CC 1 Reviewer Final

CLINICAL CHEMISTRY LEC
LECTURE 1: LABORATORY SAFETY AND

PATIENT PREPARATION
Prof. JC Louise Bandala, RMT
August 17, 2021
For updates and corrections → @mar4rii on Twitter
Laboratory Safety - rules used in every lab to keep everyone safe ● Focuses on safety related to
blood-borne pathogens that
LABORATORY SAFETY AND REGULATIONS can be present
● Safety Agencies and Organizations
○ U.S. Department of Labor’s Occupational Safety Awareness for Clinical Lab. Personnel
Safety and Health Administration (OSHA)
○ Clinical and Laboratory Standards Institute Employer’s Responsibilities Employee’s Responsibilities
(CLSI)
○ CDC, part of the U.S. Department of Health and Establish lab work methods & Know and comply with the
Human Services (DHHS), Public Health Service safety policies established lab works safety
○ College of American Pathologists (CAP) methods
○ The Joint Commission (The Joint Commission Provide supervision & guidance Have a positive attitude toward
on Accreditation of Healthcare Organization) to employees supervisors, coworkers, facilities
■ Formerly known as JCAHO & safety training
● Each of these organizations has its own rules Provide safety info, training, Give prompt notification of
● CLSI - formerly known as NCCLS (National Committee PPE & medical surveillance to unsafe conditions or practices to
for Clinical Laboratory Standards) employees the immediate supervisor and
○ Provides excellent general laboratory safety ensure that unsafe conditions
and infection control guidelines and practices are corrected
○ They already created 2 documents
■ The first is about clinical laboratory Provide and maintain equipment Engage in the conduct of safe
safety and lab facilities that are work practices and use of PPE
■ Second is the protection of laboratory adequate for the tasks required
workers from occupationally acquired ● Employer and employee should share safety
infections responsibility
● CAP - publishes an extensive inspection checklist as ○ The individual employee has on obligation to
part of the lab accreditation program which includes follow safe work practices, and be attentive to
section dedicated to lab safety potential hazards in the place their working at
● TJC - publishes a yearly accreditation manual for ○ Employers has the ultimate responsibility for
hospitals and the accreditation manual pathology and safety and delegates authority for safe
clinical laboratory services which actually includes a operation to laboratory managers and
detailed section on safety requirements supervisors
● If we summarize their roles, it's basically more on setting ○ No matter how careful we are, if the workplace
guidelines and accreditation of different laboratories in itself do not have the correct policies while
relation to if it reached the required safety present in a working = it disregards the safety
workplace ● Being an employer, you are liable to your employees.
○ Whatever may happen to employees means
Occupational Safety and Health Act that the workplace is harmful
● Public Law 91-596 ● Employees should be aware and should understand how
● Enacted by US Congress in 1970 everything works
● Goals: Provide all employees with a safe work ○ Identify the different threats (electrocution, etc.)
environment
● OSHA (Occupational Safety and Health Administration) Laboratory Hazards Prevention Strategies
○ Inspection ● Engineering Controls
- Authorized to conduct on-site ● Administrative Controls
inspections to determine whether an ● Work Practice Controls
employer is complying with the ● Personal Protective Equipment (PPE)
mandatory standards in terms of
safety OSHA’s Three Lines of Defense
- Safety is no longer a moral obligation
but also a federal law
○ Accreditation
● There are a lot of standards under OSHA
○ There are different standards that is explained
■ Blood-borne pathogen standard
■ Hazard communication standard
■ Respiratory protection standard
○ Basically, it pertains to safety specific to those
standard
■ Ex. blood-borne pathogen standard
1
● Hierarchy of Controls Color of container/bag Type of waste
○ Elimination Black Non-infectious dry waste
■ Most effective way to ensure safety
Green Non-infectious wet waste (kitchen, dietary,
■ To completely remove the hazard and
etc.)
if not possible the next thing we can
do is substitution Yellow Infectious and Pathological waste
○ Substitution Yellow with black band Chemical waste including those with heavy
■ Replace the hazard or at least find metals
safer alternatives to those existing
hazards Orange Radioactive waste
○ Engineering controls Red Sharps and pressurized containers
○ Administrative controls
Purple Cytotoxic or cytostatic waste must be
○ PPE
incinerated in a licensed or permitted facility
4. Splash guards
5. Volatile liquid carriers
- Designed to keep intact chemicals
6. Centrifuge safety buckets
● Example on how we plan and ensure safety to the

workplace
● Ex. for people checking packages
○ Involves the use of utility knives
■ Hazard: prone to certain accidents
like cutting yourselves
○ If we think about elimination, we are telling
ourselves that we need to stop our workers in
performing a task that involves using utility 7. Biological safety cabinets
knives - HEPA filtration of air intake and exhaust
○ If we can't eliminate such hazards (using utility - Recirculates filtered air into laboratory
knives), the next thing we can do is look for - Ensure sterility
alternatives. 8. Fume hoods
■ Safe utility knives - No filtration of air
○ Engineering control - Exhausts chemical fumes outside the
■ Machines do the work (to avoid being laboratory
exposed to certain chemicals) - Suitable for chemicals and non-sterile work
○ Administrative Control - Never used for infectious agents
■ Trainings to ensure that the employee 9. Mechanical pipetting devices
has lesser chance of getting injured 10. Computer wrist/arm pads
○ PPE 11. Sensor-controlled sinks
■ Cut resistant gloves 12. foot/knee/elbow-controlled faucets
13. Eyewash station
Engineering Controls 14. Chemical Storage Cabinet
● Engineering controls remove the hazard from the - In terms of storage, corrosive and flammable
workplace create a barrier between the worker and the chemicals must be in small quantities as much
hazard i.e., installing physical barriers such as as possible
bullet-resistant enclosures 15. Safety showers
● Preferred among others because they make permanent - Installed where corrosive chemicals are being
changes that reduce exposures to hazards and do not used
rely on the worker’s behavior - Readily available to all personnel
Examples of Engineering Controls Administrative Controls

1. Puncture-resistant containers ● Are those that modify workers’ work schedules and tasks
2. Safety needles in ways that minimize their exposure to workplace
3. Biohazard bags hazards
○ Biohazard- refers to biological substances that ● Examples:
pose a threat to the health of living organisms ○ Developing a chemical hygiene plan
primarily that of humans ○ Developing SOP for chemical handling
2
○ Warning alarms ● Employers of health care workers must establish and
○ Labeling systems implement an infectious waste program
○ Trainings ● All biomedical waste should be placed in a bag marked
with the biohazard symbol and then placed into a
Work Practice Controls leak-proof container that is puncture-resistant and
● Practices that reduce the risk of exposure altering the equipped with a solid, tight-fitting lid. All containers must
way in which a task is performed to make it safe be clearly marked with the word biohazard or its symbol.
● Intended to reduce the likelihood of exposure by ● All sharp instruments, such as needles, blades, and
changing the way a task is being performed glass objects, should be placed into special
puncture-resistant containers before placing them inside
that bag and container
Laboratory Hazard Prevention Strategies
● All biomedical waste must then be disposed of, according
Work practice controls ● Hand washing after each patient to one of the recommended procedures
(general procedures or contact ● Highly pathogenic waste should undergo preliminary
policies that mandate ● Cleaning surfaces with disinfectants treatment on-site.
measures to reduce or ● Avoiding unnecessary use of ● Potentially biohazardous material, such as blood
eliminate exposure to needles and sharps and not products and contaminated laboratory waste, cannot be
hazard) recapping directly discarded. Contaminate noncombustible waste,
● Red bag waste disposal such as glassware, should be autoclaved before being
● Immunization for hepatitis discarded. Special attention should be given to the
● Job rotation to minimize repetitive discarding of syringes, needles, and broken glass that
tasks could also inflict accidental cuts or punctures.
● Orientation, training, and continuing Appropriate containers should be used for discarding
education these sharp objects.
● No eating, drinking, or smoking in
the laboratory FIRE HAZARD
● Warning signage ● Four factors causing fire: (Fire Tetrahedron)
○ Fuel
Personal Protective Equipment ○ Heat
○ Oxygen
1. Gloves
○ Uninhibited Reaction
2. Lab gown
■ The continuous reaction of fuel, heat,
3. Eyewash stations
and oxygen
4. Protective eyewear
● In order for fire to start, the four must be completed
5. Face shield
6. Face mask
7. Safety showers
8. Appropriate footwear
HAZARDS CLASSIFICATION: TYPES OF HAZARDS
Safety Hazards General Classification

1. Biological
2. Fire
3. Electrical
4. Chemical
5. Mechanical (physical)
6. Radiation
7. Compressed gasses
8. Cryogenic Materials
9. Ergonomic
BIOLOGICAL HAZARD
● Medical Waste - “may transmit infectious diseases”
○ Blood and body fluids, stool, urine, body
tissues, etc.
○ 3 types of disposal
■ Incineration
■ Chemical treatment
■ Autoclaving
● Discard sharps in puncture-resistant containers located
within the work area
● Needles should NOT be transported, recapped, bent,
or broken by hand
3
● The type of fire extinguisher hat can be used for all
classes is the Dry Powder
ELECTRICAL HAZARD
● Direct effect:
NFPA (National Fire Protection Agency) Hazard Label
○ Shock
○ Burns ● Each diamond represents different hazards
○ Death ● Flash Point
● Indirect effect: ○ Lowest temp. at which a liquid can give off
○ Explosion vapor to form an ignitable mixture in air near
○ Fire the surface of the liquid
● What to look out for to prevent hazards ○ The lower the flashpoint, the easier it is to ignite
○ Free cords
○ Removed grounding prongs sa plug
○ Any type of grounding shock being used
CHEMICAL HAZARD
● Employees must be notified of the potential health
hazards of the handled chemicals
○ Chemical storage equipment must be arranged
& labeled accordingly
○ Take note of storage requirements
○ MSDS (Material Safety Data Sheets)
■ Compiled information about the
chemicals being handled
■ Must be on file and available for every
chemical in the lab
■ Control measures in case of exposure
MECHANICAL HAZARD
● A.k.a Physical Hazards
● Hazards created by the use of or exposure to either
powered or annually operated equipment, machinery,
and plant
○ Centrifugation lapses
○ Lab glassware
RADIATION HAZARD
● Ionizing radiation can damage living tissue in the human
body
● Strips away electrons from atoms and break some
chemical bonds
● When these particles come in contact with organic
materials like the human tissue, it damages them causing
burns and cancer
● To reduce radiation exposure remember:
○ Distance
■ The farther from the source of
radiation, the lesser exposure
○ Shielding
■ Wearing pieces of equipment that
shield us from it
○ Time
■ The longer the exposure, the more
hazard
4
COMPRESSED GASES ● 10-14hrs: lipids and lipoproteins
● All compressed gases are hazardous because of the ● 48 hrs fasting- increase serum bilirubin
high pressures inside the cylinders. ○ Increase clearance of bilirubin which decreases
● Mixture of gases n a container having a certain pressure during fasting.
● We are putting pressure in the container ● 72 hrs fasting-increase triglycerides while glucose
○ Pressure: 40 psi at 21.1 degree celsius decreases in women to 45 mg/dL.
● All are hazardous because of the high pressure inside ● Basal state collection: glucose, cholesterol, triglyceride
the cylinders. and electrolytes
○ Can become certain missile Note: Basal state collection is early morning blood
collection, 12 hrs after the last ingestion of food.
● Requires fasting specimen: FBS, GTT, TAG, Lipid Profile
CRYOGENIC MATERIAL
test, gastrin and insulin.
● Liquid Nitrogen
● Cryogens DIET
○ Substances used to produce low very ● High protein diet-increase urea
temperature ○ Urea- breakdown products of proteins
■ Low as -153 degree celsius ○ Ammonia can be further converted into urea
● Ex: liquid nitrogen ● Glucose, lipids and catecholamines may show variation
○ Major hazard of liquid nitrogen are associated postabsorptive hormonal effects.
with the properties of extreme cold evaporation. ● High protein, low carbohydrate diets- increased ketones
in urine.
ERGONOMIC HAZARD ○ Ketones- comes breakdown of fats
● Factors in our environment that can harm our ○ High protein, low carbohydrate→ fats used as
musculoskeletal system energy
● Causes strain disorders ● Fat-rich food may increase potassium, ALP, TAG, and 5
● Primary contributing factors: HIAA
○ posture/ position ○ HIAA- 5-hydroxyindoleacetic acid
○ Applied force
■ Breakdown product of serotonin
○ Frequency of repetition
■ Serotonin- a hormone in the brain that
Prevention strategies
can affect our mood.
● Job rotation to minimize repetitive tasks (work practice
● Serotonin-rich food (banana, pineapple, tomato, and
controls)
avocado) increase the urinary excretion of 5-HIAA.
● Computer wrist/ arm pads (engineering controls)
● Caffeine increases concentrations of glucose; it promotes
● Placing comfortable seats
the release of catecholamines from the adrenal medulla
and brain tissue.
"Safety begins with the recognition of hazards and is achieved
○ Catecholamines- increases glucose,
through the application of:common sense,a safety-focused
decreasing release of insulin
attitude,good personal behaviour,good housekeeping in all
■ Flight and fight hormones.
laboratory work and storage areas,and above all, the continual
practice of good laboratory technique." ● Increased in obese person: LD, cortisol and glucose.
POSTURE AND POSITION

PATIENT PREPARATION
● Preferred position during phlebotomy: upright position
Pre-analytical Variables/ Factors contributing to the variation of
or supine
results:
● Changing from supine to sitting or standing position-
● Exercise
increased levels of albumin, enzymes, and calcium
● Fasting
○ Causes constriction of blood vessels and
● Diet
reduction of plasma volume
● Posture and Position
● Changing from sitting to supine- increase levels of
● Tourniquet application
proteins, lipids, BUN, iron, and calcium
● Tobacco Smoking
○ Since there will be a shift of water and
● Alcohol ingestion
electrolytes into your tissues, it causes
● Stress (anxiety)
hemoconcentration
● Drugs
● Changing from standing to supine position- decreased
levels of cholesterol, triglycerides, and lipoproteins
EXERCISE
○ Since this causes exovascular water to transfer
● Volume shifts between the vascular and interstitial
to your vascular systems and dilutes your non
compartments, volume loss by sweating and changes in
diffusible plasma constituents
hormone concentrations.
● Prolonged standing for more than 30 minutes- increased
● Transient increased in lactate, fatty acid, ammonia.
potassium
○ Lactate- produced when there is a lack of
○ due to the release of potassium to your
oxygen
muscles
○ Ammonia- waste product of your amino acids
● Prolonged bedrest- decreased plasma albumin
and proteins; product of deamination of ATP
○ Due to fluid retention
● Long-term increased in CPK, AST, LD and aldolase.
○ Enzymes found in skeletal muscle
Thus, it is advisable that we need to let patients rest for at least 15
● Increased in prolactin, testosterone and luteinizing
minutes to regulate and stabilize their bodily substances
hormone (LH)
● Elevated levels of proteins in urine (proteinuria)
TOURNIQUET APPLICATION
● Vigorous hand exercise (fist clenching) increases
● 1 minute application is recommended
potassium, lactate and phosphate.
● Prolonged application= hemoconcentration and
● Decreased plasma levels of FSH and LH in long distance
anaerobiosis
athletes.
○ Anaerobiosis - increase in the substances
○ FSH and LH- reproductive hormones
present in our body that does not need oxygen
● Increase levels: potassium, proteins (albumin), enzymes,
FASTING
lactate, cholesterol, and ammonia.
● 8-10 hrs: glucose
5
● Prolonged use of tourniquet with fist exercise- increase ○ Alkaline, phosphatase, cholesterol, and
potassium 1mmol/L phosphorus
● For measurement of lactate - tourniquet use should be ● Affected by gender (increased levels):
minimal and the patient should not clench his or her fist ○ Male: Albumin, ALP, creatinine, uric acid,
otherwise will result to elevated levels of lactate cholesterol, BUN
○ Female: HDL, iron, and cholesterol
TOBACCO SMOKING ● Affected by recent food ingestion:
● Increased in plasma catecholamines and cortisol ○ Increased levels: glucose, TAG, astrin, free
● Increased in glucose, growth hormone, cholesterol, calcium
triglycerides, ammonia, urea, lactate, insulin and urinary ○ Decreased levels: electrolytes (Cl-,K+,P+), ALP,
5- HIAA. AMS
● Increased plasma non esterified acid concentration
● Decreased plasma levels of vitamin B12 and elevated
thiocyanate
○ Tobacco has nicotine, which has an endocrine
effect on our body, thus elevating endocrine
hormones
ALCOHOL INGESTION
● Increased level of urate, triglycerides, and gamma
glutamyl transferase (GGT).
○ GGT= liver enzyme; test requested by the
physician since it is a serum marker for alcohol
related diseases
● Hypoglycemia (chronic alcoholism)
○ Alcohol consumption causes an increase in
insulin secretion which leads to lowering your
blood sugar level causing hypoglycemia
STRESS (ANXIETY)
● Affects adrenal hormone secretion
● Increased: catecholamines, cortisol, ACTH, prolactin,
insulin, albumin, glucose, and lactate
● Total cholesterol has been reported to increase with mild
stress, and HDL cholesterol to decrease by as much as
15%.
● Hyperventilation - affects acid-base balance
○ Conserving or needing of increased oxygen
● Since there is body tension, we begin to breathe a little
more shallow and lower our oxygen levels in the blood,
which sends signals to our brain that we are at stress.
DRUGS
● Hepatotoxic drugs can elevate liver function enzymes.
● Diuretics can cause decreased plasma sodium and
potassium.
○ Diuretics
■ also known as water pills
■ Common treatment for high blood
pressure
● Opiates cause increases in liver and pancreatic enzymes
○ Pang high na drugs
○ 1 content - Acetaminophen which has a direct
effect to our liver causing toxicity
PHYSIOLOGIC VARIATION
● changes that occur within the body such as cyclic
changes (diurnal or circadian) or those resulting from
exercise, diet, stress, gender, age, drugs, posture or
underlying medications.
● Affected by diurnal variation:
○ increased in AM: ACTH, aldosterone, cortisol,
and iron
○ decreased in PM: Acid phosphatase, Growth
hormone, Parathyroid hormone, Thyroid
stimulating hormone
- There is fluctuation in our body
- Most substances with diurnal variation are hormones
- That is why it is very tricky when we are ing requested to
collect samples for hormonal testing
- Timing is needed for collection Ex. ACTH - highest peak
is at 8AM
● Affected by age (increased levels):
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LECTURE 2: EXPRESSING CONCENTRATIONS

PARTS 1-2
Prof. Fritdey Jad Doctolero, RMT, MLS(ALCPi)CM
August 23, 2021
DEFINITION OF TERMS
● Solution - a homogeneous mixture of two or more This formula is usually used when the problem asked for the
substances and can be in a form of solid, liquid, ot gas needed weight of a solute and the given is the percent
○ Solute - substance being dissolved concentration and either the weight or the total volume of the
○ Solvent - substance that dissolves desired solution :
● Concentration - how much of the solute is present per
unit of volume 𝑥 (𝑔)
𝑔 = 100 𝑚𝐿
[𝑥 (𝑚𝐿) 𝑑𝑒𝑠𝑖𝑟𝑒𝑑 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛]
2. %v/v
● Both solute and solvent are liquid
● Units: % v/v = mL per 100mL
This formula is used when the problem asked for the needed
volume of the solute given that the percent concentration and
desired solution is given in the problem:
Which among the 4 is very concentrated and the least 𝑥 (𝑚𝐿)

concentrated? 𝑚𝐿 = 100
● A = very concentrated (more solute)
● D = least concentrated/most diluted (less solute)
3. %w/w
How to express the concentration of solutions? ● Use the weight of the final solution rather than the
● Percent concentration volume
○ 0.9% Normal saline solution ● Units % w/w = grams per 100 g
● Molarity
○ 0.109 M buffered sodium citrate 𝑥 (𝑔)
● Molality 𝑔 = 100 𝑔
[𝑥 (𝑔) 𝑑𝑒𝑠𝑖𝑟𝑒𝑑 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛]
○ 5 moles/kg sucrose solution
● Normality
○ 2 N NaCl REMINDERS
○ 1 N = eq/L
● Having another way of solving given problems is okay as
Percent oncentration long as it arrives at the same answer
● Means per 100 or parts per 100 ● Take note of the units, make sure that before you
● Relationship substitute values, you always check the units
○ Solute/Solution ● Box your final answers every time
3 ways to express the concentration of a solution through % ● Do not round off answers in the middle of the solution,
concentration: always round off final answers to the hundredths place
1. % w/v (weight/volume) or two decimal places unless a specific instruction is
2. % w/w (weight/weight) given
3. % v/v (volume/volume) ● If the answer is not in two decimal places, it will suffice
● If whole number, hindi na lagyan ng two zeroes after
These 3 formulas are applicable when you want to determine the ● For solving and writing your answers, you can abbreviate
percent concentration especially in units of measurement bu make sure na
tama yung abbreviation na ginagamit
𝑤𝑒𝑖𝑔ℎ𝑡 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
𝑣𝑜𝑙𝑢𝑚𝑒
% = 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
𝑥100
What weight of glucose is needed to prepare 250 mL of a
𝑤𝑒𝑖𝑔ℎ𝑡 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 15% w/v solution?
𝑤𝑒𝑖𝑔ℎ𝑡
% = 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
𝑥100
𝑣𝑜𝑙𝑢𝑚𝑒 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 𝑥 (𝑔)

𝑥100 𝑔 = 100 𝑚𝐿
1. % w/v Solution:
15𝑔
𝑔 = 100 𝑚𝐿
𝑥 250 𝑚𝐿
● Used as a unit of measurement when the solute is a solid
and the solvent is a liquid 𝑔 = 37. 5 𝑔
● Grams of solute per 100mL of the total solution
● Example: 5% w/v
○ This means that there is 5grams of solute per ● It is asking for the needed volume of the solute
100mL of the total solution
● Units: %w/v = grams/100mL
1
● What you need to use is yung second na formula ng ● Preparation of the solution: Put 22.5 mL of ethanol in
w/v 127.5 mL of distilled water to make 150 mL of 15% v/v
● Substitute the values na given sa problem solution
● Remember when we say 15% w/v is the same as Countercheck
saying 15g/100mL times the volume of the desired ● Your solute and solvent should sum up to 150 mL
solution
PERCENT CONCENTRATION
A solution contains 24g of solute in 300mL solution. What What is the %w/w if 8.0 grams of copper is added to zinc to
is the percent concentration? produce 100 grams of an alloy?

