondary adrenal insufficiency
Procedure:
a. Measurement of baseline cortisol
b. Administration of cosyntropin by IV/IM
c. Another sample collected after 30 and 60 minutes post stimulation
2. Hypoglycemia (<20 µg/dL after stimulation)
3. Metyrapone test- confirmatory for secondary adrenal insufficiency
a. Metyrapone is administered orally at midnight
b. 11B-hydroxylase is blocked while 11-deoxycortisol (>7µg/dL) and cortisol (<5 µg/dL) decreases
Treatment:
- Florinef- for aldosterone and Hydrocorticosterone or prednisone- for cortisol
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2. Aldosterone(Aldo)- derived from corticosterone by 18-hydroxysteroid dehydrogenase

Action: Increase Na reabsoprtion, Increase renal K and H secretion
∑ Renin-Angiotentensin-Aldosterone System (RAAS):

Stimuli of RAAS: low blood volume/ pressure and low sodium

Compensatory organ: Kidney (releasing renin via juxtaglomerular cells)
Stimuli of Aldosterone Release: Angiotensin II, ACTH, Elevated potassium
Inhibitors of Aldosterone Release: Progesterone and Dopamine
Suppressors of Aldosterone: ANP, Intracellular calcium, Ketoconazole, ACE inhibitors, NSAIDs and herapin
Disorders:
1. Primary Hyperaldosteronism (CONN’s disease)- caused by aldosterone-secreting adrenal adenoma
-low plasma renin(fails to increase with volume depletion) and high aldosterone level (fails to decrease with saline or
angiotensin inhibition)
Symptoms: hypertension, hypokalemia, mild hypernatremia and metabolic alkalosis
Screening test:
a. Plasma Aldo Concentration/ Plasma Renin Activity (PAC/PRA) ratio- (>25/>30- suggestive; >50- diagnostic)
Confirmatory tests:
a. Saline Suppression test- infusing 2 liters NSS over 4 hours/ administering 10-12mg NaCl tablets daily for 3 days (>10 ng/dL
PAC- positive)
b. Oral Sodium loading test
c. Fludrocortisone Supression test (0.1 mg Fludrocortisone oral intake)
d. Captopril challenge test (25-50 mg Captopril oral intake)
2. Secondary Hyperaldosteronism- excessive production of renin (Increased plasma renin and aldosterone)
-conditions with hypokalemia- renal artery stenosis, diuretic therapy, malignant hypertension, and congenital defects in renal
salt transport (Bartter’s and Gitelman’s syndrome)
Associated Disorders:
A. Liddle’s syndrome (pseudohyperaldosteronism)- increased epithelial sodium channel activity in the absence of
aldosterone (low renin and aldosterone without hypertension)
B. Bartter’s syndrome (bumetanide-sensitive chloride channel mutation)- rare potassium losing disorder caused by defective
NACl reabsorption in the ascending loop of henle (high renin and aldosterone)
C. Gitelman’s syndrome (thiazide-sensitive transporter mutation)- defect in NaCl reabsorption in the distal convoluted
tubule (high aldosterone)
3. Hypoaldosteronism- due to adrenal destruction and glucocorticoid deficiency (also associated with 21-hydroxylase
deficiency)
Symptoms: hyperkalemia, metabolic acidosis

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Diagnosis:
a. Furosemide stimulation test or upright postural stimulation test- (low aldosterone- positive)
b. Saline suppression test (high aldosterone-positive)
3. Weak/Adrenal Androgens- produced as by-product of cortisol synthesis
Precursors: pregnenolone and 17-OH pregnenolone
∑ DHEA- the principal adrenal androgen (converted to estrone)
∑ Excessive production leads to virilization (pseudohermaphroditism)
∑ Excess levels are confirmed by measuring total and free testosterone and DHEAS
- Adrenomedullary hormones: composed primarily of chromaffin cells that secreates CATHECOLAMINES
Precursor: L-tyrosine
1. Norepinephrine (Primary Amine) – neurotransmitter of both CNS and SNS
-Highest concentration found in the brain (produced by symphathetic ganglia)
Major metabolite: Vanillylmandelic acid (VMA); (3-methoxy-4-hydroxyphenylglycol- MHPG- (in CNS) CSF and urine)
2. Epinephrine (Secondary Amine; Adrenaline)- neurotransmitter of both CNS and SNS
-Produced only in adrenal gland from norepinephrine; flight or flight hormone, released in response to physiologic and
psychologic stress
-Most abundant medullary hormone
-Best collected in indwelling catheters
Major metabolite: Vanillylmandelic acid (VMA)- (60% in urine)
Minor metabolites: HVA, norepinephrine, metanephrine
3. Dopamine (Primary Amine)- produced from decarboxylation of DOPA (dihydroxyphenylalanine)
Major metabolite: Homovanillic acid (HVA)
-Highest concentrations in theregions of the brain
-The major intact cathecholamine in urine
∑ Cathecol-0-methyltransferase (COMT) converts epinephrine to metanephrine, norepinephrine to normetanephrine
and dopamine to methoxytyramine, all of which can be oxidized to vanillylmandellic acid by monoamine oxidase
(MAO)
∑ N:E ratio in serum is 90%; 50% of the hormones are protein bound
Methods:
1. Chromatography- HPLC or GC-MS (VMA and metanephrines)
2. Radioimmunoassay- sensitive screening for total cathecholamines (>2000 pg/mL- diagnostic for pheochromocytoma)
3. Pisano- urinary metanephrines and normetanephrine
Disorders:
1. Pheocytochroma- adrenal medulla or sympathetic ganglia tumor
Screening test: Plasma metanephrines and normetanephrines by HPLC (four-fold increase)
Diagnostic test: 24-hr urinary excretion of metanephrines and normetanephrines (increased levels)
Patient preparation: 8-hour fast, avoid caffeine, nicotine, alcohol, acetaminophen, MAO inhibitors and TCAs for at least 5
days prior testing
Pharmacologic test:
a. Clonidine tests- (0.3 mg oral clonidine) differentiates pheochromocytoma (not suppressed) to neurogenic hypertension
(50% decreased)
-Used only when catecholamines are >1000 pg/mL (5.9 nmol/L)
b. Glucagon Stimulation test- highly suggestive of pheocytochroma
-Used for individuals with normal blood pressure and when catecholamines are only modestly elevated (three-fold increase)
2. Neuroblastoma- fatal malignant condition in children (excessive norepinephrine production)
-High urinary excretion of HVA or VMA or both and dopamine
Specimen: 24-hour urine or plasma
Patient preparation: 8-hour fast, avoid caffeine, nicotine, alcohol, acetaminophen, MAO inhibitors and TCAs for at least 5
days prior testing
-Affected by body positioning (Decreases when supine) – 20-30 minutes stable reclining position prior to collection in
prechilled EDTA

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-Urine preservation: 10 mL of 6N HCl

4. PARATHYROID GLAND- posterior to the parathyroid gland; regulate serum calcium (RAISES) and the recurrent of
laryngeal nerves that innervate the vocal cords
a. Bone- stimulates bone resorption
b. Kidney- increase renal reabsorption of calcium and to convert 1A-hydroxylation of the 25-hydroxy Vit.D to the active
metabolite 1,25(OH)2D
c. Intestines- via activated Vit. D, increase calcium absorption
ÿ Parathyroid calcium-sensing receptors determines the release of PTH in response to low or high blood calcium

PTH Vit.D Calcitonin

Stimulus Decreased serum calcium Decreased serum calcium Increased serum calcium
Decreased serum
phosphate
Increased PTH
Action
Bone Increased resorption Increased resorption Decreased resorption
Kidney Increased calcium Increased calcium
reabsorption reabsorption
Decreased phosphate Increased phosphate
retention retention
Intestine Increased calcium Increased calcium
absorption absorption
Serum calcium Increased Increased Decreased
Serum phosphate Decreased Increased