𝑥100
𝑥100 solution:
solution: 𝑤𝑒𝑖𝑔ℎ𝑡 8.0 𝑔
% = 100 𝑥100
𝑤𝑒𝑖𝑔ℎ𝑡 24 𝑔
% = 300
𝑥100 = 8% w/w or 8 g/100 mL
8𝑔
=
100 𝑚𝐿
x100 or 8%
How would you make 200 mL of a 15% salt in a water
solution?
𝑥 (𝑔)
𝑔 = 100 𝑚𝐿
Describe how to prepare 200 mL of a 5% v/v solution of
methanol. solution:
15 𝑔
𝑔 = 100 𝑚𝐿
𝑥 200 𝑚𝐿
𝑥 (𝑚𝐿)
𝑚𝐿 = 100
[𝑥 (𝑚𝐿) 𝑑𝑒𝑠𝑖𝑟𝑒𝑑 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛] = 30 𝑔
Solution: Final answer: Place 30 grams of salt and add water up to
5 𝑚𝐿 200 mL.
𝑚𝐿 = 100 𝑚𝐿
x 200 mL
= 10 𝑚𝐿
Make 300 grams of a 20% w/w aqueous solution of sodium
10 mL of methanol is added to distilled water until 200 mL chloride.
final volume of the solution is prepared.
𝑥 (𝑔)
How much ethanol is needed to prepare 150 mL of 15% v/v
𝑔 = 100 𝑚𝐿
solution? How much dH2O is added in preparing the Solution:
solution? How to prepare the solution? 20 𝑔
𝑔 = 100 𝑔
𝑥 300 𝑔
𝑥 (𝑚𝐿)
= 60 𝑔
𝑚𝐿 = 100
Solution: Final answer: 60 g of NaCl is needed to make 300 g of a
15 𝑚𝐿 20% w/w solution.
𝑚𝐿 = 100 𝑚𝐿
x 150 mL
= 22. 5 𝑚𝐿 ethanol→ solutes volume
Volume of solution= volume of solute + volume of solvent

150 mL = 22.5 mL + volume of solvent
127.5 = volume of solvent which is dH20
Addressing the first question

● Use the second formula introduced for %V/V
○ Substitute the values
● If you forget about the final unit, apply cross units.
(pag mag cancel na)
● 22.5 mL volume of solute
Finding the volume of the solvent (second question)
● Use the formule:
○ Vol of soln = vol of solute + vol of solvent
● Substitute
● 150 mL = 22.5mL + vol. Solvent
● 127.5 = volume of solvent
Third question (How to prepare the solution?)
2
PART 2
Molarity 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒
𝐹: 𝑀𝑜𝑙𝑎𝑟𝑖𝑡𝑦 = 𝑀𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝐿𝑖𝑡𝑒𝑟 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
● MOLE
○ SI unit for the amount of a substance 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
○
23
1 M = 6. 02𝑥10 pieces
6 = 58𝑔/𝑚𝑜𝑙 𝑥 2 𝐿
■ Pieces - atoms, molecules or particles 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 = 6 𝑥 58 𝑥 2
23
■ 6. 02𝑥10 = Avogadro’s number = 696 grams
○ (mass of substance/MW)
○ Atomic weight (Atomic mass)
○ Seen beside the chemical symbol in the A solution contains 3.5 grams of hydrochloric acid in 1L.
periodic table How many mmol does it contain? (H: 1; Cl: 35)
○ ATOMIC/MOLECULAR WEIGHT
○ is the actual mass of the chemical particle
relative to the mass of the carbon atom 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑢𝑏𝑠𝑡𝑎𝑛𝑐𝑒
○ Sum of all atomic masses in a molecule 𝑚𝑜𝑙𝑒 = 𝑀𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡
Ex. What is the molecular weight of water?
1. Determine the atomic mass of oxygen and hydrogen 3.5𝑔
- O = 16 ; H = 1 (2) - bc water has 2 hydrogen = 36 𝑔/𝑚𝑜𝑙
- 16 + 2 _
- = 18 g/mol = 0. 0972 𝑚𝑜𝑙
- (H20 = 18 g/mol) _
1000𝑚𝑚𝑜𝑙
How many moles will 5 grams of water have? = 0. 0972 𝑚𝑜𝑙( 1 𝑚𝑜𝑙
) = 97. 33 𝑚𝑚𝑜𝑙
- (mass of substance/MW)
- 5/18
- 0.28 moles pr 0.28 mol ● Hindi involved ang formula for molarity
● The number of moles of solute in one (1) liter of solution ● Do no forget the cancellation of units
2 formulas: ● Yung number 2 may symbol sa taas which means na
yung number 2 ay repeating
𝑀𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑡 𝑥 𝑚𝑜𝑙𝑎𝑟𝑖𝑡𝑦 = 𝑔𝑟𝑎𝑚𝑠 / 𝑙𝑖𝑡𝑒𝑟 ● We need to convert to mmol because yan ang
required sa problem
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒 ● Para malaman mo kung saan ilagay yung 1 mol or 1
𝑀𝑜𝑙𝑎𝑟𝑖𝑡𝑦 = 𝑀𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝐿𝑖𝑡𝑒𝑟 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 mmol dapat alam mo yung required na units and yung
Unit: units na dapat icancel
● moles/L ● Since the two numbers are in the same level, you
● M multiply tthem both
● mol/L
There are 20g NaCl in 400mL of solution. What is its Make 300 mL of 6M NaCl
molarity? (Na: 23; Cl: 35)
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒

𝑀𝑜𝑙𝑎𝑟𝑖𝑡𝑦 = 𝑚𝑤 𝑥 𝐿 𝑜𝑓 𝑠𝑜𝑙𝑛
𝑀𝑜𝑙𝑎𝑟𝑖𝑡𝑦 = 𝑀𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝐿𝑖𝑡𝑒𝑟 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
20𝑔
6𝑀 = 58 𝑔/𝑚𝑜𝑙 𝑥 0.3 𝐿
= 58𝑔/𝑚𝑜𝑙 𝑥 0.4 𝐿
= 104. 4 𝑔
= 0.86 M 104.4 grams of NaCl is needed to make 300 mL of 6M NaCl or
104.4 grams is added to a solvent to make 300mL of 66M NaCl
● The final answer should be in a sentence form

How many grams of Sodium Hydroxide are contained in ● Remember liters of soln ang kailangan but the given is
500 milliliter of a 4 molar solution? (Na: 23 ; O: 16 ; H: 1) in mL so we convert it to liters

𝑀𝑜𝑙𝑎𝑟𝑖𝑡𝑦 = 𝑀𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝐿𝑖𝑡𝑒𝑟 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 Molality
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 ● The number of moles of solute in one thousand grams
4 𝑚𝑜𝑙/𝐿 = 40𝑔/𝑚𝑜𝑙 𝑥 0.5 𝐿 (1000 g) or 1 kg of solvent rather than the final solution
80g = weight of solute NaOH 𝑚𝑜𝑙𝑎𝑙𝑖𝑡𝑦 = 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝑘𝑖𝑙𝑜𝑔𝑟𝑎𝑚 𝑜𝑓 𝑠𝑜𝑙𝑣𝑒𝑛𝑡
Unit: moles/kg
If NaCl has a gMW of 58g/mole, how many of the solute is
added to 6 molar solution of NSS to come up with 2L? What is the molality of a solution if 127 grams of NaCl is
dissolved in 1000 grams of dH20?
3
= 58 grams of NaCl is needed to make 300g of a 2N NaCl
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒 solution
𝑚𝑜𝑙𝑎𝑙𝑖𝑡𝑦 = 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝑘𝑖𝑙𝑜𝑔𝑟𝑎𝑚 𝑜𝑓 𝑠𝑜𝑙𝑣𝑒𝑛𝑡
Calculate the barium chloride concentration in mEq/L of a

= 2. 19 𝑚𝑜𝑙𝑒/𝑘𝑔 solution prepare by diluting 25 grams of barium chloride
2L.
How many grams of NaCl would be in a 100g of a 1 molal

solution? ● Barium = 137.33; Chloride = 35.45
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒 𝑚𝑤 137.33 + 35.45 (2)

𝑚𝑜𝑙𝑎𝑙𝑖𝑡𝑦 = 𝐸𝑤 = 𝑣𝑎𝑙𝑒𝑛𝑐𝑒
= 2
𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝑘𝑖𝑙𝑜𝑔𝑟𝑎𝑚 𝑜𝑓 𝑠𝑜𝑙𝑣𝑒𝑛𝑡
1 𝑒𝑞
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 = 104. 115𝑔/𝐸𝑞 ( 100 𝑚𝐸𝑞 )
1 𝑚𝑜𝑙𝑎𝑙 = 5.8𝑔/ 𝑚𝑜𝑙 𝑥 01 𝑘𝑔 𝑔
= 0.104115 𝑚𝐸𝑞
5. 8 g = weight of Nacl 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒
𝑁𝑜𝑟𝑚𝑎𝑙𝑖𝑡𝑦 = 𝑒𝑞𝑢𝑖𝑣𝑎𝑙𝑒𝑛𝑡 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝑙𝑖𝑡𝑒𝑟 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
Normality 25𝑔
= 0.104115 𝑔/ 𝑚𝐸𝑞 𝑥 2𝐿
● Number of equivalent weights per Liter of solution
𝑁𝑜𝑟𝑚𝑎𝑙𝑖𝑡𝑦 = 𝑒𝑞𝑢𝑖𝑣𝑎𝑙𝑒𝑛𝑡 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝑙𝑖𝑡𝑒𝑟 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 𝑚𝐸𝑞
= 120.06 𝐿
● Equivalent Weight
○ The mass of an element or compound that will
combine with or replace one mole of hydrogen How many milliequivalents are in 1L of a 4.5 N HCl
○ Is dependent on the total charge of the positive solution?
ion, or the valence, of the element
○ UNIT: g/ eq
𝑀𝑊 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒
𝐸𝑊 = 𝑉𝑎𝑙𝑒𝑛𝑐𝑒 𝑁𝑜𝑟𝑚𝑎𝑙𝑖𝑡𝑦 = 𝑒𝑞𝑢𝑖𝑣𝑎𝑙𝑒𝑛𝑡 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝑙𝑖𝑡𝑒𝑟 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
● If the compound is an acid, an equivalent is the quantity 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒
of substance that contains one replaceable hydrogen. 4. 5 𝑁 = 𝑔
36.45 𝑥 1𝐿
● If it is a base or a salt, an equivalent is the quantity of a 𝐸𝑞
substance that will react with one replaceable hydrogen. 164. 025𝑔 = 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝐻𝐶𝐿
○ Tingnan lang ang number ng H
○ Ex: sulfuric acid H2SO4 Another formula to get the equivalence:
■ Valence: 2 𝑔𝑟𝑎𝑚𝑠 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
𝐸𝑞 = 𝐸𝑤
NORMALITY 164.025 𝑔
Valence EW = 36.45 𝑔/𝐸𝑞
NaCl 1 58.5 = 4.5 Eq
BaCl2 2 104.12
K2CO3 2 69 convert:
1000 𝑚𝐸𝑞
CaSO4 2 68 𝑚𝐸𝑞 = 4. 5 𝐸𝑞 ( 1 𝐸𝑞
)
AlCl3 3 44 = 4500 mEq
Fe2(SO4)3 6 66.6
What is the normality of a solution that contains 150 grams

of sodium chloride per liter?

150 𝑔
= 58 𝑔/ 𝑒𝑞 𝑥 1𝐿
= 2.59 N or 2.59 Eq/L
How do you make 500 mL of a 2N NaCl?

2 𝑒𝑞 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
𝐿
= 58 𝑔/ 𝑒𝑞 𝑥 0.5𝐿
= 58g
4
LECTURE 3: CONVERSIONS
August 23, 2021
CONVERSIONS
● Molarity to normality
● Normality to molarity Convert 3N H2SO4 to a percentage (MW: 98)
● Molarity or normality to %w/v
● mg/dL to mmol/L or vice versa 𝑒𝑞 𝐿 𝑚𝑜𝑙𝑒 𝑔
𝑥 𝑥 𝑥 𝑥100
● mg/dL to mEq/L 𝐿 1000𝑚𝐿 2𝑒𝑞 𝑚𝑜𝑙𝑒
Molarity to Normality and Normality to Molarity N = eq/L % = g/100 mL valence = eq/mole mw= g/mol
● N = (M)(valence)
● M = N / valence How would we be able to get g/100 mL from eq/L?
Valence - easy to know if hydrogen and hydroxide is present 3𝑒𝑞 𝐿 𝑚𝑜𝑙𝑒 98𝑔
3𝑁 = 𝐿
𝑥 1000𝑚𝐿
𝑥 2𝑒𝑞
𝑥 𝑚𝑜𝑙𝑒
Convert 6N NaOH to molarity ● When we compute for normality, kailangan ang valence =
eq/mole
𝑁 ● Cancel units
𝑀= 𝑣𝑎𝑙𝑒𝑛𝑐𝑒
6𝑁 98 𝑥 3 294 𝑔 0.147 𝑔 100
𝑀 = 1 1000 𝑥 2
= 2000 𝑚𝐿
= 𝑚𝐿
𝑥 100
=
= 6𝑀 14. 7% 𝑤/𝑣 𝐻2𝑆𝑂4
● We already have the 294/2000
● Remember mL ni siya dili na siya 100
● Multiply both numerator and denominator by 100
Convert 10M H2SO4 to normality
𝑁 = 𝑀 𝑥 𝑣𝑎𝑙𝑒𝑛𝑐𝑒 CONVERSIONS:
● mg/ dl to mEq/L
= 10𝑀 𝑥 2 ● And vice versa
= 20𝑁
A sodium concentration is reported as 250 mg/dL. What is
Conversions its concentration in mEq/L? (MW = 22.99)
● % to N or M and vice versa
● Recall that when we say percent concentration that is N= ?
equivalent to grams per 100mL Valence = 1 eq/ mole
Example: 250 𝑚𝑔 10 𝑑𝐿 𝑔 𝑚𝑜𝑙𝑒 1𝑒𝑞 1000 𝑚𝑒𝑞

𝑑𝐿
𝑥 𝐿
𝑥 1000 𝑚𝑔
𝑥 22.99 𝑔
𝑥 1 𝑒𝑞
250 𝑥 10 𝑥 1000
Convert 30% NaCl to molarity (MW: 58.44) 1000 𝑥 22.99
= 108. 74 𝑚𝐸𝑞/𝐿
% = g/100mL 𝑚𝑔 𝑑𝐿 𝑔 𝑚𝑜𝑙𝑒 𝑒𝑞 𝑚𝑒𝑞

Molarity = moles/L 𝑑𝐿
𝑥 𝐿
𝑥 𝑚𝑔
𝑥 𝑔
𝑥 𝑒𝑞
= 𝑚𝐸𝑞/𝐿
MW = g/mole
30𝑔 1000𝑚𝐿 𝑚𝑜𝑙𝑒
100𝑚𝐿
𝑥 𝐿
𝑥 58.44
= 5.13M NaCl A sodium concentration is reported as 250mg/dL. What is
its concentration in mEq/L? (MW = 22.99)
Valence = eq/mole
MW = g/mole
Convert 30% NaCl to molarity (MW: 58.44)
10 𝑚𝑔 10 𝑑𝐿 𝑔 𝑚𝑜𝑙𝑒 1000 𝑚𝑚𝑜𝑙
% = g/100mL 𝑑𝐿
𝑥 𝐿
𝑥 1000 𝑚𝑔
𝑥 40.08 𝑔
𝑥 1 𝑚𝑜𝑙
Molarity = moles/L
MW = g/mole = 2.495 mmol/L
30𝑔 1000𝑚𝐿 𝑚𝑜𝑙𝑒
100𝑚𝐿
𝑥 𝐿
𝑥 58.44
= 5.13M NaCl
1
A calcium concentration is reported as 10 mg/dL. What is
its concentration in mmol?liter? (MW = 40.08) g/mol
10 𝑚𝑔 10 𝑑𝐿 𝑔 𝑚𝑜𝑙𝑒 1000 𝑚𝑚𝑜𝑙

𝑑𝐿
𝑥 𝐿
𝑥 1000 𝑚𝑔
𝑥 40.08 𝑔
𝑥 1 𝑚𝑜𝑙
= 2.495 mmol/L
Summary
● When converting, be mindful of the units
● Remember how to compute Normality and Molarity
because its units will help you convert one to the other
2
3
4
LECTURE 4: OPTICAL TECHNIQUES

Prof. Bernardo Ordaneeza Jr., RMT
August 31, 2021
PART ONE
■ wavelengths = how far away the
ELECTROMAGNETIC RADIATION (EMR) crests and the troughs of each wave
● Different types of electromagnetic:
○ Radio waves = longest
○ Microwaves
■ used in satellite broadcasting
■ Cellphones signals = bordering
between radio and microwaves
■ Microwave oven
○ Infrared
■ Remote controls
■ Being radiated by heat
■ Heat sensitive cameras
○ Visible spectra
■ Sensitive sa atong eyes
○ Ultraviolet - beyond the visible spectra
○ X-rays
○ Gamma Rays/ cosmic rays - most powerful
● Can be described as discrete packets of energy

(photons) or waves
● Sinusoidal oscillation of both electrical and magnetic
fields at right angles to each other and to the direction of
propagation
● Main principle of spectrophotometry involves light
● Light is a form of EMR
○ Oscillation = wave
○ Disturbance in the electrical and magnetic field
■ Field in physics is everywhere
■ Whenever there is a disturbance in
that field, then electrons will exist
■ According to physics, whenever there
is a disturbance in the electrical field,
there is always a corresponding
disturbance in the magnetic field at
right angles to each other
● Remember: it is not necessary that it’s automatically
parallel to the Y axis and the magnetic field is
automatically parallel to the X axis
● Often times, the oscillation comes at the right angle or at
a certain angle compared to the Y axis and X axis ● We have different parameters or dimensions of a wave
● MAIN TAKE AWAY: ● Electromagnetic radiation is just an escalation (?) of the
○ Electromagnetic radiation is an oscillation/ electromagnetic field
wave of both electrical and magnetic fields at ○ It has 3-dimensional structure
right angles to each other ● Wavelength - peak to peak
○ Can also be from valley to valley
○ Just arbitrary because we can flip this around
○ Waves do not really perfectly orient themselves
to the X or Y axis
○ Peak to peak of the oscillation or from one point
until it returns to that same point is called
wavelength
○ Symbolized by the greek letter lambda (λ)
○ One cycle = one wavelength
● Amplitude - from midline to its peak (how high or how
deep the valley is
● FM or AM (Radio)
○ AM - amplitude modulation
○ Because the way you switch the stations, the
different radio stations will broadcast at the
● Spectrum same frequency but different amplitude
○ Gradient
○ A family of electromagnetic radiation of varying
wavelengths
1
𝑓𝑟𝑒𝑞𝑢𝑒𝑛𝑐𝑦 (𝜈) = 𝐻𝑧 𝑜𝑟 𝑐𝑦𝑐𝑙𝑒/s ● If you see an equation where one element is something
● Another dimension of the electromagnetic radiation that is equal to something that is constant divided by the
● Symbolized by nu = ν wavelength, if it’s in the denominator, it is inversely
○ Pertains to number proportional
○ Cycles per sec ● That’s why decreasing the frequency, increases the
● Unit for frequency is Herts (Hz) wavelength
● Frequency - no. of oscillations in one second ● The shorter the frequency, the longer the wavelength
● Ex. propagates in 5 seconds ○ In reality, it’s not the case
● How to determine frequency? ○ C = speed of light = vacuum
○ How many cycles in one second? ○ A vacuum is a space where there are no
○ How many cycles was produced by the wave in particles in it
one second? ● What if light encounters a material like glass?
● 1 Hz or 1 cycle/ second ○ The speed decreases
○ Because particular electromagnetic energy
holds particular energy, so it could not really
change the frequency
○ Because the photon or the light holds an
energy that cannot be created nor destroyed
Summary:
● For particular electromagnetic radiation, there is a
corresponding energy
● The higher the frequency, the higher the energy
𝑠 1 ● That energy cannot be created nor destroyed. It could
Period (p) = 𝑐𝑦𝑐𝑙𝑒
= 𝑣 just transform into other forms of energy
● Frequency = speed of light/ wavelength which means the
● period - inverse of frequency
frequency is inversely proportional to the wavelength
● Periodicity - seconds/ cycle
● When electromagnetic radiation reaches a material, it
● How many seconds for a cycle to occur?
doesn’t change its energy
● Frequency remains constant
What will happen if we have electromagnetic radiation with a
● Electromagnetic radiation changes its speed, it slows
shorter wavelength?
down when it encounters a material
● When it changes its speed, let’s say it slows down, but
the frequency remains the same, how do you
compensate for the speed?
○ The wavelength increases to compensate for
the change of light
Important things to know:

● Still propagates the same speed (5s) ● Wavelength
● The behavior of the wave becomes more active ● Frequency, how many cycles per second?
○ Due to its shorter wavelength ● Periodicity- reciprocal of the frequency
● Shorter wavelength = higher frequency ○ How many seconds will it take for one cycle to
○ Shorten the wavelength by 2 fold, the frequency occur
will also increase by 2 fold
○ From 1 hz it becomes 2 cycle/sec (2hz)
● The energy of electromagnetic radiation is directly
proportional to the frequency
○ The higher the frequency, the more energetic
the electromagnetic radiation is (which shows)
○ Oscillation is much more active
■ Still travels the same way and speed,
but the frequency changed due to
higher energy
● It means that it would take ½ second for a cycle to occur

● The same is true for a wavelength with a frequency of
4hz, the periodicity is ¼
Radiofrequency
● Decrease the wavelength
● The waves will still travel at the same speed
● It will still take 5 seconds for it too reach one end to the
other
● There is more oscillation
● The shorter the wavelength, the higher the frequency
● Application
Relationship between energy and frequency ○ Broadcasting
● Directly proportional ○ Cellphone signal
● Represented by ○ Television
○ E= hv ○ Walkie talkies
● Very wide wavelength
𝑐 (𝑠𝑝𝑒𝑒𝑑 𝑜𝑓 𝑙𝑖𝑔ℎ𝑡) ○ 1 feet to the other would take 100,00 km to 1
Frequency (v) = meter
λ (𝑤𝑎𝑣𝑒𝑙𝑒𝑛𝑔𝑡ℎ)
● 1-meter wavelength of electromagnetic radiation o
2
100,000km, they are all under radiofrequency Visible Light (Blue)
λ v λ v
100,000 km 3 Hz 490 nm 610 THz
1m 300 mHz 450 nm 670 THz
● 3 Hz means 3 cycles per second
● Mega is 10^6 = 300,000,000 cycles per second
Visible Light (violet)
● Our eyes are not sensitive to radio frequencies
● Radiofrequency could travel large distances the reason
why they are used in radio broadcasting ● More energetic than your red
λ v
Microwave 450 nm 670 THz
400 nm 750 THz
● Wavelengths ranging from 1mm to 1m

● Frequency is 300 MHz to 300GHz
● Giga is 10^9 = 300,000,000,000
● More energetic than radio frequencies
● We can’t see microwaves
λ v
1 km 300 MHz
1 mm 300 GHz
Infrared
● Different textbooks will give different wavelengths
● 1 mm--10 μm wavelength
● Infra= below
● Infrared= below red
λ v
1 mm 300 GHz
10 μm 300 THz
● THz is 10^12 = quadrillion
● 30 quadrillion cycles per second because it’s very short
● Relatively more energetic then radiofrequency and
mircowave
Visible Light (Red)

● No clear cut boundary between colors because it is
subjective
● Eyes are sensitive ● Infrared is being radiated by heat
● No color in reality ○ Ex. cameras with thermometers
λ v ○ Expressed in nanometers (10^-9)
700nm 430 THz
635nm 480 THz
Visible Light (Orange)
λ v
635 nm 480 THz
590 nm 510 THz
Visible Light (Yellow)
λ v
590 nm 510 THz
560 nm 540 THz
Visible Light (Green)
λ v
560 nm 540 THz
520 nm 580 THz
3
● The only visible range is from red-violet. Anything that ● When you change the wavelength, there is a change in
goes beyond his range is invisible to our eyes the color or in the type of electromagnetic radiation
●
Ultraviolet
● Insects can be sensitive to UV light. Ex. butterflies.
○ Flowers reflect UV light
● Causes mutation that could lead to cancer
● Ionizing radiation
○ very energetic
● Visible light to low radiofrequency: non-ionizing
○ Thus, mutations due to being near to a cell
tower or having cellphones in your pocket is a
myth ● If you go for the intermedite, its yellow
λ v
100 nm 3 PHz
10 nm 30 PHz
● Longer wavelenth - red
X-Rays ● The longer the wavelength, the lower the frequency
● Gets more energetic than ultraviolet
● The limit for radiography: twice a year
● Can cause mutations
● Hexa - 10 to the power of 18
● it is so energetic that it can penetrate flesh
○ The reason why we use x-rays to have image
on the human body, although bones are
opaque
○ Flesh is transparent
● Wavelength is ranging to 1 nm to 10 picometer ( ten to
negative 12)
● If you combine the different lenghts in a spectra, you’ll
see white
● White light
○ combination of different wavelengths of visible
spectra
○ Polychromatic - many colors
λ v
1 nm 300 PHz
10 nm 30 EHz
Gamma Rays
● Most energetic
● Produced by radioactive materials, neutrons stars,
pulsars, quasars, and black holes
○ Because the universe is littered with energetic
stars, we are actually experiencing a ring of
gamma rays right now General Rule:
○ So energetic, it could penetrate the whole
planet
λ v
10 pm 30 EHz
1 pm 300 EHz
Electromagntic Spectrum
● What your eyes perceive, the thing absorbs

● When you see a red apple, does it mean that the apple is
red?
○ No, because it just reflects the red light
In the visible spectra, there is a change in color ○ If you shine it with a white light (which is
● If you shorten the wavelength, its violet composed of different colors of visible spectra),
the apple absorbs all the wavelengths of visible
spectra except red
4
○ That's why you are seeing red (being reflected)
● In a test tube, you added the serum and reagent
○ the color reaction that is apparent after reacting
the serum with the reagent is red
○ In spectrophotometer, we’re going to choose
the color or the wavelength that the particular
solution absorbs, and not the one reflected
○ If red, the best wavelength that we use for
spectrophotometer is green
● In spectrophotometry, what were after is not the light that
is reflected by the solution, but the light that is absorbed
by the solution
● If the solution is red, the wavelength that is being
absorbed is green ● Light bends at different corners.
● If colorless, it uses a different wavelength ● Light bending of light as it passes around the end of an
object.
Summary: ● Amount of bending depends on the relative size of the
● The visible spectra is composed of electromagnetic wavelength of light to the size of the opening .
radiation with varying wavelengths, energy, and ● If the opening is much larger than the lights wavelength,
frequency the bending will be almost unnoticeable.
○ different wavelength corresponds to different ● Slit
color ○ If we have a wide opening in relation to its
wavelength, there is a bending of the wave but
● Wavelength of interest we are going to use is the one the bending is much narrower.
being absorbed by the solution. ● The smaller the slit, the bending becomes more
Constructive Interference noticeable; bends more.
● property of the wave to bend around corners or slit.
waves are in-phase

● They align
● They travel together and they collide but in collision, the
peaks and the troughs are aligned.
● Points where it coincide with each other, once it reaches
the screen it is much brighter
○ Why? -- constructive interference
■ Waves coming from slit 1 and 2
coincide.
● Peaks where it coincide with the troughs of the 2 slits
○ They become dark.
○ Destructive interference.
● Interference pattern = Bright, dark, bright, dark
○ Combination of diffraction interference.
● The wave constructs itself.
● Amplitude of your wave increases because it reenforces.
Destructive Interference
waves are out-of-phase

● Out of phase ● Bending of diffraction depends on the
○ They collide, the peaks aligned with the troughs wavelength.
- it destroys itself. ○ The longer the wavelength the
more it bends
● Using diffraction grading
○ RED bends more
○ Purple bends less
Pic left: true interference pattern of white light.