5. REPRODUCTIVE HORMONES
1. Testosterone- the most potent male androgen (dominant androgen on brain, pituitary, kidney and testes)
Actions:
-Development of male characteristics
-Spermatogenesis in Sertoli cells (paracrine effect)
2. Estrogen- Important for the promotion of breast development and maturation of external genitalia
Actions:
-Development of male characteristics
-Maintains pregnancy
Types:
1. Estrone (E1)- predominant estrogen in post-menopausal women
2. Estradiol (E2)- most potent estrogen; predominant estrogen in pre-menopausal women
3. Estriol (E3)- major estrogen found in maternal urine
Laboratory Methods:
1. Urinary Estrogen-Kober
2. Serum Estradiol- Immunoassay
3. Progesterone- marker of ovulation women; used primarily for the evaluation of fertility in female
PHASES for Menstrual Cycle:
I. Follicular phase:
Increase estradiol for oogenesis
II. Ovulation phase (day 14)
Increased LH induced by estrogen
III. Luteal phase
Development of corpus luteum but if no fertilization, it will regress resulting to a menstrual period, thus decreased
synthesis of estrogen and progesterone
ÿ Test for menstrual cycle dysfunction and anovulation: FSH, LH, Estrogen, Progesterone
ÿ Test for female infertility: TSH, FT4, PRL, βHCG

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ÿ Pregnancy:
1st trimester- Increased βHCG
2nd trimester- Increased estrogen (estriol in placenta)
3rd trimester- Increased progesterone (pregnanediol released from non-pregnant women in luteal phase)
TEST ANALYTE REAGENT POSITIVE REACTION
Kober Urinary Estrogen Hydroquinone- H2SO4 Pink
Porter-Silber 17- Hydroxycorticosteroid 2,4 Dinitrophenylhydrazine Yellow
Zimmermann 17- Ketosteroid M-Dinitrobenzene in alcoholic Purple
KOH

II. THERAPEUTIC DRUG MONITORING- involves the analysis, assessment and evaluation of circulating concentrations of
drugs in serum, plasma or whole blood.
Purpose 1. To ensure that a given drug dosage is within a range that produces maximal therapeutic benefit
2. To identify when the drug is above or below a therapeutic range which may lead to either inefficacy or
toxicity (minimal side effects)
Basis Route of administration, rate of adsorption, distribution of drug within the body , and the rate of elimination
Indications 1. Identifying non-compliance in patients
2. Preventing the consequences of overdosing and underdosing
3. Maximizing therapeutic effect, particularly when there is a narrow dose range between the therapeutic and
toxic dosages
4. Optimizing a dosing regimen based on drug-drug interactions or a change in the patient’s physiologic
state that may unpredictably affect circulating drug concentrations
Influences Drug levels Patient age, gender, genetics, recent food consumption, prescribed drugs, self-administered over-the-
and efficacy counter drugs, naturopathic agents
II.1. PHARMACOLOGY- body of knowledge surrounding chemical agents and their effects on living processes
a. Pharmacotherapeutics- application of administration of drugs to patients for the purpose of prevention and treatment of
disease. It requires the knowledge of pharmacodynamics and pharmacokinetics.
b. Pharmacodynamics- (WHAT DRUG DOES TO THE BODY)- process of interaction of pharmacologically active substances
with target sites and the biochemical and physiologic consequences that lead to therapeutic or adverse effects. (drug
concentration and their responses to the tissues)
c. Pharmacogenomics- testing inter-individual genetic polymorphisms of the patient’s drug
metabolism pathway (study of genes that affect a performance of a drug in an individual)
∑ CYP450 family- one of the most prominent gene families that affect drug metabolism under
the MFO system. The three most linked to differences in the degrees of drug metabolism are
CYP2D6, CYP2C9 and CYP3A4.
∑ Pharmacogenomic profiling can be used to predict drug-drug interactions or as an
indicator if the drug will provide any therapeutic benefit at all. For example, if the CYP450

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profile indicates a faster rate of metabolism, then a LOWER DOSE of drug would be given to avoid toxic serum
concentrations. Otherwise, if the patient has a slower rate of metabolism, an INCREASED DOSE is needed to
maintain therapeutic serum drug concentration.
∑ The effectiveness of a drug over the population that uses it can be divided into two categories:
1. Responders- patients benefiting from the therapeutic and desired effects of the drug
2. Nonresponders- do not demonstrate a beneficial and desired therapeutic effect from the initiation of a given drug regimen
d. Pharmacokinetics- (WHAT BODY DOES TO THE DRUG) mathematical expression of the relationship between drug dose
and drug blood level (process undergone by the drugs in the body from its uptake to its excretion)
Five pharmacological parameters that determine serum drug concentration: (LADME)
INPUT PROCESSES:
1. Liberation- release of from its dosage formulation
2. Adsorption- transport of the drug from the site of administration to the blood circulation
Bioavailability (Bioavailable fraction) – unchanged fraction of the administered dose as it enter the systemic circulation
and eventually reaches its site of action (rate or extent of drug input)
Route of Administration Bioavailability Time until effect
1. ENTERAL Ingestion (Oral) 5 to <100% 30-90 minutes
Sublingual 100% 3-5 minutes
2. PARENTERAL Intravenous 100% 30-60 seconds
Intramuscular 75 to ≤100% 10-20 minutes
Subcutaneous 75 to ≤100% 15-30 minutes
3. OTHERS Transdermal (Topical) 80 to ≤100% variable (mins to hrs)
Rectal (Suppository) 30 to <100% 5-30 minutes
Inhalation 5 to <100% 2-3 minutes

ÿ Standard dosage- a dosage assumed in an healthy individual

∑ Intrevenous (IV) administration- the most direct route with effective delivery to their sites of section
∑ Oral administration (Ingestion)- the most common route of delivery
The effiency of absorption from the gastrointestinal tract (GIT) is dependent on many factors such as: 1. Dissociation from
the administered form, 2. Solubility in gastrointestinal fluids, 3. Diffusion across gastrointestinal membranes
∑ Drugs are absorbed either by active transport mechanism or mostly via passive diffusion from the GIT.
∑ This process requires that the drug be in a hydrophobic (nonionized state)
-Weak acids are efficiently absorbed in the stomach
-Weak bases are preferentially absorbed in the intestines
∑ Changes that may dramatically alter absorption rates: intestinal motility, pH, inflammation, food or drug intake
(e.g., inflammatory bowel syndrome, antacids and anti-ulcer medications, kaolin, sucralfate, cholestyramine,
morphine)
OUTPUT PROCESSES:
3. Distribution- delivery of the drug to the tissues (dissemination)
∑ Hydrophobic drugs- easily traverse cellular membranes and partition into lipid compartments
∑ Polar, nonionized drugs - cross cellular membranes only
∑ Ionized drugs diffures out of vasculation in a slow rate
Distribution space (Volume of distribution index)- represent a dilution of the drug after it has been distributed in the body.
It is used to estimate the peak drug blood level after a loading dose is given. It is the principal determinant of the dose.
(relationship between tissue and blood levels)
∑ Most drugs in circulation are subject to binding with serum constituents. Most common potential species are drug-
protein complexes. The free active fraction is the one that interacts with is target site and results in a biologic
response.
∑ At a standard dose, total plasma content may be within therapeutic range but the patient experiences toxic adverse
effects (HIGH FREE FRACTION) or does not realize a therapeutic benefit which may be secondary to serum protein
content changes(LOW FREE FRACTION)