● White light - combination of all the colors.
Diffraction
5
PART TWO
light is low, then the computer in the
SPECTROPHOTOMETRY spectrophotometer will translate that to high
● Spectrophotometry = principle concentration
○ Principle = how it works, why it works?
● Spectrophotometer = Instrument
● Measurement of the light transmitted by a solution to
determine the concentration of the light-absorbing
substance in the solution
● If the concentration of the solution is low, and when u

shine a high intensity green light, gamay lang na green
light ang ma absorb. Thus, the transmitted light is
relatively high.
● So, the photon detector detects that there is a high
● Cuvette - sample/ reagent holder used in intensity of transmitted light. Thus, the commuter will
spectrophotometer translate it to low concentration.
● Incoming light = incident light Basic Components:
● Outgoing light = transmitted light ● Light source
● Spectrophotometry is the measurement of this ● Entrance slit
transmitted light coming from this cuvette ● Monochromator
● Concentration = main reason why we do clinical ○ White light is a polychromatic light
chemistry ○ Polychromatic - composed of many
○ To know the concentration of a given analyte colors/different wavelengths
● Exit slit
● Sample holder (cuvette)
● Photodetector
○ Detects the transmitted light at the other side of
cuvette
● Read-out device
● All spectrophotometer have these basic components
● Glucose with Dinitrosalicylic acid (DNS)

● If u want to know the concentration of glucose in a given
serum or plasma, u must add a reagent (in this case
DNS)
● It gives off a color reaction. In this case, the color is red.
So, we use the green wavelength in spectrophotometer
● But before that, take a look at the 5 cuvettes, which do
you think has the highest amount of glucose in it?
○ The darker the color, the higher the
concentration of a substance
○ Lighter color - lover concentration of glucose
● Colorimetry - use our eyes and compare it with a
standard; eyes are subjective
● Spectrophotometer = instrument that measures
objectively the intensity of the color
○ It makes use of a light to determine the ● Single beam spectrophotometer
concentration of the light absorbing substance ● Monochromator = diffraction grating
in a solution ● Ex. (high concentration) 5% is being read by
● Ex. if a substance contains high concentration and once photodetector and is interpreted by the
u shine green light (high intensity), it absorbs the green spectrophotometer as high concentration
light, and because it is higher concentration, much of the ● The lower the transmitted light, the higher the
green light is being absorbed, And what is being concentration.
transmitted is gamay nalang na light.
○ Photodetector at the end

■ Detects the photons
■ Detects the intensity of light that is ● If the concentration is low, and has 95% transmitted light,
transmitted the spectrophotometer will interpret that one as low
○ If the photodetector detects that the transmitted concentration.
6
● We could not really measure directly the amount of light ● Temperature
that is absorbed by the sample ○ Depending on the type or source of radiant
○ We cant put an instrument inside the sample production, temperature might be a problem
because: ○ Especially if its a metal filament heat up may
■ It could interfere with the reaction of affect testing process
■ Impractical
● Incident light - should be constant Spectrophotometer
○ Light source should give the same intensity of ● Big instrument, has a robotic arm that mixes the samples
light at all times and the reagent, which puts it in the cuvette and into the
● Absorbed light - absorbed in the sample spectrophotometer
○ If the absorbed light is low, then the transmitted ● One thing that you must remember, in all of these
light is high and if the absorbed light is high, spectrophotometers it has same components
then the transmitted light is low ○ Those components are aligned in the same
○ We indirectly measure this light by measuring way
the transmitted light ○ Light source → entrance slit → monochromator
● Transmitted light → exit slit → cuvette → photo detector →
read-out device
LIGHT SOURCE ● When it comes to temperature, there are certain smaller
(Radiant Energy Source, Henry) spectrophotometers where the light source heats up
● Provides the energy (in the form of light) that the sample which affects the overall environment inside that
will modify or attenuate by absorption spectrophotometer
● The light produced is polychromatic ○ Chemical reactions are dependent on
○ I.e., visible wavelengths are present in one temperature
white light ○ You have to pick a light source that would not
● Must be powered by a regulated power supply give off that much heat
○ If no regulator, any fluctuation in the electricity ● Spectrophotometers in the laboratory (airconditioned) to
will result in the flickering of the light which is regulate the outside temperature and avoid it from
not good overheating
○ Constant and steady stream of light (must have ○ Aircon inside the lab is for the instruments
steady voltage)
● 2 types of Light Source (according to the light produced
Light Source (Radiant Energy Source)
by that light source)
1. Continuum Source
2. Line Source
→ difference is the characteristic of light being produced
by that light source
● Spectrum of visible light

● From far ultraviolet to near infrared
● Continuum source - much of the light given
peaks sa blue (doesn't mean it gives off blue
light, it also gives off other colors) ● Incandescent tungsten or tungsten-iodide lamp
○ Continuum source of colors ○ ~15% visible, the rest near IR (Bishop)
○ X axis = intensity ○ Typical clear bulb with a filament inside
○ Intensity of the light that is given off ● Tungsten or tungsten-halogen lamp
by this light source slowly decreases ○ Visible region in the EMS (Henry)
as a function of the wavelength ○ Follow kay henry
● Line source - much more limited range of colors ● Deuterium discharge lamp
that it can give off ○ Routinely used to provide UV Radiation
○ Ex. it gives off near infrared (continuous emission down to 165 nm)
● Continuum source is wider, line source is ○ Hydrogen with an extra neutron
narrower (spectrum of light that it can give off) ○ Heavier hydrogen
● A continuum source emits radiation that ● Hydrogen lamp
changes in intensity very slowly as a function of ○ UV radiation
wavelength. ● Xenon discharge lamp
● A line source emits a limited number of discrete ○ UV and visible range
lines of radiation which has limited range of ○ Noble gas
wavelength ● Mercury arc lamps or low-pressure mercury and sodium
vapor lamps
Factors in choosing a light source: ○ Sharp lines in the UV and visible regions
○ Routinely used to provide UV radiation
● Range
○ Low-pressure pertains to the pressure of the
○ Range of wavelengths that it could give off
gas inside the bulb
○ Range of colors the light source is capable of
○ It doesn’t mean na pag line source isa lang, it
giving off
could also give off different wavelengths of light
● Spectral distribution within the range
○ Used in fluorometry
○ What is the intensity of the different
○ Yellowish in color since sodium gives off a
wavelengths within that range
yellow color
● Source of radiant production
● Medium and high-pressure mercury lamps
○ Source of production of light (is it metal or
○ Continuum form UV to the mid-visible region
excitation of halogen gas inside (neon light)?)
● Hollow cathode lamp
○ Why does it light?
○ Use in AAS
● Stability of the radiant energy
○ AAS- Atomic Absorption
○ How stable and consistent the light source
Spectrophotometry
gives off a particular energy?
7
● Nernst glower nd Globar (SiC) violet
○ IR ○ In that way, we are dissecting the white light
○ Nernst- electrically heated rod off rare earth into different monochromatic light
element oxides ● Nominal wavelengths
○ Globar- uses silicon carbide is heated up ○ The wavelength (in nm) at peak transmittance
1200°C ○ Wavelength at the highest level
○ Heat emits infrared ● Spectral bandwidths
○ Range of wavelengths above one-half peak
transmittance
○ Half-powerpoint
○ Full width at half peak maximum
○ Range of wavelengths above ½ peak
transmittance
● Bandpass
○ Total range of wavelengths transmitted
○ Much wider
Entrance and Exit Slits

● Entrance Slit
○ Reduces stray light
○ Prevents scattered light from entering the
monochromator
○ Because it gives off noise
○ As much as possible, the light passing from the
light source to the photodetector must be
consistent
○ If you have noise, there might be problems in
your read-out
○ Allow a single beam of polychromatic light to
enter the monochromator
○ Related to the reason why spectrophotometerry ● A - Peak transmittance
components must be in the dark ● The narrower the spectral bandwidth and the
higher the peak transmittance, the better
Bandpass or Bandpass width

● Range of wavelength
● determines the efficiency of monochromator
● generally, the narrower the range, the more desirable
● prisms & gratings <5 nm
● interference filters = 10 to 20 nm
TYPES OF MONOCHROMATOR
● Prism
● Exit Slit ● Diffraction Gratings
○ Allows only a narrow beam of the spectrum to ● Filters
pass through the cuvette
○ Narrows down the light coming from the a. Prism
monochromator ● A wedge-shaped piece of glass, quartz, or NaCl
○ Choosing the wavelength of light that passes ● a narrow beam of light is refracted as it enters
through the sample by adjusting the exit slit up the prism (more dense material)
and down ● can be rotated, allowing the desired wavelength
○ If you bring it down, the red light becomes to pass through an exit slit
yellow or orange light or rotate the
monochromator
Monochromator
● A device that produces light of specific wavelengths from
a light source
● Produces monochromatic light
○ Light radiation of a single wavelength
○ Choose a single wavelength of light to measure
the light-absorbing substance in the cuvette
● Prism is the simplest monochromator

● In refraction, the red disperse at an angle lower than the
8
and allow only a limited domain of this
spectrum to pass through to the sample
II. Interference Filters

● simple, least expensive
● Semi-transparent
○ 50% passes through, 50% is reflected
● made by placing semi-transparent silver films
● If you adjust the exit slit, you should also adjust the on both sides of a dielectric field such as MgFl2
cuvette ● produce monochromatic light based on the
principle of constructive interference; eliminates
b. Diffraction Gratings other wavelengths by destructive interference
● most commonly used monochromator ● wavelength depends on the thickness of the
● better resolution than the prism gap between films
● made by cutting parallel grooves or slits into an
aluminized surface of a flat piece of crown
glass
● wavelengths are bent as they pass a sharp
corner
● Cloud have slits or just a reflected material;
essentially the same
● The polychromatic light is coming from this direction, but

our goal is the monochromatic light (red light)
○ We adjust the layers so that it is conducive for
constructive interference of red light
○ Once the red light enters, half of it is reflected,
half of it is scattered
○ Until such time that the red light has a higher
amplitude or being reinforced or more intense
in the other side of the interference filter
● For blue wavelengths, once it is reflected back, it is

disruptively interfere
○ So it will be eliminated as long as other colors
○ Less intense than the red light
● Filters have a higher bandpass than prisms and gratings
because of this path that some of the wavelengths might
leak (semi-transparent silver layer)
● Principle of IF: some wavelengths are constructively
c. Filters interfere while other disruptively interfere because the
● isolate monochromatic light, or at least as way the light interacts between the two semi-transparent
narrow a range of wavelengths of light as silver layer
possible, and direct it to the sample or to the ● Out of phase: disrupt, in phase: construct
photodetector
● Easiest way
● two types:
○ absorption filters
○ interference filters
I. Absorption Filters
● Ex. parol
● absorb regions of the electromagnetic spectrum
9
■ If the path length is wider, the higher
the light-absorbing substance will be
CUVETTES hit by the incident light
■ Length of the cuvette doesn't change
because it is solid
■ 10 cm cuvette pathway - sensitive
● Even small amounts of
concentrations of light
absorbing substance will be
detected by the
spectrophotometer
Types of Cuvettes
● fused silica or quartz
○ for UV (𝜆 < 350 nm)
○ Could still be used for visible light, but ideal for
UV light
● analytical cell or sample holder

● is used to hold the solution in the instrument whose
concentration is to be measure
● must be made of material that is transparent to radiation
in the spectral region of interest
● factors considered
○ ideal shape
● silicate glass (alumina silica and borosilicate)
■ Oftentimes square, but may be
○ 350 nm < 𝜆 < 2000 nm (visible region)
circular (not ideal) or rectangular
○ Visible spectra, UV light, par infrared region
■ The other two sides is transparent
○ Borosilicate glass for specrophmety analysis of
while the other two side is frosted
alkaline solutions
● Frosted side- part of the
○ For acidic, use tough glass
cuvette that you are going to
● Plastic
handle using your hands
● If you are going to handle it
on its transparent side, you
may leave fingerprints that
may give noise to your
analysis using the
spectrophotometer
■ Why square and rectangle, but not
circle?
● Circle - when you shine a
light, there would be
different angles that the light
travels
➢ this would cause a
problem since there ○ Most commonly used
may be an interaction ○ visible and UV range
between a light and ○ can present problems related to tolerances,
the glass that could cleaning, etching by solvents, and temperature
give off different deformations
readings ○ especially used for automated analyzers
● If square, the lights travels because they are disposable
in one direction ○ problem: once you clean it, you must be careful
■ Square cuvettes have plain parallel not to put scratches on it
optical surfaces and a constant light ■ since spectrophotometer is sensitive
● They have an advantage ■ Any scratch could lead to different
over round cuvettes that refraction of light that could give noise
there’s less error in the lens to your reading
effect , orientation in the
spectrophotometer, and Cuvettes
refraction
● must be clean and optically clear
● If circular cuvettes, you can
○ because etching or deposits on the surface
orient it in any orientation
affect absorbance values
while in square cuvette, you
○ scratched optical surfaces scatter light and
can orient it in one way or
should be discarded
another
● Cuvettes used for measurements in the UV region should
○ path length
be handled with special care
■ Generally, cuvettes are small (1 cm in
○ Especially fused silica or quartz
size)
● How to clean?
■ The smaller the path length, the better
○ Use tap water or distilled water (much better)
■ The amount of light-absorbing
○ Alkaline solutions should not be used because
substance that is being subjected to
it might dissolve the glass
incident light depends on the path
○ May be cleaned in a mild detergent or soap in a
length
mixture of concentrated hydrogen chloride to
10
water to ethanol in a ratio of 1:4
PHOTODETECTORS
● Converts transmitted light energy into an equivalent

amount of electrical energy
● We measure transmitted light through photodetector
Types of Photodetectors
Barrier-Layer Cell (Photocell)

● Also known as photovoltaic cell.
● composed of light-sensitive materials (e.g. selenium on
iron plate with transparent layer of silver).
● when exposed to light, electrons in the light sensitive
material are excited and are released into the highly
conductive silver.
● no need for power source.
● Electricity - stream of electrons.
○ Electricity is produced in Barrier layer cells is
once the light hits the light sensitive material,it
is excited. It jumps and is captured by highly
conductive silver.
● slowest response time.
● difficult to amplify electrical energy..
● inexpensive and durable,
● but temperature sensitive and nonlinear at very low or
very high levels of illumination ○ Cathode = photoemissive material
● thin metal film (transparent)
Photomultiplier (PM Tube)
● detects and amplifies radiant energy
○ incident light strikes the coated cathode,
emitting electrons
○ electrons are attracted to a series of anodes
(dynodes), each with successively higher
positive voltage
● dynodes give off many secondary electrons when hit by a
single electron
○ Dynodes - intensify the single light that is being
absorbed.
● Even at very low intensities of light, it will still give a
signal.
● used in instruments designed to be extremely sensitive to
very low light levels and light flashes of very short
duration
● rapid response time (10-15 dynodes); not subject to
fatigue
● Problem: Do not expose in room light as it will burn out.
Phototube
● A.k.a photoemissive tube
● also has photosensitive material that gives off electrons
when light energy strikes
● composed of a vacuumed glass that encloses:
○ positively-charged anode
○ negatively-charged cathode (e.g., Rb or Li)
● emitted electrons jump over to the positively charged
anode, where they are collected and return through an
external, measurable circuit
● Similar to barrier cell but requires an external source of
energy
11
○ Acts as a gap between p and n type
○ If there's a gap, the circuit is open→ no flow of
electricity.
● The amount of electrons is proportional to the amount of
photons hit
Photodiode (PDA)
● Arrangements of several photodiodes
● absorption of radiant energy by a reverse-biased
pn-junction diode (pn, positive-negative) produces a
photocurrent that is proportional to the incident radiant
power
● not as sensitive as PM, but with excellent linearity, speed
and small size
● A specialized diode which is sensitive to light. ● Specially used in post sample filter.
● It has excellent linearity ● If the monochromator is located before the cuvette =
○ Linearity - even at low light, the voltage also pre-sample filter
constantly increase in a linear fashion. ○ If after the cuvette = post sample filter
● Excellent response time ● U could arrange the arrays so that each of the colors are
● Small size being hit with only one wavelength.
● Spectral analysis.
Read-out device
● displays the amount of light transmitted
● examples:
○ Analog
■ needle along a scale
○ Digital
■ microprocessor to display results
using light-emitting diode (LED) of
liquid crystal display (LCD)
○ Recorder
■ strip chart or an integrator giving out
tracings
● Depletion Or intrinsic region

PART THREE
can now deduce if the spectrophotometer is
QUALITY ASSURANCE IN SPECTROPHOTOMETRY giving off tama na wavelength
● Wavelength or photometric accuracy ○ Accuracy = the closeness of a measurement to
● Absorbance check its true value
● Stray light
● Linearity Absorbance Check
● Making sure that the spectrophotometer is working ● Performed using glass filters or solutions that have
properly known absorbance values for a specific wavelength
● How to measure?
Wavelength or Photometric Accuracy ○ Use a specific glass filter again or a solution
● Implies that a photometer is measuring at the wavelength that has a known absorbance to check if the
that it is set to spectrophotometer corresponds to the
● Using special glass-type optical filters absorbance value as stated by the
(Examples:didymium and holmium oxide) manufacturer’s solution
● How do we know that spectrophotometer is really giving

off 340 nanometer?
○ We have to calibrate
○ We measure it against a standard
■ Didymium glass
● Which has a peak
absorbance at 600
nanometers
■ Holmium oxide
● Has multiple absorption
peak with a sharp peak
occurring at 360
nanometers
○ Using these special glass type optical filters, we
12
Linearity ● Remember: Percent transmittance is always less than
● Ability of a photometric system to yield a linear 100
relationship between the radiant power incident upon its
detector and the concentration (i.e., Beer’s law)
● There should be a 1 to 1 correspondence absorbance
and concentration
○ As the concentration of the analyte in the ● Transmitted light should always be less than or equal to
solution increases, the absorbance must also the incident light j
increase
● Linearity is ideal but we could not achieve linearity in Absorbance
practical sense ● Amount of light absorbed
○ If the concentration is too much, then the ● It cannot be measured directly by the spectrophotometer,
absorbance could not anymore increase but but rather is mathematically derived from the percent
rather it will plateau transmittance
■ Plateauing = non-linearity; does not ○ A = -log(%T)
anymore follow the line ○ A = log 100% - log (%T)
○ A = 2 - log (%T) → formula
■ This is already programmed in your
spectrophotometer
■ Spectrophotometer will determine the
percent transmittance and will derive
the absorbance
■ Mathematical relationship of the
absorbed light and the transmitted
light
● You can’t directly measure absorbed light because you
cant put an instrument inside the cuvette
● If the analysis is linear, the better
Beer’s Law
● But at some point, if the analyte is too much, it might
deviate from that linearity ● Absorbance is directly proportional to the concentration
● In some textbooks, it's actually A = abc
○ A - absorptivity
Stray Light
○ But, if the unit is in moles (molar), we use the
● Any light that impinges upon the detector that does not greek letter eta
originate from a polychromatic light source ● Absorbance is directly proportional to the concentration
○ Ex. if the spectrophotometer’s light is leaking (vice versa)
○ Make sure that the compartment that houses ○ The higher the concentration, the higher the
the spectrophotometer is protected from light absorbance and the lower the concentration,
bcs your photodetector might detect that stray the lower the absorbance
light and will create noise sa imong signal.make
deviations from the true value A = 𝜀𝑏c
● EMR that reaches the detector but is outside the narrow Where:
bandpass set by the monochromator ● 𝜀 - molar absorptivity
● Have a significant impact on any measurement made ● b - length of light path
○ Because we are measuring the concentration of ● c - concentration
the analyte by measuring the transmitted light
○ If we have additional light that reaches the ● Molar absorptivity - characteristic of analyte; ability of
photodetector, it might be miscomputed by your analyte to absorb light; measure it numerically
spectrophotometer as additional concentration ○ Molar absorptivity of a given analyte is constant
or false decrease in the analyte ○ Ex. Glucose of DNS
■ glucose has its own molar absorptivity
PRINCIPLES OF SPECTROPHOTOMETRY which is constant regardless of the
concentration of glucose -- even if
Beer’s Law (Beer-Lambert Law) con. is high or low, MA is constant
● The concentration of a substance is directly proportional ● Length of light path - the path of light; path that light
to the amount of light absorbed and inversely travels inside the cuvette; coming from the incident light
proportional to the logarithm of the light transmitted and when the transmitted light first goes out of the wall of
● Law that guides the principle of spectrophotometry the cuvette
● 3 lights of concern: ○ In any given analysis of spectrophotometer, the
○ Incident light - coming from the light source to length of light path is also constant because it
the cuvette doesn't make sense that cuvette will change in
○ Absorbed light shape or length (1cm forever)
○ Transmitted light ● Concentration - not constant
● Directly proportional = the higher the concentration, the ● Absorbance is proportional to the concentration;
higher the absorbed light absorbance is dependent
● Inversely proportional = whenever the concentration is
high, the transmitted light is low and vice versa. Standard
● A solution containing a precisely known concentration of
Percent Transmittance an element or a substance
● The ratio of the radiant light transmitted divided by the
𝐴𝑢 𝐶𝑢
●
radiant energy incident to the sample
Tells u how much in percent of the incident light passed 𝐴𝑠𝑡𝑑
= 𝐶𝑠𝑡𝑑
through the cuvette
𝑡𝑟𝑎𝑛𝑠𝑚𝑖𝑡𝑡𝑒𝑑 𝑙𝑖𝑔ℎ𝑡
%𝑇 = 𝐼𝑛𝑐𝑖𝑑𝑒𝑛𝑡 𝑙𝑖𝑔ℎ𝑡
𝑥 100 ● Standard solution - solution that has known concentration
○ Ex. Glucose std = 100mg/dl
13
○ Sample (unknown) put in the ● Spits or chops the monochromatic beam of radiation
spectrophotometer, you’ll get the A u into two components:
○ Standard, put same amount reagent, and then ○ One beam passes through the sample, and the
spectrophotometer, you’ll get the A std other passes through a reference solution or
○ 100 mg/dl = Cstd blank
○ Compute for the Cu ● Two designs:
○ Double beam in space
○ Double beam in time
Ex.
● Sample (Au) = 0.5 Review:
● Std (Astd) = 0.25 ● What really happens inside the Clin Chem Lab when you
● Cstd = 100mg/dl do spectrophotometric analysis
○ Extract patient sample
𝐴𝑢 𝐶𝑢
𝐴𝑠𝑡𝑑
= 𝐶𝑠𝑡𝑑
○ Process blood sample to get serum/plasma
○ 1st cuvette: Serum + reagent → rxn
0.5 𝑥 ○ 2nd cuvette: Standard + reagent → rxn
0.25
= 100𝑚𝑔/𝑑𝑙 ○ 3rd cuvette: blank
0.5 (100mg/dl) = 0.25x ● To do it manually
200mg/dl = x ○ First, measure the absorbance of the blank
○ Assuming your blank is 0.02, depending on the
The amount of glucose in the plasma of the patient is spectrophotometer
200mg/dl. ○ There are certain spectrophotometers that after
you read the absorbance of the blank, all you
have to do is press “return to zero”
○ Everything that is being read is automatically
Blank
deducted by 0.02
● Solution containing no analyte of interest, usually used to ○ After deducting and returning to zero, you
calibrate instruments read the absorbance standard
○ Reagent blank ○ Remove the cuvette that houses the standard
○ Water blank and then you put the next cuvette with the
○ Air blank serum + reagent = 0.25
● Let's say you have a weighing scale, how will you weigh ○ Do the computation of
a baby?
𝐴𝑢 𝐶𝑢
○ You can step on the weighing scale while 𝐴= 𝐴 𝑠𝑡𝑑
= 𝐶 𝑠𝑡𝑑
carrying the baby -- 100kg
○ And weigh yourself without the baby -- 94kg ○ In a single beam spectrophotometer,becuse
○ 100kg - 94kg = 6kg weight of the baby there is only one slot ofr the cuvette, you need
○ How you measure the baby, without directly to place it one by one
measuring the baby
● Ex. 2 You want to measure fish in the timbangan
○ You can directly place the fish in the timbangan
but you have to have a holder
○ Measure the weight of the holder first, then
measure it with the fish
○ Subtract the weight of the holder with the total
weight of both the holder and the fish to know
the weight of the fish
● Ex. 3 Water blank - just water and nothing else
○ In your analysis, you have to add d. Water
■ The water itself absorbs a particular
wavelength
■ Sample + reagent (reagent contains
water)
○ In a given incident light absorbed, and the Double Beam in Space
absorbance is 0.90
○ When you use water blank, with the same
wavelength
■ Absorbance of just water is 0.01
○ To know the absorbance of the sample and the
reagent, without the water, you have to subtract
○ 0.90 - 0.01 = 0.89 true absorbance of the
sample and the reagent (w/o water)
● Ex. 4 Reagent blank - use reagent with no sample
○ Reagent - test kit; a chemical you add to your
sample to generate a reaction to measure the
analyte
○ After measuring, the absorbance is 0.56
○ Then, you do a regent blank and measured the
reagent as 0.06
○ The true absorbance of the sample is not 0.56
but minus ang blank 0.50
● In reality, once you loaded the blank in your ● Has two photodetectors
spectrophotometer, it will automatically subtract the ● There is a light source but there is a beam splitter
absorbance of the blank ○ Splits the beam into the sample and the other
○ Blank will depend on the analyte goes to the standard cuvette
DOUBLE-BEAM SPECTROPHOTOMETRY ○ Using two light sources is prove to error since
14
you cannot ensure that both sources will emit
the same intensity of light
○ After reading the absorbance, the
spectrophotometer will automatically compute
for the concentration
● used for Group 1 metals (1+ charge)

● Na+ , K+ , Li+
○ ‘excitable’ metals that emit a specific light
● Na+ – emits yellow light (589 nm)

Double Beam in Time ● K+ – emits only violet light (367 nm)
● Li+ – transmits only red light (767nm)
Components of FES
● Flame
○ breaks the chemical bonds to produce atoms
○ source of energy that will be absorbed by the
atoms to enter the excitation state
○ also serves as the cuvette
■ No cuvette
● Atomizer
○ breaks up the solution into finer droplets so that
the atom will absorb heat energy from the flame
and get excited
● There is only one photodetector ○ Does not produce atoms
● There are still double beam ○ Smaller droplets are easier to burn
● But because there is a single photodetector, we have to ● Burner
use a rotating chopper ○ types:
● It is a mirror that the spectrophotometer rotates so that if ■ total consumption burners
you want to read the sample cuvette’s absorbance, yan ● the sample is aspirated
ang i bounce sa photo detector and once it roatttes, it ill directly into the flame
block the light form the sample cuvette and read the light ● the flame can be made
from the standard cuvette hotter but there is the
production of large droplets
FLAME EMISSION SPECTROPHOTOMETRY in the flame
● Or Flame Emission Photometry ■ premix burner
● Sometimes FES or FEP ● the sample is atomized
● The measurement of emitted light when electrons in an before entering the flame
atom become excited by heat energy produced by the and does not create noise
flame ● gravitational feeding of
● Excited atoms return to the ground state by emitting light sample into the flame
energy that is a characteristic of that atom ● Interference Filter
● When light strikes an electron, when a given wavelength ○ Filter is found after the flame
of light is being absorbed by the electron, the electron ○ Na+ filter – transmits only yellow light (589 nm)
becomes excited ○ K + filter – transmits only violet light (367 nm)
● After the excitement, it relaxes ○ Li + – transmits only red light (767nm)
● Once the electron relaxes back to its original level, it
reemits the light it has taken up previously
● That particular emission of light is what we measure for
us to know the concentration of the given element
● Main parts of FES:
○ Sample injection Port
○ Acetylene Flame Source ● The more violet light, the higher the potassium is
ATOMIC ABSORPTION SPECTOPHOTOMETRY

● measures concentration of element by detecting
absorption of light (EMR) by atoms
● elements are not excited but they are dissociated from
their chemical bonds and placed in the unionized,
unexcited ground state
● AAS is used for group 2 metals
○ Calcium and Magnesium
15
● used for Group two metals (2+ charge)
● not easily excited but can absorb light
○ Higher temperatures are required for them to
be excited
● Just like Sodium, Potassium, and Lithium wherein they
can absorb light so the electrons are excited but once
they are in a relaxation state, they re-emit the same
amount of light they absorbed
● We have a:
○ Burner
○ light source (hollow cathode)
○ chopper which turns a single beam into a
pulsating beam (it closes the beam to be turned
on and off)
○ Monochromator which will select a particular
wavelength of light after the burner (just like
how we put the interference filter in FES)
○ PM tube is protected by the monochromator
● We have two sources of light going to the PM tube
○ 1st light: light coming from the excited atoms
■ But, it is unexcited if group 2 metals
● Serum aside from
containing magnesium and
calcium, it also contains
potassium and sodium
● Components ● so it is inevitable to have
○ Burner excitation in this flame (acts
■ uses a flame to dissociate the as an FES which emits light
chemical bonds and form free coming from the excited
unexcited atoms atoms)
■ serves as the cuvette ● It is a problem since we
○ Monochromator don't want that emitted light
■ selects the desired wavelength from a coming from excited atoms,
spectrum of wavelength but the absorbed light from
■ Because it is located after the burner, those unexcited atoms in
it is also a post-sample filter Group 2
■ Function: serves to protect the ○ 2nd light: Hollow cathode lamp
photodetector from excessive light ■ Light source
coming from the flame ● If the chopper closes and cuts off the light from the light
■ Why? source
● PM tube burns out ○ So, the PM tube detects (A) light emitted from
whenever exposed to the excited atoms
excessive light ● If the chopper is open, the PM now detects both lights
○ Photodetector emitted from the excited atoms and hollow cathode lamp
■ Photomultiplier (PM) tube ○ The amount of light being absorbed is
○ read-out device indirectly proportional to the transmitted light
■ The amount of light that is being ● What the computer does is to subtract the detection
absorbed by the Group 2 metals amount of light when the chopper is open (1+2) with
● Ex. The higher the when the chipper is closed (1)
magnesium, the more light ○ The readout device will only give you the
is absorbed amount of light that is emitted by the light
■ The transmitted light that reaches source (=2)
your photodetector is low ● We are basically eliminating the light coming from the
Group 1 metals (excited atoms)
● Interferences
○ Chemical
■ situation at which the flame could not
dissociate the sample into neutral
atoms.
■ Ex. calcium
● Calcium phosphate
○ U put something
into the serum first
so that it could
dissociate into
calcium molecules.
○ ionization
16
■ situation at which atoms in the flame
become excited and emits energy.
○ FES is obsolete as well as AAS (ref method).
Types of Photometric Instruments
● Spectroscope
● Colorimeter
● Photometer
● Spectrometer
Spectroscope
● an optical instrument used for visual identification of
atomic emission lines
● has a monochromator, (prism or diffraction grating)
● exit slit is replaced by an eyepiece that can be moved
along the focal plane.
● Direct it in your eye and detect the spectral line.
Colorimeter
● Matamater
● Subjective
● uses the human eye as the detector
● user compares the observed color of the unknown
sample against a standard or a series of colored
standards of known concentrations
● Ex: urine dipstick
Photometer
● consist of a light source, a filter, and photoelectric
transducer, as well as a signal processor and Readout.
○ Photoelectric transducer = photodetector
● some manufacturers use the term colorimeter or
photoelectric colorimeter
● use filters for isolation of specific wavelengths, NOT
gratings or prisms
Spectrometer
● an instrument that provides information about the
intensity of radiation as a function of wavelength or
frequency
● spectrophotometer are spectrometers equipped with one
or more exit slits and photoelectron transducer.
● Detects whole spectrum of light.
● No monochromator.
17
LECTURE 5: LUMINESCENSE
Prof. JC LOuise Bandala, RMT
September 6, 2021
LUMINESCENCE both electrons get combined, like a magnet = triplet state

● is the emission of light by a substance ● In molecule, there are several electronic levels (electrons
● occurs when an electron returns to the electronic ground are present at the ground state) in the singlet state
state from an excited state and loses its excess energy (opposite spin)
as a photon ● Fluorescence phenomenon
● Three types: ○ When molecules absorb UV or visible light, one
○ Fluorescence electron from ground state will go to an excited
○ Phosphorescence state
○ Chemiluminescence ○ Excited state is having several vibration levels ,
There are substances that when u give heat, it will absorb the heat excited electron loses energy by intermolecular
and gets excited away from its normal orbit and jump off another collision and comes to the lowest vibration level
orbit. Pero dili man niya forever i carry ang certain heat/energy na ○ It then returns to ground state by emitting the
nakuha niya. Thus, there will come a time na mubalik siya didto sa radiation or light of lower energy or higher
iyang orbit. Pagbalik niya sa original na orbit, kailangan niya i give wavelength
off ang energy. Upon giving off that energy, mao to ang light ○ Instant re-emission of light
emitted. ○ Energy of radiation emitted is always less than
the energy of radiation absorbed
FLOURESCENCE ○ Wavelength of emitted radiation will always be
● When a beam of light is incident on certain substances, higher than the wavelength of radiation
they emit visible light or radiations absorbed
● It starts immediately after the absorption of light and ● Phosphorescence
stops as soon as the incident light is cut off ○ Inversion of electron spin takes place
○ When the energy from UV or visible light is
absorbed, the electron from the ground state
jumps to excited state, at the same time the
spin of electron gets reversed and it is now in
triplet state (same spin)
○ Pairing of electron with same spin is not
favorable thus it takes more time to reverse the
spin and come back to ground state
○ Delay re-emission of radiation
○ The energy of radiation emitted is always less
● Upon directing that light into your substance, it will than the energy of radiation absorbed
immediately absorb it. ○ Wavelength of emitted radiation is higher than
● By the time na tanggalon ang light source, mawala lang the wavelength of radiation absorbed
pud ang incident light
PHOSPHORESCENCE
● Also known as Delayed fluorescence
● When light radiation is incident on certain substances,
they emit light continuously even after the incident light is
cut off, it will remain.
● By the time na naay light na irender padulong sa

substance, iabsorb to niya na energy. ● Fluorescence is immediate emission of light;
● By the time we remove the source, it will continue to glow phosphorescence is delayed
or absorb the light (longer period of time) ● Process that involves transition of electronic state (singlet
○ But if you give it some time, it will go back to its and triplet state) is called intersystem crossing
original appearance ● Having different state of spin is the reason why
○ fluorescence - immediate phosphorescence takes time (delay)
○ Phosphorescence - minutes or days
● Difference between phosphorescence and fluorescence HOW TO MEASURE FLUORESCENCE?
○ Instant re-emission of the absorbed energy is ● Fluorometry
called fluorescence ○ Measures the fluorescence or the energy
○ Delayed remission of the absorbed energy is emission that occurs when a certain compound
called phosphorescence absorb electromagnetic radiation, become
excited and then return to an energy state that
● pair of electrons have opposite spin = singlet state is usually higher than their original level
● Electrons having the same spin, magnetic moment of ○ The emitted fluorescent light has longer
1
wavelength and lower energy which is due to for both absorption and fluorescence
the energy lost between the time when the ○ Fluorescence measures the amount of light
photon is absorbed and when it is emitted intensity present over a zero background
○ Emitted light has lower wavelength and lower ○ Zero background- lesser interference occurs
energy - due to energy loss that happened Disadvantages
between the time it absorbed photon and ● Very sensitive to environmental changes
energy is emitted ● Quenching- quick disappearance of fluorescence
○ Changes in pH affect electron availability
Basic Components of Fluorometry ○ Temperature changes the probability of loss of
energy
● Light source ○ Contaminating chemicals or a change of
○ Mercury vapor lamp (commonly used) or solvent may change the structure
xenon arc lamp ○ UV light used for excitation can cause
● Excitation/Primary Monochromator photochemical changes especially if dili gina
○ Selects the wavelength that is best absorbed calibrate every now and then
by the solution to be measures ○ Fluorometry is not often used nowadays
○ Monochromator: one single color; from a wide
range of colors, it narrows down an individual CHEMILUMINESCENCE
color or wavelength selected ● Is the production of light from a chemical reaction
● Cuvette ○ From the word “chemi”- there is a chemical
● Emission/Secondary Monochromator reaction that happened inside
○ Filters out fluorescence form stray light ● Reactions are oxidation reactions of luminol, acridinium
radiation steers, and dioxetanes characterized by a rapid increase
○ Position at a right angle from the cuvette to in intensity of emitted light followed by a gradual decay
eliminate potential interference from the ○ Other examples na most common: oxygen,
excitation light hypochlorite, hydrogen peroxide
○ What we are trying to capture is the emitted ● The excitation of the substance does not involve
light already electromagnetic radiation and no monochromators are
● Photodetector needed, instead, the excitation energy comes from a
○ The commonly used photodetector is the PMT chemical or electrochemical reaction
or Photo Multiplier Tube ○ The most basic difference between
○ Because it has the highest sensitivity compared fluorescence, phosphorescence, and
to other photodetectors chemiluminescence
○ In chemiluminescence, the presence of
radiation is not necessary, in measuring light
absorbed dili kailangan ug monochromator
purely chemical reaction lang
● The light signal is measured against a completely dark
background
○ Nganong kailangan i break ang glowstick?
■ Naa duha ka compartment
■ Naay inside and outside na portion
■ Each compartment has different
chemicals inside
■ That is why it is necessary to break
the glowstick para naay interaction
between those chemicals presence
creating a glowing light
(VIDEO)
● There are reactions where light is emitted without the
emission of a considerable amount of heat. This
● The secondary filter is positioned at a 90-degree angle phenomenon is called cold light or chemiluminescence
● Attenuator - another term for entrance slit ● In the following experiments chemiluminescence
● Why is positioned at 90 degrees? accompanying the oxidation of luminol is demonstrated.
○ Remember your transmitted light, and diba dili ● A solution of ammonia water NH3+h20 with potassium
transmitted light ang atong gina try measure diri tricyanocuprate(I) K2 (Cu(CN)3) addition has been
buy tour emitted light prepared
○ The emitted light is not directly released from ● To oxidize the luminol solution of hydrogen peroxide,
your sample holder H2O2 is added to the mixture
○ Examples of substances that can absorb light ● A more generous amount of H2O2 added
and can cause fluorescence ● Luminol was oxidized by hydrogen peroxide, energy was
■ POPOP- phenyloxizolebenzene emitted in the reaction as light quanta
■ Quinine ● Several consumer goods based on luminescence are
■ Fluorescein available on the market
■ Acridine Orange ● Different light colors are obtained with different dyes
■ Rhodamine B ○ Blue- 9,10-Diphenylentracene
■ Pyridine 1 ○ Orange- 5,12-Bis(phenyl ethynyl)naphthacene
○ The term for the atom molecule that can cause ○ Red- Rhodamine B
fluorescence is a fluorophore ○ Yellow-green-1-Chloro-9,10-bis(phenyl
ethynyl)anthracene
Advantages ● Chemiluminescence can be observed in nature. For
● Increased sensitivity (1000x more sensitive than example, the green glow of the common glow-worm is
spectrophotometric methods) known
○ Emitted radiation is measured directly ● Luciferin is the dye here, activated by enzyme luciferase
● Increased specificity by selecting the optimal wavelength and air oxygen
● ADVANTAGES:
2
○ Subpicomolar detection limits
○ Speed (Fast)
■ Flash-type reactions
■ Light s measured for 10 seconds
○ Ease of use (mix of chemicals only)
○ Simple instrumentation
● DISADVANTAGES:
○ Impurities can cause a background signal that
degrades sensitivity and specificity
NEPHELOMETRY AND TURBIDIMETRY

● Light scattering is a physical phenomenon that results
from the interaction of light with particles in solution.
● Unlike fluorescence emission, the wavelength of the
scattered light is the same as that of the incident light.
NEPHELOMETRY Nephelometry Turbidimetry

● measures the amount of light scattered in a particulate Mercury arc lamp Tungsten lamp
suspension at 90-degree angle Scattered light is measured Light transmitted is measured
● useful method to determine the concentration of solutions
Measured at 90 degrees Measured in straight line
that contains particles too large for absorption
PMT is the detector Photocell is the detector
spectrometry
○ Applicable to both 4
● Too large particles but very low concentration FACTORS THAT AFFECT SCATTERED LIGHT
○ If light is introduced, it tends to scatter it out ● Particle size
● The detecting cell is placed at right angles to the light ○ The smaller the particles, the scattered the light
source, to measure light scattered by particles. The ● Concentration of particles
intensity of the scattered light savers as a measure ● Molecular weight of particles
of the turbidity. The instrument is called a ● Wavelength dependence
“Nephelometer” or a “Nephelometric Turbidimeter”. A
spectrophotometer can be used, however, a special THREE TYPES OF SCATTERED LIGHT
attachment is required for nephelometry.
Rayleigh
TURBIDIMETRY
● Turbid - cloudiness or haziness of the solution
● measures the amount of light blocked in a particulate
suspension
○ ↓ in light transmission
● Amount of light blocked depends not only on
concentration but also on the size
● Sampling handling becomes critical
○ Since the particles in the sample are too large
and tend to aggregate with each other, so dali
mag settle down ang suspension ● Wavelength of light > particle size
○ So before measuring the substances, we really ● Light symmetrically scattered around the particle.
need to mix samples well first to prevent ● Answers as to why the sky is blue.
miscalculation ● Amount or intensity of the scattered light is inversely
● Thou large particles, its scattering is extensive = hazy proportional to the 4th power of the wavelength of a
sample - high concentration wave.
○ Once light is present, it will not be able to pass ● If the wavelength increases, intensity will be lower.
through because of the haziness of the ● Lesser wavelength, more scattering.
suspension
● The amount of light passing through a solution is Mie
measured. The higher the turbidity, the smaller the
quantity of light transmitted (i.e. more light i absorbed).
Any spectrophotometer or photometer can be used as a
turbidimeter, without modification. Since property
concerns visible light the measurement is commonly
carried out at 420 nm.
● Wavelength of light < particle size

● Light scatters backward but appears forward due to
destruction out of phase background scatter
● Explains why the clouds are white
● Light scattering in all directions.
Rayleigh Debye
3
● Wavelength of light = particle size
● More forward light scatter
● Antigen-antibody reactions
● Mas daghan ang scattered light sa rayleigh debye
compared to Mie
4
LECTURE 6: CHROMATOGRAPHY AND MASS

SPECTROMETRY
Prof. JC LOuise Bandala, RMT
September 6, 2021
CHROMATOGRAPHY GAS CHROMATOGRAPHY

● is an analytical technique commonly used for separating ● separating compounds based primarily on their volatility
a mixture of chemical substances into its individual ○ Volatility = easily evaporated at normal
components, so that the individual components can be temperature
thoroughly analyzed ● is useful for compounds that are naturally volatile or can
○ All about separation be easily converted into a volatile form
○ Chrome = color ○ Thus, dili tanan compounds pwede gamiton ug
gas chromatography
BASIC COMPONENTS OF CHROMATOGRAPHY ● Two types:
● Mobile phase or carrier ○ Gas-Liquid Chromatography: based on partition
○ Gas or liquid ○ Gas-Solid Chromatography: based on
○ Solvent moving through the column adsorption
○ Carries the sample
● Stationary phase or absorbent
○ Solid or liquid
○ Where the mobile phase flows
○ Does not move; fixed
● Column – holds the stationary phase
● Eluate – separated components
● Eluent – Fluid or substance that enters the column and
moves the analyte; help in the movement of analyte
● Elution – The process of washing out a compound
through a column using a suitable solvent
● Analyte – Mixture whose individual components have to
be separated and analyzed
● Retention time or factor – The time it takes for a
compound or analyte to elute
● Butanganan = column
● Stationary phase = Materials inside (beads) ● Based on the vid, your sample is being evaporated
● Mobile phase = what will pass to ur column carrying ur ● May collector sa may detector
analyte ● FID = Flame ionization detector
● Analyte = shaded violet sa top ○ able to detect whatever sample was vaporized .
● Once u place your analyte, and then naa moy makita na ● Any changes na mahitabo sa FID is being recorded
white na part (eluent) na in order for your sample to ● Peak = referring to the concentration of your sample
move and start being separated
● After some time from your original analyte, your BASIC COMPONENTS OF GAS CHROMATOGRAPHY
components will now separate
● Columns
● In terms of who comes out first and who comes out last,
○ Packed columns or Capillary columns
it would depend on what are the substance that u used
○ Glass or stainless steel (packed) or thin-fused
● Chromatographic techniques may be classified according
silica (capillary)
to their mobile phase:
○ Packed columns are filled with inert particles
○ Gas chromatography
such as diatomaceous earth or porous polymer
○ Liquid chromatography
or glass beads coated with a nonvolatile liquid
(stationary) phase
1
○ liquid stationary phase must be nonvolatile at gas and the sample gas which produces an
the temperatures used, must be thermally electrical signal unique to the compounds being
stable, and must not react chemically with the analyzed
solutes to be separated ○ This signal is proportional to the concentration
■ May cause wrong detection of certain of the sample components provided a direct
substances means of measuring component concentrations
○ Packed column in a particular sample and form this you receive
■ Packed inside a chromatogram that represents the
■ Packed up in the hollow portion (could components found in the sample analyzed
be bead column or porous column ● Additional input on how TCD work:
layer) ○ Wheatstone bridge - measures unknown
■ Used for gas-liquid chromatography resistance values in the TCD
or gas-solid chromatography ■ Can also be used for calibration of
■ Greater sample capacity different instruments
○ Capillary column ■ Important in the TCD
■ Open column ○ Carrier gas used is helium
■ Hollow part in the column is empty ○ 2 entries
■ There's coating of the sides of column ■ One where the sample enters and the
■ Used only for gas liquid other for standard reference
chromatography ■ As your reference and sample enters
■ Can provide higher separation each compartment, once we
efficiency compared to packed introduce heat, sample components
column have lower thermal conductivity
■ Can be overloaded easily compared to reference
■ Because of difference in thermal
TWO DETECTORS conductivity, it will create changes in
● Thermal conductivity the electrical resistance detected by
○ Contains wires (filaments) that change the filament
electrical resistance with change in temperature ■ That electrical change is directly
● Flame ionization detector proportional to the concentration of
○ More sensitive that TC detectors your analytes causing peaks in your
○ Small hydrogen flame and collector electrode chromatogram
■ Collects specific particle or molecule
○ As the sample burns, ions form and move to
the charged collector
● Flame Ionization Detector