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∑ Changes in serum-binding proteins: inflammation, malignancy, pregnancy, hepatic disease, nephritic syndrome,
malnutrition and acid-base disturbances
-Acid drugs bind with albumin
-Basic drugs bind with alpha-1-acid glycoprotein
ÿ ↑ binding of these drugs in acute phase reactants: propanolol, quinidine, chlorpromazine, cocaine and
benzodiazepines
ÿ Substances that compete for binding sites- other drugs or endogenous substances such as urea, bilirubin or hormones
4. Metabolism- chemical modification of the drug by enzymatic process of the cells (biotransformation)
First pass hepatic metabolism (First pass effect/ Presystemic metabolism)- fraction of drug lost during the process
of absorption; Drugs are subjected to significant metabolism during its passage to liver before reaching the general
circulation (main factor that affect blood level of administered drug)
∑ All substances absorbed from the intestine (except rectum) enter the hepatic portal system.
∑ Hepatic Mixed-Function Oxidase (MFO) system- the biochemical pathway responsible for a large portion of drug
metabolism. The basic function of this system is to convert hydrophobic substances into water-soluble
substances which is either transported in bile or urine.
-TWO FUNCTIONAL PHASES:
ÿ Phase I reactions- produce reactive intermediates
ÿ Phase II reactions- conjugate functional groups to the reactive intermediated
Common Functional groups being attached: glutathione, glysine, phosphate and sulfate
5. Excretion- drug and its metabolites are removed from the body (elimination)
Drugs can be cleared from the body mostly in the plasma free fraction of a parent drug or its metabolites via hepatic
metabolism or renal filtration or a combination of the two.
First order elimination- represents a linear relationship between the amount of drug eliminated per hour and the blood level
of a drug. (exponential rate of loss)
∑ For those drugs not secreted or those who are subjected to reabsorption, rate of elimination directly relates to
glomerular filtration rate (GFR). (↓ GFR; ↑Half-life) (e.g., aminoglycosides, cyclosporine)
II.2. Drugs in the Human Body:
a. Therapeutic index- the ratio between the minimum toxic and maximum therapeutic serum concentration
b. Therapeutic range- the difference between the highest and lowest effective dosages; the goal of drug administration is
to achieve this level of concentration in the bloodstream which provides the optimum amount of medication for treatment
of clinical disorder.
c. Subtherapeutic dose- a blood level of medication below the therapeutic range which provides no clinical benefit
∑ Most drugs are not administered as a single bolus but are delivered on a multiple-dosage regimen. The goal of this is
to achieve peak and trough levels in the desired clinical effect.
d. Steady state (equilibrium)- Evaluation of this oscillating function cannot be done immediately after the initiation of a
scheduled dosage regimen. Approximately, 5-7 doses/ half-lives are required before a steady-state oscillation is acquired
where the rate of administration equals the rate of metabolism and excretion. The peak and trough levels can only be
evaluated after this state has been reached.
e. Half-life (T ½)- represents the time needed for the serum drug concentration to decease in half of its initial peak value
ÿ Causes of Drug Toxicity:
1. elevated concentration of drug
2. abnormal response to drug after administration
3. presence of active drug metabolites
II.3. Laboratory Assessment of Therapeutic Drugs:
SAMPLE CONSIDERATIONS:
∑ Timing of collection is the single most important factor in TDM. The laboratory result should contain:
1. Time of Last Dose
2. Time of Blood Extraction
∑ Serum or plasma is the sample preferred for TDM, although heparinized plasma or whole blood may be used
Do not use serum separator tubes and calcium-binding anticoagulated tubes for blood collection
Whole blood is the specimen of choice in cyclosporine while whole blood concentrations correlation well with therapeutic

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and toxic effects for tacrolimus

Specimens for TDM:
ÿ PEAK specimen (maximum drug level)- obtained in a certain period of time after the drug administration
but may vary on the type of drug and the route of delivery.
∑ Intravenous- 30 minutes after
∑ Intramuscular- 60 minutes after
∑ Oral- 1-2 hours after
-Specimen for screening for drug toxicity to ensure it is not on the toxic range
ÿ TROUGH specimen (minimum drug level)- obtained before the administration of the next dose (30
minutes prior)
-Easiest to collect, usually immediately prior to the administration of the next scheduled dose. Measured to ensure it stay on
the therapeutic range
LABORATORY TECHNIQUES FOR DRUG DETECTION:
1. Colorimetry:
∑ Urinary acetaminophen is detected by boiling to form p-amphenol which reacts with orthocresol to form
indophenols blue
∑ Acetaminophen by using nitrous acid (sodium nitrite plus hydrochloric acid) which forms 2-nitro-5-
acetaminophenol then turns into yellow color in alkali and can be measured at 430nm
∑ Trinder assay for salicylate by using mercuric chloride, hydrochloric acid and ferric nitrate. Mercuric slats
precipitates proteins presents in the specimen while Ferric ions form a purple colored complex which can be
measured at 540 nm.
2. Immunoassay- sensitive and specific rapid analyses of blood and urine samples. It can detect drug levels in the
nanomolar range
∑ Enzyme-Mediated (Mulitiplied) Immunologic Technique (EMIT) – amount of enzyme activity is directly
proportional to the amount of drugs present
∑ Fluorescence Polarization Immunoassay (FPIA) – drug is attached to a fluorescent label or fluorophore; binding of
marked drug to antibody can be quantified at an angle at which emission occurs
∑ Enzyme-linked Immunosorbent Assay (ELISA)
∑ Enzyme immunochromatography
∑ Radioimmunoassay
3. Chromatography:
ÿ Best specimen: Urine
∑ Thin Layer Chromatography – uses serum, urine or gastric fluid for toxicology screening; most commonly
employed technique; can demonstrate the presence of multitude abused drugs qualitatively
-Drugs are identified according to the distance of the separation and how they appear with each stain
-Extraction of drugs is pH dependent (acidic- pH4.5; alkaline- pH9.0)
∑ High Performance Liquid Chromatography – highly quantitative procedure which depends on the type of column,
solvent and detector system used.
-Ideal for separation of tricyclic antidepressant and its metabolites
∑ Gas Chromatography-Mass Spectrophotometry – gold standard- confirmatory test for drug analysis; the most
sensitive and specific for drug screening; used for the quantification of many drugs
-Drugs must be volatile or can be chemically derivatized into volatile forms
-MS can be added onto effluent end of GC, enhancing its capability to quantify drugs
∑ Gas Liquid Chromatography
4. Others:
∑ Ultravolet-visible Spectrophotometry
∑ Atomic Absorption Spectrophotometry
∑ Fluorometry
∑ Diffusion
∑ Microbiological