○ Important component: have a hydrogen supply
○ Sample inlet - where your sample enters
○ Collector electrode
○ Flame ignitor
○ Almost universally employed, where the flame
commonly is generated with hydrogen and air
○ The autosampler provides the means to
introduce a sample automatically into the inlets
■ Manual insertion is also possible
○ The column inlet or injector is attached to the
column head and provides the means to
introduce a sample into a continuous flow of
carrier gas
○ In the injector, a sample is introduced to a
● How does a TCD work? heated chamber via a syringe through septum
○ Each gas enters the gas the gas ○ Heat facilitates volatilization of the sample and
chromatograph instrument separately sample matrix
○ The sample goes into one column while the ○ Carrier gas then either sweeps the entirety
pure carrier gas goes into another column splitless mode or a portion split mode of the
○ Electrically heated resistance wires are located sample into the column
in chambers inside of the TCD ■ Split mode - a part of the sample
○ Power supply provides a current to the carrier gas mixture in the injection
resistance wires which causes the wires to heat chamber is exhausted through the
up split vent
○ The electrical circuitry shown here is a ■ Splitless mode - all the sample carrier
characteristic of thermal conductivity detectors gas mixture in the injection chamber
(TCD) which is known as the wheatstone bridge is transported through the column
○ As the gas flows through the TCD, the physical ○ 2 types of column:used in GC
properties of the reference and sample gases ■ Packed column - where the stationary
(ex. Specific heat capacities) will allow the phase is coded directly in the column
wired of the TCD to be cooled at different rates, ■ Capillary column - where the
this change in temperature will result in a stationary phase is coded with the
change in resistance from both the reference inner wall of the column
2
○ Mixture separation is based differential Types of Separation Chromatography
partitioning of components between the mobile ● Adsorption
and stationary phases ● Partition
○ The component with less affinity to the ● Steric exclusion
stationary phase, consequently less interaction ● Affinity
travels faster and diluted out first ● Ion-exchange
○ The component which has more affinity to the
stationary phase, consequently more Adsorption
interaction travels slower and diluted later
● Liquid-solid chromatography
○ In addition, other factor influence the separation
○ Adsorption- adhesion of atoms or your
of the components such as column
molecules to a certain surface
temperature, carrier gas flow rate, column
○ First word= mobile phase
length, amount of material injected
○ Second word= stationary phase
○ As compounds elute from the column, they
● Competition between the sample and the mobile phase
interact with the detector
for the adsorptive sites on the solid stationary phase
○ Different detectors can be used:
● Stationary phase can be acidic polar (e.g., silica gel),
■ Flame ionization detector
basic polar (e.g., alumina), or nonpolar (e.g., charcoal)
- Based on the detection of
○ Depends on the analyte of interest kug asa siya
ions formed during
better mag adhere
combustion of organic
● Disadvantage: strong retention of many compounds by
compounds in a flame which
the supports, making them difficult to elute from the
generated by hydrogen and
column
air
- To detect these ions, 2
electrodes are used to
provide a potential
difference
- The positive electrode
doubles as a nozzle head
where the flame is produced
- The negative electrode is
positioned above the flame
- When another compound is
mixed with the hydrogen
flame, mainly carbon ions
are generated (VIDEO)
- Current is produced ● Based n the fact that certain solid materials known as the
between the electrodes adsorbents have the ability to hold molecules on the
proportionally to the amount surface by the phenomenon of adsorption
of organic compound ● The adsorption process usually involves attracted forces
present like Van der wals interaaction and hydrogen bonding
- This current is measured ● One of the common adsorbent ifs silica
with an electrometer ● Silica has silanol, Si-OH functional group
amplified into proper voltage ● The Si-OH group interacts with other functional groups
and fed into an integrator of the sample molecules
- Number of peaks indicate ● Different sample molecules bind the absorbent with
how many components are different affiinitty
in the mixture ● Hence when the mobile phase passes the molecules
■ Thermal conductivity detector having less interaction with adsorbents are release first
■ Mass spectrometer detector while the molecules having the most interaction will be
○ X-axis of gas chromatogram shows the amount released last
of time taken for the analytes to pass through ● Adsorption, adherence, affinity to stationary phase
the column and reach the FID detector
○ Y-axis (area of the peak) - a reflection of the Partition
amount of a specific analyte present ● Liquid-liquid chromatography
● Separation of substances according to their solubility in
LIQUID CHROMATOGRAPHY an organic/non-organic polar solvent and in an
● Uses lower temperatures for separation achieving better aqueous/polar solvent
separation of thermolabile compounds ● “Like dissolves like”
○ Thermolabile- compound is unstable when ● Polar molecules remain in the aqueous solvent; nonpolar
heated and they are readily destroyed or molecules are extracted in the organic solvent
deactivated when introduced to heat
● Easier to recover a sample compared to GC
● Commonly used and suitable alternative than GC
○ Because not all compounds are not volatile.
Some are too unstable, insufficiently volatile to
be assayed using your gas chromatography
● The mobile phase can be removed, and the sample can
be processed further or reanalyzed under different
conditions
● Kaya siya tinatawag na paper chromatography because

the support used is a paper
3
● Instead of using a marker, what is used is a leaf extract in Resin Type Cation Exchanger Anion Exchanger
a paper Net charge of a
● Paper is the support and the leaf extract is the liquid molecule of interest + -
● The other liquid is the propanone (acetone) which will
serve as your mobile phase that will carry your analyte Change of resin - +
and then such time we see a reaction
● Solvent front refers to the distance moved by your Affinity
solvent from its starting point to the end ● most selective type of chromatography employed
● Observe for the solubility ● utilizes the specific interaction between one kind of solute
● The more soluble the component, the faster the molecule and a second molecule that is immobilized on a
movement, the greater the distance travelled stationary phase
● Since lahi-lahi man siya ug solubility, thuss, a separation ● Examples: antigen and antibody, enzyme and substrate,
will occur receptor and ligand, protein and nucleic acid
● Ethanol, methanol, or chloroform canalso be used as ○ Similar to lock and key
your mobile phase ○ Have specificity
Steric Exclusion
● Variation of L-S chromatography
● A.k.a. Size exclusion chromatography
● Separation based on size and shape
● Solid-phase is packed with porous material (beads) that
separates solutes according to size
● Similar to enzymes (specific)

(VIDEO)
(VIDEO) ● Affinity Chromatography occurs based on the specific
● No adsorption interaction between the two molecules
● No interaction between the molecules and the surface of ● This specific interaction can be between enzyme and the
the porous particles substrate, receptor and ligand, protein and nucleic acid,
● Size exclusion chromatography separates molecules etc
based on their size Components of Affinity Chromatography
● Smaller particles have a longer residence time in the ● Carried out in column which is filled with the supporting
column and larger molecules have a shorter residence material called matrix
time ● Matrix
● Longer column ○ Chemically inert
● SEC can be used for protein desoldering ○ Good flow
○ Salt ions are small and can enter into the pores ○ Have functional groups for the covalent binding
and gets longer residence time of the ligand
○ Proteins will have a shorter residence time ○ Agarose, polyacrylamide, polystyrene,
Residence time = retention factor cellulose, silica
● SEC vs Adsorption chromatography ● Matrix is attached with a space m which is usually made
○ SEC has no affinity up of Ch2 group
● The presence of space prevent the non-specific
Ion Exchange interaction of the ligand with the matrix itself
● use of a resin (stationary solid phase) for covalent ● Molecules that passed through the column and did not
attachment of anions or cations onto it bind will be washed away
● Solute ions of the opposite charge in the mobile liquid Removal of a molecule of interest from the column
phase are attracted to the resin by electrostatic forces ● Buffer with different pH or ionic strength is passed
● Widely used for the separation of proteins, peptides, and ○ This releases the molecule of interest and will
nucleic acids be obtained in a pure form
○ Proteins have charge ● Competitive inhibitor
○ Can be removed by dialysis or by changing the
pH od ionic strength
4
● As a result, efficiency of separation increases giving high
CHROMATOGRAPHIC PROCEDURES resolution
● Thin-layer chromatography ● Components;
● High-performance liquid chromatography
○ most recommended because it has highest
sensitivity
THIN-LAYER CHROMATOGRAPHY
● Variant of column chromatography
● A thin layer of sorbent, such as alumina, silica gel,
cellulose or cross-linked dextran, is uniformly coated on a
glass or plastic plate
● Most commonly used as a semiquantitative screening
test
○ column: made up of stainless steel which can
withstand a very high pressure upto 50 MP;
length can vary from 5 - 25 cm and have an
internal diameter of 4.5 mm
■ The flow rate from the mobile phase
to the column is usually 1-3 mL/min
● Almost the same with paper chromatography

● Mobile phase: where we put the analyte and after some
time through capillary action, there is separation
happening ○ Stationary phase: made up of an absorbent
9 material has a very small particle size and kept
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY uniform to obtain a better performance
● Uses pressure for fast separations, controlled ■ Usually chemical modified silica,
temperature, in-line detectors and gradient elution divinyl benzene and etc. are used as
techniques a SP
○ Mobile phase
■ Mixture of different solvents (polar or

nonpolar) which depends on the type
of molecule
■ Usually kept solvent reservoir
● attached to the pump which
pumps the mobile phase in
the column with high
pressure
■ Injector
● Just before the HPLC
column which introduction of
the sample into the column
■ Detector
● In order to detect the
● Modified column chromatography molecules of the sample
● A column is with speck with an absorbing material like that is coming out from the
silica and the mobile phase is pressed down the column column
because of gravity ● Types: UV, iode,
● But in HPLC, a high pressure pump is attached with a fluorescence, reflective
column which can generate a pressure up to 40 MP index detectors and
● The column is filled with an absorbing material which has spectrometers
a very small particle size and this gives a large surface ● Working: as the sample molecules get separated, they
area for the molecules to interact are detected and the peak is obtained from on the
5
computer which is plotted with respect to the retention
time
● To identity the components, we need to have standards
(serves as a basis)
○ Ex: if we run glucose as our sample and its
peak is obtained at 5 minutes. Next we run
sucrose, and its peak is gained at 8 minutes.
Now we analyze the unknown sample for the
detection of sugars present on it.
● Detectors
○ Monitor the eluate as it leaves the column
○ Produce an electronic signal proportional to the
concentration of each separated component
○ Spectrophotometers – detect absorbances of
visible or UV light
○ Photodiode array – used for spectral
comparisons and compound identification and
purity and for drug analysis in urine
○ Amperometric or electrochemical detector –
measures current produced when the analyte of
interest is oxidized or reduced at some fixed
potential set between a pair of electrodes
● Recorders
○ Used to record detector signal versus the time
○ This indicates that there is glucose but no mobile phase passed through the instrument,
sucrose. starting from the time of sample injection
○ The graph is called chromatogram
BASIC COMPONENTS OF HPLC ■ Peak area is proportional to
● Pump concentration of the compounds that
○ Forces the mobile phase through the column at produced the peaks
a much greater velocity
○ Helps in hastening separation MASS SPECTROMETRY
○ Types: ● always coupled to liquid or gas chromatography.
■ Mechanical reciprocating pump – ● Considered as the gold testing
most widely used ● It doubles the sensitivity and specificity = the better.
■ Pneumatic pumps – for preoperative ● based on fragmentation and ionization of molecules
purposes using a suitable source of energy
■ Hydraulic amplifier pumps – no longer ● charged particles moving through a magnetic or an
commonly used electrical field can be separated from other charged
● Columns particles according to their mass-to-charge (m/z) ratios.
○ Long stainless steel
○ Silica gel – most common BASIC COMPONENTS OF MS
● Sample injectors
○ Can be used to introduce the sample into the
path of the mobile phase that carries it into the
column
○ Loop injector
■ Best and most widely used
■ High reproducibility
■ Used at high pressures
● Sample inlet
● Ionization source
● Mass analyzer
○ Actual measuring of your mz ratio occurs when
the gas phase ions pass into
○ It will generate into an electrical field that can
manipulate the charge molecules to sort them
now according to mass ratios
● Ion detector
Electron ionization
- A method that requires a source of electrons into form a
filament to which an electric potential is being applied.
MAJOR STEPS:
● conversion of the parent molecule into a stream of ions
(usually singly charged positive ions)
● acceleration of ions in a magnetic or electrical field
● separation of the ions by mass/charge ratio (m/z)
● counting of the number of ions of each type or
measurement of current produced when the ions strike a
transducer
6
TWO TYPES:
Quadrupole mass spectrometer
● direct electrical current and radiofrequency voltages of

selected magnitudes are applied to two pairs of metallic
rods
● Only ions of specific mass/charge ratio can pass
undeflected to the end of the rods, where they are
detected
● Separation is based on the mass charge ratio; if it's not
the analyte that u are looking for then it is being deflected
into different direction and the only thing that could pass
thru is your analyte of interest.
Iron Trap Mass Spectrometer
● Three electrodes, in a ring shape and two end caps,

produce ions in the cavity until selectively ejected to the
ion detector
● ability to get full mass spectra at very low sample
concentrations
● Trapping electrons using electrodes.
TANDEM MASS SPECTROMETRY
● GC/MS/MS and LC/MS/MS

○ Common used in drug testing
○ Tandem - 2 mass spectrometry ang present
● used for greater selectivity and lower detection limits
● link three quadrupoles in series – triple quad
○ Q1 – used to scan across a preset m/z range
and select an ion of interest
○ Q2 – functions as a collision cell
○ Q3 – serves to analyze the product ions
generated in Q2 (full product ion scan or
selected reaction monitoring)
7
LESSON 5: Quality Control and Quality Assurance

PROF. BERNARDO ORDANEZA JR., RMT
OCTOBER 3, 2021
What is Quality? patient receives the result

● It is the conformance with the requirements of users or ■ Ex. Instrument is not properly
customers calibrated
● Refers to the satisfaction of the needs and expectations
of users or customers ● External failure costs
● Do not use term “customer” when it comes to health care ○ Much more catastrophic than internal failure
institution costs
○ Refer as “clients” or “patients” ■ Ex. Wrong result -> wrong treatment
-> death
Quality ○ Costs that are associated with errors after the
● Is important to customers. It can be assessed and patients receive the test results
monitored. It can be improved.Quality’s benefits exceed ● Remember: The costs of nonconformance is always
its cost. (Clambird, 1990) greater than the costs of conformance
○ Quality is not free
○ In order to improve services or laboratory tests, Errors are costly…
it requires u to invest ● Majority of errors are in the pre-analytic (32%-75%) and
post-analytic (9-55%) phase
Quality and Cost ○ If u have errors, it will cost you money, time,
● Improvements in quality (which requires cost) can lead to and work
long-term reduction of cost ● Analytic phase, which was the focus of earlier Quality
○ Invest in quality and later on u will have no Management schemes, accounts for onoy 4-32% of
problems when it comes to errors errors.
■ Errors are most costly than Review:
investments in quality ● 3 phases of laboratory analysis
● A balance must be obtained so that a net reduction in ○ Pre-analytical phase
cost and an improved quality in services can be obtained ■ things that u do inside the laboratory
just before testing
■ Ex. Patient preparation, competency
of the personnel, receiving of
samples, sample transport
○ Analytic phase
■ Testing proper
■ Start and end of the test
○ Post-analytic phase
■ Releasing of result
Quality over Quantity

● Useless ang quantity without quality
● Make sure that your laboratory test results are of high
quality; They are accurate and precise
Quality cost
● How much money, time, personnel/work hours Total Quality Management (TQM)
● Highest in the hierarchy bcs it encompasses all concepts
Cost of conformance of quality
● If u conform to the quality of the patients/clients, what are ● Process improvement as a means to meet a set of
the costs? standard
● Unsa ang imong iinvest na money, time or work ○ These are series of processes to improve your
hours/effort workflow as a means to meet a specific set of
○ Prevention costs standard
■ Preventive costs
■ Costs that are associated with
activities designing to prevent defects
■ To prevent errors
○ Appraisal costs
■ Related to the detection of those
defects
○ Prevention and appraisal costs will work hand
in hand; will cost u money, time, and work
Cost of Nonconformance
● Internal failure costs
○ Associated with errors found before the
customer receives the product/service
○ In the case of the lab, these are errors that are
happening inside the laboratory just before the
1
5Qs of Total Quality Management
Quality Laboratory Process

● includes analytical process and general guidelines on
how the work is done
○ E.g: Standard Operating Procedures
■ Serves as the general guidelines and
procedures inside the lab
■ Encompasses everything from the
pre-analytical to post-analytical phase
■ Ex, waste disposal, how to set sched,
how to document errors, testings,
steps
○ How do u maintain quality in QLP?
■ Make sure that the SOP are correctly ● From the QLP, follow guidelines inside laboratory and try
outlined and formulated to improve by checking if there are errors in the analytical
■ Must be strictly followed phase, assess for any defects in the workflow (specially
in the pre to post analytical).d determine root cause and
Quality Control improve on those errors, lastly document those solutions
● Detect and repair defects to prevent errors and integrate back to the QLP
● Both statistical and nonstatistical control procedures ● The overall total quality management (5Qs)
○ E.g: Levey-Jennings chart, temperature ● TQM is similar to PDCA (Plan, Do, Check, Act)
monitors, westgard rules, delta check ○ QLP = Plan and Do
■ Delta check - checking previous result ○ QA & QC = Check
in order for u to know if there are ○ QI & QP = Act
abrupt changes in the result Ex.
Platelet results Quality Assurance
■ Check if the temp of the refrigerator is ● Quality from the perspective of end-user
well maintained (temp monitors) ○ In every service oriented industry, it's always
■ Nonstatistical = temperature monitor two: provider of service and customer (client)
and delta check ○ No matter how you’ve managed to create a
○ Under sa umbrella sa total quality management quality service, if the customer can’t feel it,
○ Used only in the analytical phase TQM is useless
● Requires that causes of problems be identified through
Quality Assessment QI and eliminated through QP
● Broader measures and monitors of lab performance ● Also requires for QC to detect problems in order to
○ E.g: TAT, Specimen ID, Patient ID prevent them
○ Encompasses preanalytical to postanalytical ○ Detect problems in the testing itself to prevent
■ Ex. checking if specimen ID and them because the lab results given to patients
patient ID is correct (not duplicate comes from the analytical phase
■ Monitoring TAT ○ Analytical phase must also be protected, and
ensure that test results are high quality
Quality Improvement ● Planned and systematic activities to provide adequate
● Determine and address root cause of problem confidence that requirements for quality will be met (ISO
● Structured problem-solving process 8042,3,4; CLSI)
● After detecting errors in QC & QA, you have to assess ○ CLSI - Clinical Laboratory Standards Institute
the root cause of the problem and then address them ○ ISO - International Organization of Standards
(quality improvement) ● Measurement of the broader dimension of quality from
● Tools for root cause analysis the perspective of the end-user/client (Bishop)
○ Ishikawa or fishbone diagram
○ Problem tree analysis
○ Failure mode and effects analysis
● Quality Planning
● Standardize the remedy
○ Document those remedies to the new QLP
○ Integrate those solutions back to your standard
operating procedure
● Establish performance monitoring
● Total Quality Management has 5Qs
○ After identifying the error, try to monitor if that
○ Quality Control
error occurs again and how often it occurs
○ Provider side
again
● In some sense, quality assurance is on the end-user side
● Ensure that quality performance requirements are
● However, some textbooks are saying that QC is under
achieved
QA
● Document these to the new QLP
Why is QA of testing important?
● Public expects high quality
○ If patient has a bad experience inside the
laboratory (bad service, test results inaccurate),
may be detrimental to the name of the
laboratory
● Defines quality goals & parameters
○ Quality - minimize the errors in the result or
make sure that the test results are released
within the TAT (within 1 hour)
● Evaluation & improvement system
2
○ Evaluating and improvement of process
● Assures reliability and comparability of results
○ Reliability - results is accurate and precise over
time
○ Comparability - maski pa nag change ug shift
ang 2 medtechs, they still have comparable
results (no debate sa result)
● Cost-effective
○ Investing in quality management tools, you can
assure that everything you do is cost-effective
● Even the simplest of testing is not foolproof
○ If the lab test requires several process, it is
vulnerable to errors
Benefits of Laboratory Quality Assurance

● Provides evidence of good performance ● The laboratory has a bigger role in clinical diagnosis
● Laboratory mistakes are prevented
● Significant improvements in testing performance can be Quality Assurance: Quality Control
achieved* ● Involves the systematic monitoring of analytic processes
to detect analytic errors and to ultimately prevent the
Quality Assurance Model reporting of incorrect patient test results (Bishop, Fody, &
● staffing/personnel component Schoeff, 2013)
● Quality control (QC) ● A testing designed to assess the “health” of an analytical
● Proficiency testing (PT) aka external quality assurance method (Henry’s)
(EQA) ○ If you want to assess the health of the patient,
do laboratory tests
In some textbooks, quality assurance encompasses quality control. ○ To assess the health of the laboratory test (if its
working properly or gives accurate and precise
Quality Assurance: Staffing/Personnel results), do quality control
● Able to execute responsibilities ○ QC assesses the health of the test itself
○ Director, supervisor, testing personnel
● Appropriate educational credentials So why do quality control?
○ Graduates, passed board exams ● Error detection
○ Makes sure that the minimum knowledge are ● Error prevention
met ● Measure performance
● Appropriate experience ○ Bias, imprecision, total error
● Receive training ○ 2 types of bias
○ Rationale behind CPD units ■ Positive bias
■ In the professional world, CPD units ■ Negative bias
are units you must earn from ■ Eg.: FBS, let’s say that consistently,
seminars and trainings to renew your all patients tested have high results
license where in fact, only some should be
■ CPD (Continuing Professional high and some should be normal, this
Development) is termed as a positive bias
● Competency assessments ■ If all of the results are lower than what
○ Every after seminars and trainings, there will be is intended or the true value, that is a
a test negative bias
○ If you pass the test, you can earn those units ○ Total error can
○ Ex. Medtech Licensure Exam, ASCP ○ Precision can be computed by knowing the
Standard deviation and by doing the Levey
Three phases of the Testing Process Jennings Chart
● Pre-analytic phase ■ If you plot a chart and the lines are
● Analytic phase erratic, then it is imprecise
○ Quality control ● Monitor performance
● Post-analytic phase ● Validate performance
○ Most important part
○ Validated lab methods ensure tat test results
are also valid
Quality Control
● QC results in lab are used to validate (confirm) whether
the instrument is operating within pre-defined
specifications, concluding that patient test results are
reliable
○ Simply put, operating within normal limits
○ Once test results are validated and px results
are reliable they can now be used in the
treatment, diagnosis and prognosis of the
patient
How does basic QC work?