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II.4. Classifications of Therapeutically Monitored Drugs:

CARDIOACTIVE/ CARDIOTROPIC ANTICONVULSANTS/ ANTIEPILEPTIC
Digoxin Phenobarbital
Digitoxin Phenytoin (Dilantin)
Lidocaine (Xylocaine) Valproic acid (Depakene)
Quinidine Carbamazepine (Tegretol)
Procainamide(Pronestyl) Ethosuximide (Zarontin)
Disopyramide Gabapentin (Neurontin)
Propanolol Topimarate
Amiodarone Lamotrigine (Lamictal)
Verapamil Felbamate
ANTIPSYCHOTIC MAJOR TRANQUILIZER/ NEUROLEPTICS IMMUNOSUPPRESIVE
Phenazothiazines (Chlorpromazine, Thioridazine, Cyclosporine
Fluphenazine) Tacrolimus (Prograf/FK-506)
Butyrophenones (Haloperidol) Rapamycin
Risperdal Mycophenolate mofetil
Olonzapine (Zypreza) Leflunomide (LFXM)
Quetiapine (Seroquel) Prednisone
Aripirazole (Abilify) Cyclosphosphamide (Cytoxan)
ANTIDEPRESSANT/ PSYCHOACTIVE ANTIBIOTICS
Lithium Aminoglycosides
Tricyclic Antidepressants (TCAs)- Imipramine, Vancomycin
Amitryptiline, Nortriptyline, Doxepin, Trazadone, Chloramphenicol
Desipramine Trimetophrim lactate
Fluoxetine (Prozac) ANTI-INFLAMMATORY/ ANALGESICS
Acetylsalicylic acid (Aspirin, Salicylates)
Acetaminophen (Tylenol)
BRONCHODILATORS/ ANTIASTHMA ANTINEOPLASTIC
Theophylline Methotrexate
Theobromine Busulfan

III. TOXICOLOGY- study of poisons and toxic agents, their origin, physical, chemical properties, physiologic action, treatment
of their noxious effects and method of detection.
Six Major Disciplines in Toxicology:
1. Mechanistic Toxicology- cellular, molecular and biochemical affects of toxins
-Provides a basis for rational therapy design and development of tests to assess the degree of exposure in individuals
2. Descriptive Toxicology- uses results of animal experimentation to predict level of exposure that will harm humans; this
process is known as risk assessment
3. Regulatory Toxicology- combined data of mechanistic and descriptive toxicologies that is used to establish standards that
define a level that will not pose a risk to public health for safety.
4. Forensic Toxicology- medico-legal consequences of exposure to toxins; Major focus is to establish and validate analytic
performance of methods used to generate legal evidences
5. Environmental Toxicology- evaluation of environmental chemical pollutants and their impact on human health; growing
area of concern focused on chemical’s mechanism of action, monitoring of occupational health, and increasing of public health
biomonitoring efforts
6. Clinical Toxicology- studies the relationship between toxin exposure and disease state (diagnosis testing and therapeutic
intervention
-“Any substance can cause potential harm if given at a certain dosage”
-ED50: EFFECTIVE DOSE- Dose that would be predicted to be effective or have therapeutic benefit in 50% of the population
-TD50: TOXIC DOSE- Dose that would be predicted to produce toxic response in 50% of the population
- LD50: LETHAL DOSE- Dose that would predict death in 50% of the population
Kinds of Toxicities:
a. Acute toxicity- single short-term exposure to a substance which is sufficient to cause a toxic effect (0-14 days)
b. Chronic toxicity- repeated exposure for extended period of time, usually at doses that are insufficient to cause an acute
response which accumulates (>365 days)
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c. Intermediate- (15-364 days)