● Run a control sample
● Compare the result with expected range of values
○ Each of the controls as an expected range of
values
○ E.g: glucose - 100-120 mg/Dl
3
○ If you run that control, the values given by the calibrating the spectrophotometer
instrument should be within the expected range ● In higher, state-of-the-art instruments, calibrations are
of values more complicated
● Check to see if the result is right ● Run prior to QC
○ If Yes → system is working → report good ○ Manually or by the laboratory analyst
patient results ○ Or automatically by the microprocessors
○ If No → system is not working → do not report controlling the instrument
any bad data
● Perform quality control at the start of the shift Absorbance Curve
○ In clinical chemistry, you can do it at the start of
the day
○ In hematology, at the start of the shift, or
depending on protocols or on how often you
want to make sure that your lab results are
working properly and how much money do you
invest on those controls
● If you perform QC at the end of the shift, meaning you
performed testing on patient samples before running a
QC, how can you make sure that those results are
correct?
● QC should never be done last
Quality Control Inside the Laboratory

● Application of multirule systems (Westgard Rule)
○ Used to assess the Levey-Jennings chart
● Plotting Data (Levey-Jennings Chart) ● In some machines, this is called Calibration Curve
● Statistical concepts ● How do you do calibration?
○ Central tendency (Mean) ○ Calibrators are solutions that contain a known
○ Range amount of concentration
○ SD ○ Let;’s say you performed a calibration with 100
○ CV mg/L concentration of an analyte and the
○ Central tendency (mean), and SD are used in absorbance is 0.5
the Levey-Jennings chart ○ You perform 500 mg/L and the absorbance is
around 2.4
Calibration vs. Quality Control ○ 200mg/L and the absorbance is 1
○ Then, you draw the best line which is
Calibration represented by the formula y= mx + b
● “Setting” the analyzer to give correct results ○ r^2= Pearson r
● Uses calibrators (standards) ■ How fit our plots are within the line
● Let’s say you want to create or make sure that your ○ If you calibrate ad the plots are scattered., you
weighing scale is correctly calibrated you may use cannot make a straight line out if it so the
standard weight. Let’s say muadto kag DOST and you Pearson r has a low correlation
will borrow a 1kg weight and you will check if the dial ○ There are certain criteria by which your
goes to 1kg, if not, then you have to calibrate the spring calibration is correctly performed, it should
inside so that the 1kg weight will produce a 1 kg result satisfy a certain r^2
● For instruments that are as complicated as ○ After drawing the absorbance line which should
Beckman-Coulter Analyzers, there is a gap between be linear
calibration and quality control ○ Once you perform a test and the absorbance is
● In order to ensure that it is working properly you need to 1.7, you can confidently say that the
calibrate concentration is around 360 mg/L
○ That is setting your analyzer to give correct
Quality Control results
● “Checking” ● Calibration is done only once every after opening a new
● If the analyzer (after calibration) is producing correct batch of reagents to make sure that your instrument is
results (expected values) well-calibrated with respect to the new batch of reagent
○ Expected values are represented by intervals of
acceptable values
○ How to compute for acceptable values? Controls
■ Do the Levey-Jennings Chart ● QC materials or solutions used to monitor the
● The instrument’s calibration and other analytical performance (precision and accuracy) of an assay
processes method once it has been calibrated
● How do you check your weighing scale? ● Run alongside patient samples
○ Every day you weigh 1kg ● Results are calculated from calibration data in the same
● Checking after setting manner that patient results are calculated
● Every day, you do quality control to check if it is still ○ You perform the testing of controls the same
working properly way you run the sample
● Run as samples
Rule of thumb: If the instrument has more parts, the more reasons
for it to fail. You must make sure that all the parameters
contributing to the conditions of the instruments are working
properly by doing quality control every day.
Calibrators
● Solution that contains a known amount (standard) of
analyte used to calibrate an assay method
● Using the standard in spectrophotometry, that is
4
● Abnormal control (high and/or low)
- In hematology, there are 3 levels of control:
normal, high, and low
6. Convenient packaging for easy dispensing and
storage
● Usually contained in low actinic glass to prevent
degradation from light
Types of Control Material

● Assayed and unassayed are manufacturer made
● homemade/in-house means the laboratory itself creates
that control
1. Assayed
● Target value predetermined
○ Not advisable; you must create your
● This is for QC own values
● Performed every day ● Verify and use
● X-axis: Number of days ● More expensive
● Y-axis: Values
○ Compute the mean and standard deviation 2. Unassayed
● See if the control is still inside the boundaries and it is ● Target values not predetermined
usually within +2, -2 ○ Determine it yourself
● You also have to apply for Westgard rules because not ● Full assay to be established
all the time that it is within +2, -2 siya nga limit is within ● Less expensive
control
● There are Westggard rules that needs to be followed 3. Homemade or In-house
● If you have outliers, or controls that are beyond +2 or ● Pooled sera collected in the laboratory
-2, that is a possible error ● Full assay, validation needed
● If it’s an error, you troubleshoot ● Disadvantage: pool sera degrades overtime
Assayed
Criteria for Selection of Control Materials ● If assayed materials are used, the values stated on the
assay sheets should be used only as guides
1. Closely mimic (same matrix) the characteristics of ● Actual values and standard deviation must be
the patient’s sample being tested established by serial testing in the laboratory
- Same matrix refers to the substance or base ● Every lab should establish their own range (setting
form from which the control material is prepared control limits)
● Matrix
○ Refers to the substance or base form Setting Control Limits
which the control material is prepared ● The Clinical Laboratory Standard Institute (CLSI)
○ Characteristics of a sample (serum, recommend that at least 20 measurements should be
plasma, urine) made on “separate” days when the measurement
○ If the sample is serum, then the system is known to be stable
control must be as serum like as ○ Compute for the mean and the SD and then
possible your own chart using those values
○ Urine degrades over time. ○ 20 days because the measurement system is
known to be stable
2. Stable for prolonged period (at least a year) without
interfering preservatives of special storage needs\
Terminologies Relevant to Quality Control
- Controls are manufactured at factories of the
supplier 1. Accuracy
- Special storage needs = dili need ifreezer at 2. Precision
-40; refrigerator is enough 3. Reliability
- Most controls are stored at 2-8 degrees celsius 4. Repeatability/Practicability
- Preservatives can be present as long as it does 5. Reproducibility
not interfere with the test itself 6. Analytic Sensitivity and Specificity
● Lyophilized control material 7. Diagnostic Sensitivity and Specificity
○ Dehydrated to powder
○ Reconstitute to use 1. ACCURACY
■ Add distilled water and mix ● Describes the closeness of a test value to the
thoroughly to become a actual/target/true value
liquid control ● Closeness to the mean value
○ Lyophilizing the control makes it ● Ex. the true value is FBS = 126mh/dl
stable for prolonged periods ○ If your test result is 99mg/dl it is not accurate
3. Inexpensive ○ To determine if you are wrong or right, perform
● cost-effective QC
4. Available in aliquots convenient for daily use ■ The closer the values of your control
● Aliquots are small portions of a sample measurement to the mean, it is
● Most controls are 1ml-5ml only accurate
○ Amount needed everyday is very ● Can be measured in three ways:
small ex. 0.5ml 1. Recovery study
○ Control will destabilize if the - Changing the parameters of your
temperature changes so frequently sample and seeing if the results also
5. Include at least 2 levels of controls (for clinical change
chemistry lab) 2. Interference study
● Normal control - Deliberately adding interference and
see if the test results ares till correct
5
3. Comparison of methods study Imprecise and inaccurate
- Comparing your test method to your ● The capacity of a method to maintain both accuracy and
reference method and see if it is the precision into the futureRepeatability/Practicability
same ● Capacity to produce the same results on one sample
2. PRECISION again and again when performed by the same individual
● The consistency (degree of replication) of a series of using the same lot numbers on the same instruments
tests results
● The closeness of the agreement between independent 3. RELIABILITY
test results obtained under prescribed condition ● The capacity of a method to maintain both accuracy and
● Ability of an analytical method to give repeated results on precision over time
the same sample that agree with one another
4. REPEATABILITY/ PRACTICABILITY
ACCURACY vs. PRECISION ● Capacity of the method to produce the same results on
one sample again and again when performed by:
○ Teh same individual (MT) using..
○ The same lot numbers on..
■ Lot number - series of numbers that
pertains to when it was produced or
the batch it was produced.
○ The same instruments
● There are certain conditions in the laboratory wherein
repeatability will be done.
○ Example: fluctuating temperature of the
instruments which will render diff results so
troubleshoot
Example: FBS 5. REPRODUCIBILITY

● You have a standard solution of 200mg/dl ● Capacity of the methods to produce the same results on
● Accurate results would be 201, 199, 202, 195 one sample again and again when performed by
○ Close to 200 ○ Different individuals on…
○ Average would be close to 200 ○ Different days using…
○ Different sets of reagents
● Imprecise since the values are not close ● Ideally, all medtechs should perform it in the same way
● Inaccurate but precise results: close values but far from but there are minute differences on the way we perform
the true value things
6. SENSITIVITY (ANALYTICAL SENSITIVITY)

● Ability of an analytical method to measure the smallest
concentration of the analyte of interest
● Example:
imprecise/inaccurate
● Precision can be easily related to the standard deviation.
That is why in the Levy Jenning’s chart, aside from the
mean, there is also the SD.
○ the more erratic ang plot, it’s imprecise
○ If it’s close to the mean, accurate
○ Close values but layo sa mean, precise but not
accurate
○ X axis- sample A,B,C

○ Y axis - level of analyte X
precise but inaccurate ■ presence of the analyte means that
○ Close values but so far from the mean you have the disease; a presence of a
single malarial parasite means that
you have malaria
○ What does it mean that the test is sensitive?
method A can only detect a specific level of
malaria, but even if sample B has malaria, it
cannot detect that (so not sensitive)
precise and accurate ○ Using Method B, it can detect the smallest
○ Close to the true value, not erratic in the Levey concentration of analyte of interest thus, more
Jenning’s chart sensitive than Method A
● Do all of the tests must be as sensitive as possible?
How do you judge the values in terms of accuracy & precision? ○ Sometimes it helps that is not very sensitive
because not all analytes require that
6
○ ability of the test to detect the proportion of
individual without the disease who test
negatively for the disease
○ If negative sa test, this is the proportion of the
individual without the disease.
○ Element x - has a certain value where it is

normal in the bloodstream
■ Normal - 0.1 ng/dL
■ Abnormal - >0.1 ng/dl
○ Method A - can detect 0.1 (level of sensitivity)
○ Method B - 0.02
■ If the test is very sensitive, then it
might give you false-positive results
● Out of 100, 10 women has breast cancer; 990 has no
6.1 SPECIFICITY (ANALYTICAL SPECIFICITY)
breast cancer.
● Ability of an analytical method to measure only the
● Blind
analyte of interest
○ We don't know which among the women has
● Termed as analytical to differentiate from the diagnostic
breast cancer
specificity/sensitivity
○ You have to perform a screening test
● After screening test
○ 10 = ( +)
○ 990 = (-)
■ Because of the various analytical
● Which of the three methods is the most specific? sensitivity and specificity of the test,
○ Method A: proteins you cant have a too sensitive or too
○ Method B: enzymes (subset of proteins) specific because u might come up
○ Method C: CkMB - most specific because it with false positive and negative result
only detects the analyte of interest ● How do you compute for Dx sensitivity and specificity of
● Can we have a test that is too specific? yes that screening method
EXAMPLES:
Method C Method D
𝑡𝑟𝑢𝑒 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒
CK-MB Specific isoenzymes of Dx sensitivity=
CK-MB 𝑡𝑟𝑢𝑒 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒 + 𝐹𝑎𝑙𝑠𝑒 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒
● If what you're after is just measuring CKMB in general,
then don't use method D. Sensitivity
● Ty = positive
● sensiT = true
Protein levels 9
Method A Method B =
9+1
= 90%
proteins enzymes
𝑇𝑢𝑟𝑒 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒
● Do not use method B because it's too specific for your Dx specificity = 𝑡𝑟𝑢𝑒 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 + 𝑓𝑎𝑙𝑠𝑒 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒
test.
● Choose the right method depending on what you want to Specificity
measure ● Ty = positive
● Don't use specific methods if you want to have a general ● speciFI = false positive
measurement of an analyte. 901
= 901 + 89 = 91%
Sensitivity and Specificity
Analytic VS diagnostic ● Is the test accurate or not?
● Analytic ○ Sensitivity = 90%
○ Focuses on the test itself ○ Specific = 91%
○ Focuses on the ways the analytes are detected ■ Fairly good test
by the test ■ Accurate but accuracy is not
● Diagnostic necessarily predictive
○ Pertains to how the disease is detected by the ● What are the chances that you have breast cancer?
test itself ○ Compute for the positive predictive value (PPV)
7. DIAGNOSTIC SENSITIVITY ○ Although accurate, the PPV is low.
○ ability of the test to detect the proportion of
individuals with that disease who test positively 𝑇𝑃
with the test PPV = 𝑇𝑃 + 𝐹𝑃
○ If you test positive, what is the proportion of the
individuals with the disease
9
7.1 DIAGNOSTIC SPECIFICITY PPV= +89
= 9% chances of u having the disease if u tested
positive.
7
● The predictability of your result is more than just its
sensitivity and specificity.
Quality Assurance Model

● Staffing/personnel component
● Quality control (QC)
● Proficiency testing (PT) aka external quality assurance
(EQA)
Proficiency testing (PT) aka external quality assurance (EQA)

● central organization sends out challenge specimens for
testing
○ NRL
● laboratories results are evaluated
○ Clin chem
■ Lung center of the Philippines
○ Microbio
■ RITM
■ Blood banking
○ Hematology and Blood banking
■ NKTI
○ Drug testing
■ EAMD
○ HIV, Hepa B, syphilis
■ San Lazaro Hospital
● early warning-system for problems
● measure of laboratory quality valuable benchmarking tool
○ for standardization and traceability
● indicator of where to direct improvement efforts
● monitor of changes in technology and testing practices
○ evaluation component
METHOD EVALUATION
● Evaluating the test method itself.
● Formulating the method itself.
● Establishing a test.
● Evaluating the new method u come up with.
8
LECTURE 6: CARBOHYDRATES: BIOCHEMISTRY AND

CLINICAL SIGNIFICANCE
Prof. Fritz Von T. Gella, RMT, MD
October 5, 2021
PART 1 2. The size of the base of the carbon chain
General Description
● Most abundant organic molecules in nature
● Carbohydrates are the major food and energy source of
the body and are stored primarily in the two most
important systems:
○ Liver and muscle glycogen
● Function:
○ Major energy source= glucose
○ The storage form of energy= glycogen
○ Component of the cell membranes =
glycoprotein
○ Structural components in plants, bacteria, and
insects ● The number of carbon atoms
● Compounds containing C, H, and O ● Trioses: 3 carbons
● Contain C=O, and -OH (hydroxide) functional groups ● Tetroses: 4 carbons
● Pentoses: 5 carbons
● Hexoses: 6 carbons
● Heptoses: 7 carbons
Glucose
Mono-saccharides Fructose
(Hexoses) Galactose
Mannose
● Different structure/projections of carbohydrates Ribose
● Left structure: Fischer projection Mono-saccharides
○ Show a linear structure Ribulose
(Pentoses)
● Middle: Haworth projection Xylulose
○ Shows a cyclic structure as viewed from the
side showing the stereochemistry or location of ● There are monosaccharides that are hexoses and some
the attached molecules to the monosaccharide are pentoses
ring
● Right: Chair confirmation 3. Location of the CO functional group
○ Possible to have a boat type confirmation ● Aldose vs Ketose
where C1 is tilted upwards in the same ○ 2 forms of carbohydrate are aldoses and
direction as the C4 which is less common ketoses.
● Aldose
Classification
The classification of carbohydrates is based on four different
properties
1. The number of sugar units

● Monosaccharides: Glucose, Fructose, Galactose
○ One sugar unit
● Disaccharides: Maltose, Lactose, Sucrose (MLS)
○ Two sugar units
■ A combination of glucose, fructose, ○ Functional group is an aldehyde
and galactose ○ Carbonyl carbon at the end
○ Each sugar molecule is held together by ○ Ex. glucose, galactose, mannose
glycosidic bonds ● Ketose
■ Type of covalent bond that joins
carbohydrate molecule to
another group which may or may
not be a carbohydrate
● Oligosaccharides: chaining of two or ten sugar units
○ Disaccharide belongs to oligosaccharides
● Polysaccharides: Starch, glycogen
○ Functional group is a ketone
○ More than 10 units of sugar molecule
○ Carbonyl carbon at any other position
○ Ex. fructose
1
○ Example
■ L-glyceraldehyde and
● Glucose = carbonyl group is at the end D-glyceraldehyde
● Ketoses’ carbonyl group is located in any other position ■ L-glucose and D-glucose
except the terminal or end part
● Always look for the carbonyl group to identify whether an ● Anomers
aldoses or ketoses ○ In aqueous solutions, monosaccharides with
● Aldose = tip; ketose= any position except terminal five or more carbon atoms in the backbone
occur predominantly as cyclic (ring) structures
4. Stereochemistry of the compound ○ Furanose: monosaccharide structure with a
● Sturdy of spatial arrange of an atom; 3D or 4D five-membered ring
configuration of a carbohydrate; how the molecules are ○ Pyranose: monosaccharide structure with a
being arranged in a 3D or 4D configuration six-membered ring
● Isomers ○ Rotation around the carbonyl carbon produces
○ Compounds that have the same chemical anomers, which are labeled a (alpha) and b
formula (beta) anomers
○ Ex. glucose, fructose, galactose, and mannose ○ Unlike epimers, anomers can undergo
are all isomers of one another because they interconversion (from a to B, and vice versa)
have the same formula C6H12O6 without energy expenditure or the need for
● Epimers enzymes, in a process called mutarotation
○ Isomers that differ in configuration around only
one specific carbon atom (except the carbonyl
carbon)
○ Ex. glucose and galactose (differ only in
position of -OH in C4) glucose and mannose
(differ only in position of -OH in C2
■ As long as they differ in configuration
around only 1 specific carbon atom
● Anomere can be identified by identifying the carbonyl

atom
● Label alpha (if below ang OH) or beta (if above ang OH)
REVIEW:
● Isomers
○ Same chemical formula regardless of structure
● Epimers
○ Same itsura but naiiba lang ang position OH ng
isang carbon.
● Glucose and Galactose = differ only -OH in C4 ● Enantiomers
● Glucose and Mannose = differ only -OH in C2 ○ Mirror images
○ Na flip lang ang position ng OH
● Enantiomers ● Anomers
○ Optical isomers or stereoisomers ○ Iba ang position ng OH sa anomeric na carbon
○ Pairs of structures that are mirror images of ■ Alpha
each other ● Baba ang OH
○ The enantiomers are designated as a D-sugar ● “Ababa”
(Dextrorotatory) and an L-sugar (Levorotatory) ■ Beta
○ D-sugars are more common ● Nasa taas ng anomeric
● “betaas”
2
METABOLISM CARBOHYDRATES ■ Transfer fructose towards epithelial
cells
■ Only fructose
○ GLUT-2
■ Transfer all types of
monosaccharides (glucose, galactose
and fruc) inside SI.
Ingestion of food:
1. Salivary amylase will be stimulated and begins digestion
in the mouth
○ Initial digestion in the mouth
2. Salivary amylase is denatured by stomach acids.
3. Food will now go to your SI and stimulate release of
pancreatic amylase. Pic above: lumen of SI
4. Enzymes at the microvilli break down carbohydrates to
monosaccharides
○ What is being absorbed in SI is not PATHWAYS IN GLUCOSE METABOLISM
disaccharide, oligo or polysaccharide, it must
be digested to become monosaccharide for it to
be absorbed by the body.
5. Resistant starches and fibers are digested and fermented
in the LI and the colon.
Absorption
● Only Monosaccharides are absorbed
● Luminal Side:
○ Different transporters:
■ Will transfer your monosaccharide
going to your system or to be
absorbed in the body.
■ SGLT-1
● (secondary active transport):
for glucose and galactose
■ GLUT-5
● (facilitated diffusion): for ● NOTE:
fructose ○ 2 terminologies:
● Basolateral Side: ■ Well fed state - increased sugar level
■ GLUT-2 ■ Fasting state - decreased sugar level
■ (facilitated diffusion): all ● Glycolysis
types of monosaccharides ○ Metabolism of glucose molecule to pyruvate, or
lactate for production of energy.
○ Well fed state
● Gluconeogenesis
○ Formation of Glu-6-phosphate from non
carbohydrate source = Lactate, glycerol and
amino acid
○ Fasting state
● Glycogenolysis
○ Breakdown of glycogen to glucose for energy
○ Fasting state
● Glycogenesis
○ Conversion of glucose to glycogen for storage
○ Well fed state
● Lipogenesis
○ Conversion of carbohydrates to fatty acids
○ Well fed state
● Lipolysis
Pic above: lumen of SI ○ Decomposition of fats
● Paano sha makakapasok sa ating epithelial cells of SI
bago makapasok sa blood?
○ SGLT-1
■ Will transfer glucose and galactose
■ Transport sodium
○ GLUT-5
3
Blood glucose level declines to a set point; stimulus for insulin
release diminishes
↓
Low blood glucose (due to skipping a meal)

↓
Alpha cells of the pancreas stimulated to release glucagon into the
blood
↓
Glucagon
↓
Liver breaks down glycogen and releases glucose into the blood
↓
Blood glucose level rises to set point; stimulus for glucagon
release diminishes
1. INSULIN
● Beta-cells of islets of Langerhans
● Stimulus: Hyperglycemia
○ Increase in blood glucose level
● Actions:
○ Promotes glucose cellular entry
HORMONE REGULATION ○ Muscles and adipose tissues
● Brief fast ○ Increases glycogenesis, lipogenesis, and
○ Glucose is supplied to the ECF from the liver glycolysis (metabolism of glucose as a source
through glycogenolysis of energy)
● Fasting period longer than 1 day ○ Inhibits glycogenolysis
○ Glucose is synthesized from noncarbohydrate
sources (gluconeogenesis) How is insulin produced?
Control of blood glucose is under 2 major hormones:

1. Insulin
2. Glucagon
- Both produced by pancreas and their action
opposes each other
Hormone Regulation
1. Insulin
2. Glucagon Initially synthesized as a precursor polypeptide: Preproinsulin
3. Epinephrine ↓
4. Cortisol Subsequent protolytic processing removes the amino-terminal
5. Growth hormone signal peptide giving rise to the Proinsulin
6. ACTH ↓
7. Thyroxine After few processing, Insulin and C peptide is released
8. Somatostatin
9. Incretins ● The mature insulin molecule and C- peptide are stored
together and co-secreted from secretory granules in the
Glucose Homeostasis beta cells.
● Because C peptide is cleared more slowly than insulin, it
is a useful marker of insulin secretion.
Pancreas
● Exocrine
○ Enzyme: Amylase and Lipase
● Endocrine
○ 4 hormones from different cells in the Islets of
Langerhans:
■ Glucagon (alpha cells)
■ Insulin (Beta-cells)
■ Somatostatin (delta cells)
■ Pancreatic polypeptide (PP or F cells)
If there is a high glucose level (after eating)

↓
Beta-cells of the pancreas stimulated to release insulin into the
blood
↓
Body cells will take up more glucose and Liver takes up glocse and
stores it as glycogen
↓
4
5. GROWTH HORMONE
● Anterior pituitary gland
● Actions:
○ ↑ blood glucose level
○ ↓ entry of glucose into the cells
○ ↑ glycolysis
6. ACTH
● Anterior pituitary gland
● Stimulus: decreased cortisol levels
● Actions:
○ Stimulates cortisol release thus increases
plasma glucose
○ ↑ glycogenolysis and gluconeogenesis, ↑ blood
glucose level
7. THYROXINE
● Insulin will attach to the insulin receptor
● Glucose channel will enter once the insulin has attached
● Glucose enters cell through glucose channel (basic
function of insulin)
2. GLUCAGON
● Produced in the Alpha-cells of islets of Langerhans
● Stimulus: during stress, fasting states
● Actions:
○ Enhances glycogenolysis (cause breakdown of
glycogen forming glucose) and
gluconeogenesis (formation of glucose from ● Thyroid gland (follicular cells)
another non-carbohydrate source) ● Stimulus: Release of Thyroid Stimulating Hormone (TSH)
○ ↑blood glucose level ● Actions:
○ ↑ glycogenolysis,
3. EPINEPHRINE ○ ↑ gluconeogenesis
○ ↑ intestinal absorption of glucose
● Flight or flight hormone
● Produced in Adrenal medulla
● Stimulus: Released during stress 8. SOMATOSTATIN
● Actions: ● Delta-cells of islets of Langerhans of the pancreas & GI
○ ↑ blood glucose level cells
○ Inhibits insulin secretion ● D cells of duodenum
○ ↑ glycogenolysis ● Actions:
○ promoting lipolysis ○ INHIBITORY HORMONE to Insulin, glucagon,
growth hormone, and other endocrine
hormones
○ “somatoSTOPin”
9. INCRETINS
● Gut hormones secreted by the enteroendocrine cells
minutes
● after eating
● Examples:
Tissue area Hormones released Examples ○ Glucose-dependent insulinotropic peptide (GIP)
○ Glucagon-like peptide-1 (GLP-1)
Adrenal Zona Mineralocorticoids Aldosterone
cortex glomerulosa (regulate mineral
balance)
Zona Glucocorticoids Cortisol,
fasciculata (regulate glucose corticosterone,
metabolism) cortisone
Zone Androgens (stimulate Dehydroeplands
reticularis masculinization) terone
Adrenal medulla Stress hormones epinephrine ,
(stimulate norepinephrine ● Stimulate insulin release → inhibit glucagon
sympathetic ANS) release → lowering of blood glucose level
PART 2
4. CORTISOL
Clinical Correlation: Hypoglycemia
● Adrenal cortex (zona fasciculata) ● Insulin overdose (reactive hypoglycemia)
● Actions: ● Postprandial hypoglycemia
○ On stimulation by ACTH ↑ blood glucose level ○ GI surgery
○ Decreasing entry of glucose into the cell ○ Mild diabetes
○ Increasing gluconeogenesis, liver ● Fasting hypoglycemia
glycogenolysis, and lipolysis ○ Insulin-producing pancreatic islet tumor
5
(insulinomas) Table: Etiologic Classifications of Diabetes Mellitus
■ The body creates more insulin
I. Type I diabetes (immune-mediated beta cell destruction,
resulting to hypoglycemia
usually leading to absolute insulin deficiency)
○ Hepatic dysfunction II. Type II diabetes (may range from predominantly insulin
○ ROH consumption resistance with relative insulin deficiency to a predominantly
■ Excessive alcohol consumption, insulin secretory defect with insulin resistance)
drinking heavily without eating can III. Specific types of diabetes
block your liver from releasing stored A. Genetic defects of beta cell development or function
glucose into the bloodstream causing characterized by mutations in:
hypoglycemia 1. Hepatocyte nuclear transcription factor (HNF) 4ɑ
(MODY 1)
Clinical Correlation: Hyperglycemia 2. Glucokinase (MODY 2)
3. HNF - 1ɑ (MODY 3)
Diabetes Mellitus (DM) 4. Insulin promoter factor-1. HNF - 1β, NeuroD1,
● It is not just one disease but a group of metabolic and others leading to other forms of MODY
disorders 5. Insulin, subunits of ATP-sensitive potassium
channel leading to permanent neonatal diabetes
● It refers to a group of common metabolic disorders that
6. Mitochondrial DNA
share the phenotype of hyperglycemia 7. Other pancreatic islet regulators/proteins such as
● Depending on etiology, factors contributing to KLF11, PAX4, BLK, GATA4, SLC2A2 (GLUT2),
hyperglycemia include reduced insulin secretion, RFX6, GLIS3
decreased glucose utilization & increased glucose B. Transient neonatal diabetes
production C. Diabetes of the exocrine pancreas - pancreatitis,
● All of these factors leads to hyperglycemia → diabetes pancreatectomy, neoplasia, cystic fibrosis,
mellitus hemochromatosis, fibrocalculous, pancreatopathy,
● Several types of DM are caused by a complex interaction mutations in carboxyl ester lipase
of your genetic and environmental factors D. Genetic defects in insulin action, including type A
● The metabolic dysregulation associated with DM causes insulin resistance, leprechaunism, Rabson-Mendenhall
secondary pathophysiological changes in multiple organs syndrome, Lipodystrophy syndromes
system and imposes a tremendous burden to the E. Endocrinopathies - acromegaly, Cushing’s syndrome,
individual with DM and to the healthcare system glucagonoma, pheochromocytoma, hyperthyroidism,
● DM will have different complications somatostatinoma, aldosteronoma
○ Has many complications F. Drug or chemical-induced - glucocorticoids, vacor (a
rodenticide), pentamide, nicotinic acid, diazoxide,
○ Leads to several disorders
β-adrenergic agonists, thiazides, calcineurin and
○ Through time it affects your eyes like cataracts, mTOR inhibitors, hydantoins, asparaginase,
liver problem, kidney problem, complex system ɑ-interferon protease inhibitors, antipsychotics
problem (atypicals and others), epinephrine
G. Infections - congenital rubella, cytomegalovirus,
coxsackievirus
H. Uncommon forms of immune-mediated diabetes -
“stiff-person” syndrome, anti-insulin receptor antibodies
I. Other genetic syndromes sometime associated with
diabetes: Wolfram’s syndrome, Down’s syndrome,
Klinefelter’s syndrome, Turner’s syndrome,
Friedreich’s ataxia, Huntington’s chorea,
Laurence-Moon-Biedl syndrome, myotonic dystrophy,
porphyria, Prader-Willi syndrome
IV. Gestational diabetes mellitus (GDM)
Classification
● Diabetes can be classified into the following general
categories:
1. Type 1 diabetes (due to autoimmune B-cell
destruction, usually leading to absolute insulin
deficiency, including latent autoimmune
● Type I through time, you would need insulin for survival diabetes of adulthood)
● Type II pwede maging impaired then normal na naman a. Autoimmune beta cell destruction,
but could lead to insulin required for control your own cell is attacking the beta
● Gestational is similar with Type II cell; one cell is attacking the beta cell;
● A timeframe showing the progression of glucose no beta cell = insulin deficiency
problem/failure will happen leading to your Diabetes 2. Type 2 diabetes (due to progressive loss of
Mellitus adequate B-cell insulin secretion frequently on
● 3rd row: different lab criteria to diagnose for diabetes, the background of insulin resistance)
pre-diabetes, or just a normal glucose tolerance a. Meron kang insulin pero nag reresist
● FPG: Fasting Glucose ang katawan. Therefore, wala parin
● 2-h-PG: 2-hours Post prandial glucose effect at tumataas ang sugar level.
● HbA1C Dahil di makakapasok ang glucose sa
● Take note of the normal values cell
3. Specific types of diabetes due to other causes,
e.g., monogenic diabetes syndromes (such as
neonatal diabetes and maturity-onset diabetes
of the young), diseases of the exocrine
pancreas (such as cystic fibrosis and
pancreatitis), and drug- or chemical-induced
diabetes (such as with glucocorticoid use, in the
treatment of HIV/AIDS, or after organ
transplantation)
6
4. Gestational diabetes mellitus (diabetes
diagnoses in the second or third trimester of
pregnancy that was not clearly overt diabetes
prior to gestation)
TYPE 1 DIABETES Complete or near Total Insulin

MELLITUS Deficiency
Heterogenous group of disorders
characterized by variable degrees of
TYPE 2 DIABETES
insulin resistance, impaired insulin
MELLITUS
secretion, and increase glucose
production
Insulin resistance related to
GESTATIONAL DIABETES
metabolic changes of late
MELLITUS
pregnancy
Maturity-onset diabetes of the Bakit sinisira ng ating own cells ang beta cells?
young, pancreatic exocrine disease, ● Because of genetic predisposition and environmental
SPECIFIC TYPES
cystic fibrosis related DM, viral factors.
infection Autoantigens form insulin-producing beta cells and circulate in the
bloodstream and lymphatics.
● Type 1 = Nasisira ang beta cell possible complete or near ↓
total insulin deficiency Processing and presentation of autoantigen by antigen presenting
● Type 2 = a lot of variables leading to insulin resistance cells (macrophage→ present to T helper cells)
● GDM = insulin resistance related to pregnancy ↓ ↓
Activates T helper 1 lymphocytes activation of T helper 2 lymphocytes
● Type 1 Diabetes Mellitus ↓ ↓ ↓
○ Results from the complete or near-total insulin IFN gamma IL-2 IL4
deficiency
● Type 2 Diabetes Mellitus IFN gamma
○ A heterogeneous group of disorders ● Activation of macrophage with release of IL-1 and TNF a
characterized by variable degrees of insulin IL-2
resistance, impaired insulin secretion, and ● Activation of autoantigen-specific T cytotoxic (CD8) cells
increased glucose production. Type 2 DM is IL-4
preceded by a period of abnormal glucose ● Activation of B lymphocytes to produce islet cells
homeostasis classified as impaired fasting antibodies and antiGAD 65 antibodies
glucose (IFG) or impaired glucose tolerance ↓
(IGT) Destruction of beta cells with decreased insulin secretion
○ Type 1 and Type 2 DM are preceded by a
phase of abnormal glucose homeostasis as a Clinical Correlation:DM TYPE 2
pathologic process progresses. ● Type 2 DM is characterized by impaired insulin secretion,
○ However, there is an increasing recognition of insulin resistance, excessive hepatic glucose production,
other forms of diabetes in which the and abnormal fat metabolism.
pathogenesis is better understood ● As insulin resistance and compensatory hyperinsulinemia
○ This other forms of diabetes may share progress, the pancreatic islets in certain individuals are
features of Type 1 or Type 2 DM unable to sustain the hyperinsulinemic state.
● At early stages, glucose tolerance remains normal
Clinical Correlation: DM TYPE 1 despite insulin resistance because beta cells
● Type 1 is the result of interactions of genetic, compensate by increasing insulin output.
environmental and immunologic factors that ultimately ○ Hyperplasia of pancreas to compensate by the
lead to the destruction of the pancreatic beta cells and insulin resistance.
insulin deficiency.
○ Sinisira ng sariling katawan ang beta cells
● It can develop at any age, develops most commonly
before 20 years of age
○ Individuals with genetic susceptibility are taught
to have normal beta cell mass at birth.
○ At first normal beta cells pa sila. They have
normal pancrea at birth but they begin to lose
beta cells secondary to autoimmune that occurs
over months to years.
○ These auto-immune process can be triggered
by:
■ Infection
■ Environmental factors
Insulin enters bloodstream from pancreas

↓
Glucose enters bloodstream from digestive system and liver
↓
In response to insulin, cell takes up glucose, which is used as fuel.
↓
Healthy balance of glucose and insulin circulate in the bloodstream
IN DM TYPE 2
7
Insulin enters bloodstream from pancreas
↓
Glucose enters bloodstream from digestive system and liver
↓
Insulin is unable to bind with the cell
↓
Glucose cannot enter the cell
Management of Type 2 Diabetes
↓
Glucose returns to blood stream
↓
unhealthy balance of glucose circulate in the bloodstream:
hyperglycemia
● Glycemic control
Insulin resistance: ○ Diet/lifestyle
● Genetic predisposition ○ Exercise
● Obesity ○ Medication
● Lifestyle factors ● Treated associated conditions
Compensatory beta hyperplasia ○ Dyslipidemia
● Increase the output insulin→ normoglycemia ○ HypertensionObesity
Beta cell failure at an early stage ○ Coronary heart disease
● Lead to impaired glucose tolerance ● Screen for/manage complications of diabetes
● Dahan dahan ng tumataas ang glucose level sa body. ○ Retinopathy
Later stage: beta failure ○ Cardiovascular disease
● Diabetes mellitus ○ Nephropathy
○ Neuropathy
Clinical Correlation: DM TYPE 2 ○ Other complications
● Risk factors for Type 2 Diabetes
○ Have a family history of diabetes Clinical Correlation: CLASSICAL SIGNS AND SYMPTOMS OF DM
○ Have a BMI ≥ 23.0 kg/m2 1. Polyuria
○ Lead an inactive lifestyle - Excessive urination
○ Have high blood pressure 2. Polyphagia
○ Have an abnormal blood cholesterol/lipid levels - Increased appetite
○ Have a history of gestational diabetes 3. Polydipsia
○ Are ≥ 40 years old - Excessive thirst
○ Have impaired glucose tolerance or impared
fasting glucose ● Symptoms of Diabetes
○ Always tired
○ Always hungry
○ Sexual problems
○ Vaginal infections
○ Numb or tingling hands or feet
○ Always thirsty
○ Wounds that won’t heal
○ Blurry vision
○ Sudden weight loss
○ Frequent urination
8
CLINICAL CORRELATION: DM TYPE 1 vs TYPE 2
Characteristics of Type 1 and Type 2 Diabetes Mellitus
Type 1 Diabetes Type 2 Diabetes
Frequency 5% to 10% 90% to 96%
Age of onset Any, but most common in children and young More common with advancing age, but can
adults occur in children and adolescents
Risk factors Genetic, autoimmune, environmental Genetic, obesity, sedentary lifestyle,

race/ethnicity, hypertension, dyslipidemia,
polycystic ovarian syndrome
C-peptide levels Very low or undetectable Detectable
Prediabetes Autoantibodies (GAD65, IA-2, IAA, ZnT8) Autoantibodies absent

may be present
Medication therapy Insulin absolutely necessary; multiple daily Oral agents and/or non insulin injectable
injections of insulin pump hypoglycemic drugs insulin commonly
needed
Therapy to prevent or delay onset of None known Lifestyle (weight less and increased physical
diabetes Clinical trials in progress activity) Oral medications (metformin,
acarbose) may be helpful
CLINICAL CORRELATION: ETIOPATHOGENESIS OF DM
● Osmotic diuresis - glucose will attract more water

● Appetite is increased when glucose does not enter our cells
9
CLINICAL CORRELATION: DIAGNOSIS OF DM ● Normal values will depend on the different reference
laboratories
Criteria for the Diagnosis of Diabetes Clinical Correlation: DM TYPE 3
● Alzheimer’s disease is type 3 diabetes
FPG ≥ 126mg/dL (7.0mmol/L). Fasting is defined as no caloric ○ Related to insulin deficiency and resistance
intake for at least 8hrs*
OR
2-h PG ≥ 200mg/dL (11.1mmol/L) during OGTT. The test should

be performed as described by WHO, using a glucose load
containing the equivalent of 75g anhydrous glucose dissolved in
water*
OR
A1C ≥ 6.5% (48mmol/mol). The test should be performed in a

laboratory using a method that is NGSP certified and
standardized to the DCCT assay *
OR
In a patient with classic symptoms of hyperglycemia or

hyperglycemic crisis, a random plasma glucose ≥ 200mg/dL
(11.1mmol/L)
DCCT, Diabetes Control and Complications Trial; FPG; Fasting Clinical Correlation: GESTATIONAL DIABETES MELLITUS
plasma glucose; OGTT, oral glucose tolerance test; WHO, world ● any degree of glucose intolerance with onset or first
health organization; 2-h PG, 2hr plasma glucose. recognition during pregnancy
*in the absence of unequivocal hyperglycemia, diagnosis ○ 2nd trimester (6 months)
required two abnormal test results from the same sample or in ● Causes metabolic and hormonal changes
two separate test samples ○ Insulin do not cross the placenta but GLUCOSE
can!
● associated with increased perinatal complications and
Classical symptoms: increased risk of development of diabetes in later years
● Polyuria, polydipsia, polyphagia and others ● Infants increased risk for: respiratory distress syndrome
Criteria for defining Prediabetes ● High blood glucose levels in mother → brings extra
glucose to baby → causes baby to put on extra weight
FPG 100mg/dL (5.6mmol/L) to 125mg/dL (6.9mmol/L) (IFG)
OR Pregnancy Labor Postpartum and

Mother beyond
2-h PG during 75-g OGTT 140mg/dL (7.8mmol/L) to 199 mg/dL ↑ Pre-eclampsia ↑ induction of labor ↑ recurrent GDM
(11.0 mmol/L) (IGT) ↑ cesarean section ↑ Type 2 diabetes
↑ operative
OR deliveries
↑ labor
complications
A1C 5.7-6.4% (39-47mmol/mol)
Offspring congenital Neonatal Long term
FPG, fasting plasma glucose; IFG, impaired fasting glucose; complications outcome
IGT, impaired glucose tolerance; OGTT, oral glucose tolerance - CNS Prematurity, ↑ obesity
test; 2-h PG, 2hr plasma glucose. - Cardiac fetal perinatal asphyxia, ↑ type 1 diabetes
*For all three tests, the risk is continuous, extending below the programming respiratory ↑ type 2 diabetes
lower limit of the range and becoming disproportionately greater - ↑ LGA distress, metabolic ↑ metabolic
at the higher end of the range - ↑ Macrosomia complications syndrome
- Increased fat (hypoglycemia &
mass hypocalcemia),
polycythemia and
hyperviscosity, low
iron stores,
hyperbilirubinemia,
cardiomyopathy
10
LECTURE 2: LIPIDS: BIOCHEMISTRY AND CLINICAL

SIGNIFICANCE
Prof. Francis Ian Salaver, RMT, MD
October 11, 2021
Lipids are organic compounds

● Chemical compounds in which one or more atoms of
carbon are covalently linked to atoms of other elements,
most commonly hydrogen, oxygen, and nitrogen
○ There is sharing of atoms between carbons and ● The human cell membrane is described to be bilipid layer
the other elements mentioned ● The most abundant of which is the phospholipid and
● Examples inserted in between is the cholesterol
○ Carbohydrates
■ Several carbon atoms TYPES OF LIPIDS
attached to hydrogen
and oxygen Fatty Acids
● Linear chain of C-H bonds that
terminate with carboxylic acid
○ The reason why fatty acids
○ Proteins are named such is that
■ Made up of amino they are fats and they are
acids acidic because it contains
■ Composition: a carboxylic acid group
Carbon, Oxygen, ● Plasma - only a few exist as free (unbound) fatty acids
Hydrogen and and most are bound to albumin
Nitrogen ○ Why do they have to interact with albumin?
■ Nitrogen is an atom unique to proteins ■ The plasma of blood contains 90%
○ Lipids water and fatty acids are highly
■ Several carbon hydrophobic, they do not interact with
atoms are attached water
to oxygen and ■ For them to be transported in the
hydrogen blood they must attach to something
that interacts well with water and that
is albumin
Lipid Chemistry ● Some are found as part of triglycerides and
● Also known as fats phospholipids
● Composed mostly of carbon-hydrogen (C-H) bonds ● Fatty acids are the simplest types of lipids
● Functions ● Can be found in more complex lipids such as
○ Rich source of energy phospholipids, cholesteryl ester, and triglycerides
○ An efficient way of storing excess calories ● Good source of energy
■ Transported to the adipose cells ○ If the body runs out of glucose, the body uses
stored in the form of triglycerides fatty acids as a source of energy
○ Play an integral part of the cell membrane of ○ Fats are stored in adipose cells and if there is a
the human cells need for an additional source of energy, the fats
inside the adipose cells will be broken down to
release the fatty acids which will be utilized as a
source of energy
● Fatty acids are released and

transported through binding
with serum albumin
● Adipocytes seres as sites in the
body that would store lipids
● Excess glucose in the blood will be stored in the adipose ● Should there be a need for
cells of the body in the form of Triglyceride contributing to energy production the lipids will
the increase of the sizes of the adipose cells leading to be broken down and fatty acids
weight gain will be released into the blood
● The fatty acids along the blood
Cell Membrane Structure vessels are coated with the
white-colored structure which is
the albumin
● Some fatty acids exist as free
fatty acids in the blood but the majority are transported
by the albumin
1
● Once fatty acids are metabolized in the body, they can ● Both structures are composed of hydrocarbons that
produce acetyl-coenzyme A which will enter Kreb’s cycle terminate with the blue-colored carboxylic acid
● The products of Kreb’s cycle such as the FADH2 and ● In a saturated fatty acid, all of the carbon in the
NADH can proceed with the electron transport chain hydrocarbon chain are already bound to four bonds and
which is involved in ATP production you cannot break one bond and insert another hydrogen
atom
Fatty acids - Esterified with the glycerol backbone of ● In the case of unsaturated fatty acid, there is a presence
Triglycerides and Phospholipids of a double bond, we can still break that double bond and
insert hydrogen atoms that’s why the fatty acid is called
unsaturated because you can still add more hydrogen
atoms into it
Unsaturated fatty acids can be classified as cis or trans forms

● In order to classify hydrocarbons
as such, look at the carbon atoms
involved in the formation of the
double bonds. After identifying the
carbon atoms, take a look at the
hydrogen atoms attached to the
carbon atoms
● Left side: Triglyceride ● Cis forms- if the hydrogen atoms
○ Triglycerides have a glycerol backbone near the double bond are on the
○ It is an alcohol that has three carbons same side of the chain
○ Attached to the three carbons are three fatty ● Trans forms- if the hydrogen are
acids the reason why triglycerides are named on the opposite side of the chain
such is that they have three fatty acids (Tri) that
are attached to a glycerol backbone (glyceride)
● Right side: Phospholipids
○ Also have a glycerol backbone
○ Attached to two out of three carbons are two
fatty acids and these fatty acids are attached to
the glycerol backbones off triglycerides and
phospholipids by ester bonds
Classification of Fatty Acids

● Based on the length (number of carbons)
○ Short-chain (4-6 carbons)
○ Medium-chain (8-12 carbons) ● Take note that the presence of hydrogen atoms in the
○ Long-chain (more than 12 carbons) same side of the chemical structure on the carbon atoms
● Fatty acids have an even number of carbon atoms with the double bonds in the structure of the fatty acid
● Based on the number of C=C double bonds would cause kinking or bending
● But if the hydrogen atoms are found on the opposite
sides of the chemical structure of the unsaturated fatty
acid on the carbon atoms with a double bond, that will
not cause kinking or bending on the structure of the
unsaturated fatty acid
● If the lipid is primarily composed of unsaturated fatty
acids in their cis forms, the cis double bods would create
kinks and bends that’s why it is difficult to pack these
structures together and it would exist as liquid in room
temp
○ Saturated ● The majority of the fatty acids from plant sources are in
■ No presence of double bonds their cis forms that’s why the majority of lipids we get
○ Monounsaturated from plants are in liquid form or referred to as “Plant oil”
■ Presence of one double bond along
the hydrocarbon chain Hydrogenation
○ Polyunsaturated ● Cis unsaturated fatty acids can be converted to their
■ Number of double bonds go beyond trans forms by the process called hydrogenation
one ● The main purpose of hydrogenation is to convert liquid oil
into solid fats such as in margarine
2
Health effects ● Neutral lipid
● Fatty acids with cis configuration are typically found in ● Good energy source
natural foods ● Large lipids but light
● Most of the trans fatty acids are formed during the ○ Have large sizes but light in terms of weight
process of hydrogenation of the vegetable oils to convert ● 70%-80% of the fats in the diet
them to solid forms ○ Expect that after ingesting a meal, the majority
● These trans fatty acids are known to increase the levels of the lipids that will absorbed for the intestinal
of LDL or bad cholesterol in the blood lumen towards the blood are triglycerides
● When you have high cholesterol in your blood, there is a
high chance of developing heart attack and stroke in the Triglyceride is like a balloon: LARGE but light
future ● Big but light
● TAG are a good source of energy because they have 3
Saturated Fatty Acids fatty acids
● Saturated fat is solid at room temperature, which is why it
is also known as “solid fat”. It is mostly in animal foods Phospholipids
Triglycerides
● Glycerol backbone esterified with 3 fatty acids
○ Glycerol is an alcohol with three carbons
attached to it are three fatty acids and they are
attached to the carbon atoms via ester bonds
● The fatty acids in the triglycerides need not to be
identical to each other
○ If the fatty acid attached to carbon number 1 is
saturated, fatty acids attached to carbon 2 and
3 should not be necessarily saturated
○ It can be a combination fo fatty acids
● Each of the fatty acids can be potentially different thus ● Same structure as TAG except that they only have two
forming many possible structural forms of triglycerides esterified fatty acids
● Triglycerides that contain saturated fatty acids pack more ● Glycerol backbone with 2 fatty acid and 1 phosphate
closely and tend to be solid at room temperature - animal group on the 3rd carbon
sources ○ Choline is a chemical that contains a phosphate
● Most triglycerides from plant sources such as corn, group
sunflower seeds, etc are rich in polyunsaturated fatty ● Amphipathic (hydrophobic fatty acids and hydrophilic
acids in cis forms - oil phosphate head)
● Phosphate head groups can be choline, inositol, serine
Animal vs Plant fats and ethanolamine
● The triglycerides in the animal fat contain saturated fatty ● Most of the time, one of the fatty acids is saturated while
acids the other one is unsaturated
● While the triglyceride found in plant oil contains
unsaturated fatty acids
● Most abundant of the cell membrane - phospholipid