Common substances causing acute toxicity: alcohol, acetaminophen, salicylate, abuse substance and carbon monoxide
Most common routes of exposure: ingestion, inhalation and transdermal absorption
Associated with: suicide, homicide, accidental exposure or occupational exposure
-In cases of drug overdose, CBC, serum electrolytes, BUN, glucose, urinalysis and blood gases must be determined
-Absorption of toxins in GIT is by passive diffusion.
∑ Toxidromes- constellation of clinical signs and symptoms that suggests a specific class of poisoning, includes:
Anti-Cholinergic Cholinergic Opiod Sedative-hypnotic Sympathomimetic
Agitation Diarrhea Bradycardia Ataxia Agitation
Blurred vision Urination Decreased boewel Blurred vision Diaphoresis
Decreased bowel Miosis sounds Confusion Excessive motor
sounds Bradycardia Hypotension Diplopia activity
Dry skin Bronchorrhea Hypothermia Dysesthesisas Excessive speech
Fever Emesis Lethargy/ Coma Hypotension Hallucinations
Flushing Lacrimation Miosis Lethargy/ Coma Hypertensuion
Hallucinations Salivation Shallow respirations Nystagmus Hyperthermia
Ileus Slow respiratory rate Respiratory Insomnia
Lethargy/ Coma depression Restlessness
Mydriasis Sedation Tachycardia
Myoclonus Slurred speech Tremor
Psychosis
Seizures
Tachycardia
Urinary retention
III.1. TOXIC AGENTS:
a. Alcohols- toxic kinds are: ethanol, methanol, isopropanol and ethylene glycol
-Ethanol is the most commonly abused substance
-Whole blood- best specimen for alcohol determination
ALCOHOL Serum Metabolic Serum Urine Metabolites in Remarks
osmolal acidosis acetone oxalate the Liver
gap with anion
gap
Ethanol (grain alcohol) + - - - Acetaldehyde- Antidote:
Acetic acid Diazepam
Methanol (wood alcohol) + + - - Formaldehyde- Commonly
Formic acid used solvent;
causes
blindness and
arterial and
venous damage
Isopropanol (rubbing alcohol) + - + - Acetone Suicidal agent
Ethylene glycol (anti-freeze + + - + Oxalic acid & Commonly
agent) Glycolic Acid constituent of
hydraulic fluid

INFLUENCES OF ETHANOL INGESTION ON ETHANOL LEVELS AND BEHAVIOR

Stages of Impairment by Ethanol:
Blood alcohol Signs and Symptoms
(%w/v)
0.01 – 0.05 No obvious impairment, some changes observable on performance testing
0.03 – 0.12 Mild euphoria, decreased inhibitions, some impairment of motor skills
0.09 – 0.25 Decreased inhibitions, loss of critical judgment, memory impairment, diminished reaction time
0.18 – 0.30 Mental confusion, dizziness, strongly impaired motor skills (staggering, slurred speech)
0.27 – 0.40 Unable to stand or walk, vomiting, impaired consciousness
0.35 – 0.50 Coma and possible death

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Whiskey Blood Concentration Influence

ounces
1-2 10-50 mg/dL None to mild euphoria
3-4 50-100 mg/dL Mild influence on stereoscopic vision and dark adaptation
4-6 100-150 mg/dL Euphoria; disappearance of inhibition; prolonged reaction time
6-7 150-200 mg/dL Moderately severe poisoning; reaction time greatly prolonged; loss of inhibition and
slight disturbances in equilibrium and coordination
8-9 200-250 mg/dL Severe degree of poisoning; disturbances of equilibrium and coordination;
retardation of thought processes and clouding of consciousness
10-15 250-400 mg/dL Deep, possibly fatal coma
b. Gases
∑ Cyanide- characteristic odor of bitter almonds; binds with HGB – causing hypoxia, flushing, headache, tachypnea,
dizziness and respiratory depression
∑ Carbon Monoxide- colorless, odorless and tasteless gas; has up to 250x greater affinity for HGB compared to
oxygen
-produces cherry red color of blood
SYMPTOMS OF CARBOXYHEMOGLOBINEMIA (COHB)
COHB (%) SYMPTOMS and COMMENTS
0.5 Typical in nonsmokers
5-15 Range of values seen in smokers
10 Shortness of breath with vigorous exercise
20 Shortness of breath with moderate exercise
30 Severe headache, fatigue , impaired judgment
40-50 Confusion, fainting, exertion
60-70 Unconsciousness, respiratory failure, death with continuous exposure
80 Immediately fatal
c. Heavy Metals:
∑ Lead- blocks delta aminolevulinic acid synthetase; causes anemia, birth defects, ADHD, low IQ
∑ Arsenic- odor of garlic breath; metallic taste; strong affinity to keratin
Short-term exposure to arsenic Blood and urine
Long-term exposure to arsenic Hair and nails

∑ Mercury- can amalgamate; causes pink disease (acrodynia) and congenital minamata disease
∑ Cadmium- environmental pollutants used in electroplating and galvanizing- (+) GGT in urine
∑ Organophosphates- pesticide and insecticide poisoning; associated with decreased cholinesterase, can be
hepatotoxic
-Pesticides have organophosphates and carbamates which can inhibit the enzyme, acetylcholinesterase

REINSCH Test (Arsenic, Cadmium, Lead, Mercury, Bismuth, Antimony)

∑ Based upon the ability of heavy metals to oxidize elemental copper
∑ Positive results: heavy metals are reduced to the elemental form, plating the copper wire BLACK
(Arsenic/Selenium, Antimony and Bismuth) or GRAY/SILVER (Mercury)
∑ Negative result: unchanged copper wire

III.2. Drugs of Abuse:

Stimulants Depressants Hallucinogens/ Narcotics/ Anesthetics Other compounds
Psychedelics Opiods/ Opiates
Methamphetamine Barbiturates Lysergic acid Opium Ketamines Anabolic steroids
hydrochloride (SHABU) Benzodiazepines diethylamide Morphine Inhalants
Methylene (Ativan, Valium) (LSD) Heroin
dioxymethamphetamine Methaqualone Mescaline Codeine
(ECSTASY) (Quaalude) Psilocybin Methadone
Amphetamine sulfate Gamma Marijuana Meperidine
Dextroamphetamine Hydroxybutyrate Hashish (Demerol)

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 lOMoARcPSD|52862958

CLINICAL CHEMISTRY 3 LECTURE NOTES RAISA M. MIRZA, RMT by God’s grace

sulfate Propoxyphene
Phenmetrazine (Darvon)
Methylphenidate Fentanyl
Cocaine
Caffeine
Theophylline
Theobromine
Nicotine

III.3. Laboratory Assessment of Toxic Drugs:

SAMPLE CONSIDERATION:
1. Alcohol in blood may be analyzed even after a moment of delay provided that the samples remain sealed, because blood
lacks the enzyme that metabolize it
2. Urine temperature is a vital factor to assure that it is freshly voided and not tampered.
3. Aspiration of gastric contents or vomitus may reveal tablets or capsules from which the ingested drug can be determined
LABORATORY TECHNIQUES FOR DRUG DETECTION:
1. Enzymatic test
2. Capillary Electrophoresis
3. Homogenous Immunoassay
4. Chromatographic Methods
a. GLC- legally accepted method for ethanol testing
b. GC with IR Spectroscopy- for amphetamine detection
c. HPLC- alterantive of GC-MS for definitive identification of drugs
d. GC-MS- gold standard
e. TLC- uses serum, urine and gastric fluid; extraction is pH dependent:
-Acidic drugs (barbiturate)- pH 4.5
-Alkaline drugs (opiates, amphetamines) pH 9.0

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