● Triglycerides are composed of glycerol and three fatty
○ Hydrophilic head
acids
■ Phosphate containing group
● Left side: alcohol containing hydroxyl group which is
○ Hydrophobic tail
known to be basic. Beside the glycerol are the three fatty
■ 2 fatty acids attached to the glycerol
acids
backbone
○ In the formation of ester bonds, what will be
● Since the cell membrane of humans is composed of
used are the hydroxyl groups of the glycerol
bilipid layer = there’s a certain arrangement of
backbone and the three carboxy groups of the
phospholipids
fatty acids
○ Outer part - hydrophilic heads
○ There is the reaction between the alkaline
○ Hidden in the middle - fatty acid tails
hydroxyl group and the acidic carboxylic group
■ Hydrophobic - never interacts with
○ In the reaction, there is neutralization, therefore
■ water
triglycerides are also referred to as Neutral
Lipids
● Hydrophobic- no polar charges
or hydrophilic groups
○ Glycerol and fatty
acid components of
triglycerides are
highly hydrophobic
○ Doesn’t interact well
with water
3
Cholesterol secrete cholecystokinin hormone causing the
contraction of the gallbladder, releasing now the
bile
- Bile will be drained in the bile duct into the
duodenum to facilitate digestion and absorption
of fats
● Some cholesterol molecules are converted by endocrine

glands to steroid hormones
● TESTES will CORner the OVARY (endocrine glands the
will produce steroid hormones from cholesterol)
○ Testosterone
○ Aldosterone
○ Cortisol
○ Androgens
● Composed of 4 fused hydrophobic rings called ○ Estrogens and progesterones
perhydrocyclopentanophenanthrene and 1 hydrophilic
hydroxyl group attached to A ring
● Amphipathic
○ Contains both hydrophobic and hydrophilic
portions
● Hydroxyl groups oriented outwards while the fused rings
are buried in the cell membrane
● Aside from phospholipids, we also have cholesterol
molecules in our cell membrane
● Chemical component of testosterone, aldosterone,

cortisol, androgens, estrogen and progesterone
● How do you think cholesterol is arranged in the bilipid ○ They all have that
layer of the cell membrane? perhydrocyclopentanophenanthrene ring
○ 2 lipid components: structure
■ phospholipids and phosphate heads ○ All derived from cholesterol
(displayed on the outer part)
■ Fatty acid tails in the center Cholesteryl Ester
○ Besides the 2 components - cholesterol
■ Embedded within the hydrophobic
fatty acid tails of the phospholipids
are the 4 fused rings
■ Exposed on the outer part of the
membrane is the hydroxyl group (can
interact with water, hydrophilic)
● Exclusively synthesized by animals (humans)
○ Because we have different sterols that other
organisms would produce (ex. Ergosterol in
fungi)
● Readily catabolized by cells thus does not serve as
source of energy
● Converted by cholic and chenodeoxycholic acid
(composing bile acid) which aids in fat digestion ● Same structure as cholesterol except that the hydroxyl
group is replaced by fatty acid
○ Cholesterol attached to another lipid via the
ester bond
○ Presence of 4-fused rings
(perhydrocyclopentanophenanthrene ring)
● Cholesterol esterified with fatty acid
○ No hydroxyl group, instead an attached fatty
acid
■ Fatty acid is esterified to the 4-fused
ring of the cholesterol
● Hydrophobic
○ 4-fused rings are hydrophobic, fatty acid is also
hydrophobic
■ Cholesteryl ester is highly
hydrophobic
- Cholesterol is converted by the liver to cholic ● Good source of energy because of the fatty acid
and chenodeoxycholic acid to compose bile ○ Cholesterol component can be used to form
acid bile acid and steroid hormones
- Bile acid is known to be stored in the
gallbladder
- In the presence of fats in the duodenum, it will
4
♡ NAME THAT LIPID ૮ ˶ᵔ ᵕ ᵔ˶ ა ○ TAG have 3 fatty acid = good source of energy
Made up of glycerol backbone and 3 Triglyceride ● VLDL
fatty acids ● LDL
● HDL
Made up of glycerol backbone, 2 fatty Phospholipid ○ smallest, contains the least amount of TAG
acids and one phosphate group ○ Densest, because it contains highest amount of
Made up of 4 fused rings and is highly Cholesteryl Ester apolipoproteins
hydrophobic ■ Proteins have high molecular weight
Made up of 4 fused rings and is Cholesterol ■ More proteins = more dense
amphipathic
Lipoproteins
Take note: ● Composed of lipids and proteins (apolipoproteins)
● Majority of the lipids are hydrophobic ● Main purpose is to transport energy → the core of
● Plasma is composed of 90% water lipoprotein essentially represents the cargo transported
○ If these lipids are secreted in the blood, they by the lipoproteins.
can't interact well with the components of the ○ Some of them would fxn to transport the
blood because they are hydrophobic substrate for steroid hormone production if they
● How are these lipids transported in the blood? primarily contain cholesterol.
○ These lipids must associate themselves with a ● Size correlated with its lipid content-- higher
hydrophilic substance in order for them to ○ Large lipoproteins have large core
interact with water regions--TAG and cholesterol esters
■ That hydrophilic substance is a ○ The larger the lipoprotein, the higher the lipid
protein content than proteins, the lighter it is.
○ These lipids will form complexes with proteins
in order for them to be transported and used up Apolipoproteins
by the cells in the body ● Protein portion of lipoproteins
● Lipids bind to proteins (water soluble) = LIPOPROTEINS ● Found on the surface of the lipoproteins
○ TAG + phospholipids + cholesterol + CE + ○ Help maintain the integrity of the lipoprotein.
Apolipoprotein = LIPOPROTEIN ■ So that the lipids could be packed
together
○ Some bind to host cell receptors.
○ Some are activators or inhibitors
Proteins are hydrophilic in nature, so how can this apolipoprotein
pack themselves with hydrophobic lipids?
● Associates with the lipids because of their amphipathic
helix (protein sequences that fold once in contact with a
polar/non-polar interference.
● Assembly of this lipoprotein is for the purpose of

transporting the lipids
● It is composed of:
○ TAG
○ Phospholipids
○ Free cholesterol
○ Cholesterol bound to fatty acids (cholesteryl
ester)
● Take a look at the locations of these lipids in the
lipoprotein
○ TAG and cholesteryl ester are highly
hydrophobic - found in the core (center) to hide
away from water
○ Phospholipids and free cholesterol are
amphipathic - found in the surface because ● Non polar = hydrophobic
they can interact with water ● Polar = can interact with water
■ Phosphate head of phospholipids ● Even if proteins are generally hydrophilic, they also have
■ Hydroxyl group of cholesterol hydrophobic portions.
○ Apolipoprotein (protein component) - found in ○ Bcoz some of the amino acids found in the
the surface structure of these proteins could be non polar.
Different Lipoproteins in the Blood
● Chylomicron
○ Appears big, contains the largest lipid (TAG)
5
● Intestinal lumen→ large TAG; if that FA by the pancreatic
lipase, FA will be removed
● The purpose of removing the FA is to reduce the size of
the TAG so that it will become small and will be readily
absorbed by the intestines.
○ But once absorbed the TAG will be
reassembled.
● As soon as the monoglycerides and the two FA will be
absorbed→ inside the cytoplasm of the intestinal cells of
the enterocytes, the TAG will be reassembled.
Blue: apolipoprotein (have folds depending on whenever they are

exposed to hydrophobic or hydrophilic surfaces)
● Folds
○ Contains non polar amino acids
○ Could be inserted deep down into lipoproteins
bcoz these non polar amino acids cannot
interact well with water but can interact with
● In the action of lipase, one fatty acid could be removed
TAG and cholesterol ester of lipoproteins.
from TAG and converting into diglyceride
○ The will use other folds (composed by polar
● Further action of lipase will remove another fatty acid
amino acids) to interact with water in the
converting diglyceride into monoglyceride.
plasma.
CHOLESTEROL
Lipid Absorption
● Fatty acids are small, so they can just be easily absorbed
thru passive diffusion from the lumen of the intestine
across the intestinal cells towards the blood circulation.
● Triglycerides are acted upon by pancreatic lipase which
cleaves them into fatty acids and
monoglycerides/diglycerides.
○ Large
○ Not easily absorbed the microvilli intestinal
cells.
○ If there is fat in the diet, the pancreas will be
triggered to release its secretion→ pancreatic
lipase→ act on TAG
Pic above: intestinal lumen

● In the lumen, cholesterol molecules will be mixed with the
bile acids, mixing with the bile acids will enable the
cholesterol molecules to be absorbed via the NPC1-L1
receptor displayed on the apical surfaces of the
enterocytes.
● Part of the hypercholesterolemia
○ Is to block the NPC1-L1 receptor
○ So that the cholesterol in the diet will no longer
be absorbed and we will now reduce the blood
level of the cholesterol.
● With the action of lipase, one or two fatty acids will be ● Cholesterol is absorbed by the action of the bile
removed from the TAG ● Absorption is via the NPC1-L1 receptor
● If one FA is removed ● Cholesteryl ester acted upon by cholesteryl esterase
○ diglyceride or diacylglycerol producing cholesterol and Fatty acid
● If two FA are removed What if the lipid present in the diet is cholesteryl ester or esterified
○ monoglyceride or monoacylglycerol with FA?
● cholesteryl ester will be acted upon first by cholesteryl
esterase enzyme from the pancreas.
● cholesteryl esterase enzyme
○ Break ester bonds
■ → Liberation of the FA from the
cholesteryl ester → cholesterol→ mix
up with bile→ absorbed by NPC1-L1
receptor→ absorbed thru passive
diffusion (because its small)
6
Phospholipid cells of the body for the TAG components (
glycerol and fatty acids) to be converted into
energy.
● Because of the large size of chylomicrons, they can
reflect light making the plasma/ serum of the px turbid
● If u are going to centrifuge the blood, and since
chylomicrons are light, they will settle on the upper
portion of the plasma. Thus, the plasma would appear
turbid
● Phospholipid is acted upon by the phospholipase

enzyme releasing fatty acid and lysophospholipids
● The short chain fatty acids from the digestion of TAG,
phospholipids and cholesteryl esters are readily
absorbed and are transported by albumin in the blood.
● If this phospholipid will be acted upon by phospholipase
enzyme in the intestinal lumen, one of the FA will be
removed and the remaining portion of the phospholipid
will be called lysophospholipids
○ Lysophospholipids
■ One that has the head in a single FA ● 2 red arrows: turbid plasma of a px who have ingested a
tail meal = rich in chylomicrons
■ Simply be absorbed by intestinal ● Upon storage, these chylomicrons would appear as
cells. creamy layer on the superficial portion of the plasma bcs
● TAG it has the least density
○ Pancreatic lipase
● Cholesterol
○ Bile
● Cholesteryl ester
○ Cholesteryl esterase
● Phospholipid
○ phospholipase
● Chylomicrons = largest but lightest

○ Thus, it can make the plasma of the px who
had a meal appear turbid
Take note!!
● This TAG along with the absorbed cholesterol,
phospholipid and cholesteryl ester will be assembled in
the form of CHYLOMICRONS
○ Will only be formed if the TAG, cholesterol,
phospholipid and cholesteryl ester will be in
complex with apolipoprotein.
Component of chylomicrons
● TAG ● TAG in the diet will be broken down into smaller
● Cholesterol components in order for it to be absorbed
● Cholesteryl ester ● Once absorbed, it will be reassembled back to TAG and
● Phospholipid will be bound to cholesterol, cholesteryl ester and
● Apolipoprotein phospholipids that were also absorbed from the diet and
What component will predominate? will be formed in complex with an apolipoprotein
● 80% of them are TAG ● Together, they will be assembled to form the chylomicron
● Rich in TAG lipoprotein
● TAG are large, chylomicron are also large ● The chylomicron will be released on the basal party of
● TAG are light in terms of weight= chylomicrons would the intestinal cells towards the lamina propria and will be
have less density absorbed in the green colored lymphatic capillaries
○ Why? Chylomicrons are large lipoproteins and
Chylomicrons they cannot diffuse through the wall of blood
● Transport diet-derived lipids vessels
● Contain large amount of TAG = large but light!! ○ Also, lymphatic capillaries are more permeable
● Large size enables it to reflect light and account for the than blood capillaries
turbidity of the postprandial plasma
● Light weigh causes it to float to the top of the stored
plasma and form a creamy layer
● Good source of energy (fatty acids in Triglycerides)
○ Should be transported in the blood towards the
7
● How can they be transported towards our body so that Chylomicron lipoproteins secreted initially to the lymph
the TAG will be used up? ↓
Chylomicrons that were absorbed, will be transported via the Drained to the subclavian vein by the thoracic duct
lymphatic capillaries ↓
↓ Once in the blood, it is transported to the tissues.
These lymphatic capillaries will later transport the chylomicrons ↓
towards the thoracic duct In the capillaries of these tissues, it will interact with heparan
↓ sulfate and other proteoglycans, activating lipoprotein lipase (LPL)
Thoracic duct drains towards the subclavian vein ↓
↓ LPL will act on the TAG components of the chylomicrons; liberating
After the drainage into the subclavian vein, the chylomicron now is glycerol and fatty acids converting it to energy
in our blood circulation ↓
Chylomicrons lose its TAG components and will reduce in size
which will be referred as chylomicron remnant
↓
Chylomicron remnant will be metabolized or cleared by the liver
● Thus, hours after eating, expect that the plasma of the

patient will become clear bcs of the clearance or
metabolism of chylomicrons.
Entire process of synthesizing chylomicrons and metabolising

them is called the: Exogenous pathway
- Chylomicrons are derived from lipids from the diet
● Lipoprotein lipase hydrolyzes TAG on the chylomicrons
generating fatty acids and glycerol
● The fatty acids and glycerol are then taken up by the
cells and are sued as source of energy
Exogenous pathway ● Excess fatty acids are then stored in adipose tissues and
● The newly synthesized chylomicrons in the intestine are are stored as triglycerides
initially secrets into the lymphatic ducts and eventually
enter the circulation by the way of the thoracic duct
● In the tissues, chylomicrons bind to heparan sulfate and
other proteoglycans present in the capillaries of
different tissues, The binding to proteoglycan promotes
interaction with lipoprotein lipase.
○ Lipase - specialized in liberating fatty acids
from triglycerides
○ Chylomicron lipoproteins will interact with
lipoprotein lipase and all of the fatty acid in TAG
will be liberated and will be used up by the cells
in the tissue as source of energy
○ The cells can also use the glycerol backbone
as source of energy ● Excess fat is stored in adipocytes, which expand in size
○ The chylomicron will lose its TAG component until the fat is used for fuel
bcs it has been used up.
○ Thus, it will reduce in size and will be referred
as chylomicron remnant
● If the body is subjected to fasting/starvation, the stored
8
TAG will be broken down to release the fatty acid which
will be used up by the cells as sources of energy
VLDL synthesized in the liver

↓
Secreted into the blood circulation
↓
Exogenous pathway VLDL will be acted upon by LPL since it is also rich in TAG
● After lipolysis, chylomicrons is converted into ↓
chylomicron remnant 50% of the VLDL remnants will be transported to the liver for
● Chylomicron remnants are then rapidly taken by the metabolism
hepatocytes of the liver and are broken down by the ● What do you think will happen to the other 50%?
lysosomes into free cholesterol, fatty acids and amino
acids
VERY LOW DENSITY LIPOPROTEIN

● Involved in endogenous pathway
○ Lipids transported are produced in the (liver)
○ These lipids are synthesized by the liver in
times of fasting and starvation
● Transports hepatic-derived lipids
● Transfers triglycerides from the liver to the peripheral
tissues
● Contains large amount of Triglycerides but not as much
as Chylomicrons = Large but light!!!
○ VLDL is large but not as much as chylomicrons
○ VLDL is light but not as light as chylomicron
● 50% of the VLDL remnants will be metabolized by the
*the higher the TAG, the lighter the protein is
liver.
● Can also block light similar to chylomicrons
● While the remaining 50% will start accumulating
○ Thus, it can also make the plasma turbid
cholesterol in its structure
● Good source of energy (fatty acids in triglycerides)
● The VLDL remnant will become the intermediate
density lipoprotein (IDL)
○ This IDL will still continue accumulating
cholesterol until such time that it will become
the low density lipoprotein (LDL)
● LDL has high cholesterol bcs it was derived from VLDL
catabolism by accumulating cholesterol
○ purpose: transport cholesterol
○ LDL is good because it provides the cell
cholesterol to become part of their cell
membrane.
○ It provides the testis, adrenal cortex, and
ovaries cholesterol for them to produce steroid
hormones
● Like chylomicrons, it can also reflect light and account for ● If LDL is in excess, it can deposit cholesterol in the walls
the turbidity in fasting hyperlipidemic plasma of the arteries hence called the bad cholesterol
○ But will not form creamy layer like that of ○ Only bad if excessive
chylomicrons ● What triggers the LDL synthesis are:
○ High carbohydrate diet
■ Only chylomicrons can settle on the
○ Eating saturated fatty acids from animal
superficial portion of the plasma sources
● What will trigger the liver to produce high amounts of ○ Eating too much trans fatty acids
VLDL?
○ High amounts can be harmful Endogenous Pathway
● Excess dietary intake of carbohydrates, saturated fatty ● Lipids are all produced in the liver
acids from animal sources, and trans fatty acids from the ● VLDL, once secreted into the blood, undergo a lipolytic
hydrogenation of plant oils or cis form of unsaturated process similar to that of chylomicrons
fatty acids enhance the hepatic synthesis of triglycerides ● VLDL loses core lipids in the action of Lipoprotein lipase
and becomes the VLDL remnants
● ½ of VLDL remnants gain cholesterol and eventually
becomes the IDL and LDL
● ½ are taken up by the liver and are metabolized
9
● Where do we derive chylomicrons? hormones
○ Lipids in the diet
● Where do we derive VLDL?
○ Lipids produced from the liver
● Where do we derive LDL?
○ From the catabolism of VLDL
VLDL vs LDL
● LDL is smaller because it has lesser triglyceride and has
more cholesterol
○ Capable of transporting cholesterol to tissues
Low-Density Lipoprotein
● Transports cholesterol to the different cells in the body
○ Good because it provides cholesterol to the
cells for the formation of their cell membrane
○ Provides testis, adrenal cortex, and ovaries the
substrate for the formation of steroid hormones
● Taken up by the cells by binding to LDL receptors
present in the cells High Density Lipoprotein
● “Bad” Cholesterol ● Smallest but the densest lipoprotein
○ Excess cholesterol ○ Composition is protein
■ Proteins have a high molecular weight
● Transports cholesterol from the cells to the liver for
elimination or formation of bile acids
○ Reduces chances of stroke and acute
myocardial infarction
○ Bile is the means to excrete excess cholesterol
● Secreted by the liver and small intestine
● “Good” cholesterol
● Has APO-A1 (activator of Lecithin Cholesterol
Acyltransferase or LCAT)
○ A- angel
○ Picks up cholesterol, cholesteryl ester, and
triglycerides = HDL
● LDL transports cholesterol to different tissues of the body

through:
LDL uses the green-colored APO B-100 to bind to the LDL

receptor present on the cell membrane of the human cells
↓
Binding will cause the LDL to be internalized by the cell
↓
Lysosome will digest the engulfed LDL, release the cholesterol
from it
↓
Cholesterol will be used as part of the cell membrane or will be
converted to steroid hormones
● Once bound to the receptors, the LDL is endocytosed 2 types of HDL:

● Transported to the lysosomes for degradation 1. Discoidal HDL
● TAG component is acted upon by acid lipase to produce - Newly synthesized HDL
glycerol and fatty acids - the source of energy 2. Spherical HDL
- Mature HDL
● Cholesterol - may be used for membrane synthesis,
synthesis of steroid hormones and stored as cholesteryl
esters by the enzyme acyl-CoA: cholesterol
acyltransferase (ACAT)
○ The cholesterol part of the LDL may be used for
membrane synthesis or synthesis of steroid
10
Reverse Cholesterol Transport Pathway transporter A1 (ABCA1)
○ Absence of ABCA1 = no efflux
■ Discoidal HDL will never get
cholesterol and lipids
■ No mature HDL
■ Cannot excrete cholesterol through
the bile
● Subsequent action of lecithin-cholesterol acyltransferase
(LCAT) esterifies cholesterol in discoidal HDL particles
and converts them to mature/spherical HDL particles
● This is the discoidal HDL and it is primarily composed of
APO lipoprotein A1
Reverse Cholesterol Pathway
● The main purpose is to pick up small amounts of
cholesterol and triglycerides from the tissues to be
transported to the liver
● Yellow-colored cholesterol is now loaded on the discoidal

HDL
● This cholesterol might escape from the structure of the
HDL ● Efflux will be mediated by the body by ABCA1 receptor
○ Cholesterol must be transported to its core ● Cholesterol and lipids will be part of the discoidal HDL
region ● LCAT will convert free cholesterol to cholesteryl ester
○ Since they are amphipathic, you cannot put and store to the core region of HDL = mature HDL
them on the core region ● Cholesteryl ester is transported to the liver
○ Will be esterified with the fatty acid converting ● HDL will bind to SR-B1 receptor
them to the green-colored cholesteryl ester
■ Converted by LCAT
■ APO-A1 is the activator of LCAT
● From the discoidal form, HDL will now assume the
spherical form
● APOA1 is produced in the liver and the intestine and will

be incorporated as part of the discoidal HDL
● Cholesterol and phospholipids will efflux from the cells of
the body through the ABCA1 receptor and ABCG1
receptor
● The cholesterol and phospholipids will become part of
the discoid HDL
● With the action of LCAT, the cholesterol molecules will be
sterified to form the cholesterol ester and the discoid
● Lecithin is a phospholipid HDL will become the spherical (mature) HDL
● With the action of LCAT, the fatty acid in the second
carbon of lecithin will be esterified to the cholesterol
converting now the free cholesterol to cholesteryl ester
● Recall:
○ Exogenous = chylomicrons
○ Endogenous = VLDL (can give rise to LDL)
● Excess cholesterol from non-hepatic tissues is
transferred to the liver for metabolism and excretion into
the bile
● Lipid-poor discoid HDL particles, produced in the liver or
the intestine, initiate the efflux of cholesterol and Synthesis of the Apolipoprotein A1 by the liver and small
phospholipids from cell membranes via interaction with intestine
the adenosine triphosphate-binding cassette transporter ↓
A1 (ABCA1) Discoid HDL will pick up phospholipids and cholesterol from the
● Subsequent action of lecithin-cholesterol acyl transferase tissues
(LCAT) esterifies cholesterol in preB-particles and ↓
converts them to mature a-HDL particles With the action of LCAT, the cholesterol will be converted into
● Excess cholesterol from non-hepatic tissues is cholesterol ester, converting the discoid HDL into the spherical
transferred to the liver for metabolism and excretion into (mature) HDL
the bile ↓
● Lipid-poor discoid HDL particles (APO-A1), produced in HDL will be able to transport the cholesterol towards the liver by
the liver or the intestine, initiate the efflux of cholesterol binding to scavenger receptor Type B1 (SRB1)
and phospholipids from cell membranes via interaction
with the adenosine triphosphate-binding cassette
11
bloodstream
● Chromosome 9 has the gene that encodes for the
ABCA1 transporter.
○ If this transporters are defective, there would be
no efflux of cholesterol and phospholipids into
the blood
○ Therefore, the discoidal HDL will no longer
receive cholesterol (will not be acted upon by
LCAT enzyme) and you will not have the
formation of mature or spherical HDL
○ Hence, there would be accumulation or
deposition of cholesterol in the different part of
● Some of the spherical or mature HDL will be acted upon the body which predisposes someone to acute
by cholesteryl ester transfer protein (CETP) which will myocardial infarction
transfer the cholesterol ester of HDL into VLDL and LDL
● The LDL and VLDL might help in the elimination of the

cholesterol by binding to LDL receptors present in the
liver ● Most will have yellowish depositions in their tonsils
○ Which is a relief, because we know that LDL is which are composed of cholesterol and have an
a bad cholesterol since it transports cholesterol increased risk of developing plaque formations in the
in the tissues and if present in excess, it might arteries, resulting to peripheral vascular disease, stroke,
cause the formation of atherosclerotic plaque and coronary heart disease or acute myocardial
on the walls of the arteries infarction
● But with the CETP, some of the cholesteryl esters will be
brought to the liver for excretion by LDL Apolipoproteins
● However, not all of these LDL will transport the ● Proteins that bind lipids to form lipoproteins
cholesteryl ester towards the liver. Some will go to the
circulation and bring the cholesteryl ester and cholesterol 1. Apolipoprotein A1
towards the tissues ● Produced by the liver and intestine
● So, if the patient has high HDL levels in the blood or has ● Activator of LCAT enzyme which is very important in HDL
hypercholesterolemia, it’s better to give a drug that will ● Major protein component of HDL particles in plasma
inhibit the CETP. ● Chylomicrons secreted from the intestinal enterocyte also
○ So that HDL will no longer give or transfer the contain apo A1, but it is quickly transferred to HDL in the
cholesteryl ester to LDL and all of the bloodstream
cholesteryl ester will be transported by HDL ● ANGEL - GOOD CHOLESTEROL - HDL
towards the liver via the SRB1. ● Initially produced in the liver and small intestine and after
● Mature HDL can deliver cholesterol to the liver either its production, it will start accumulating the cholesterol
directly via: and phospholipids that have leaked out of the cells
1. The scavenger receptor type B1 (SR-B1) through the help of ABCA1 receptor
2. Indirectly by exchange of cholesterol esters to ● The chylomicrons which are assembled in the intestine
apoB containing particles for triglycerides (TG) (the origin of Apo A1), will also originally contain ApoA1
● Cholesterol esters can be exchanged but with the encounter of HDL, it will just simply transfer
for triglycerides in apoB-rich particles the Apo A1 to HDL
(LDL and VLDL) by cholesteryl ester ○ Because Apo A1 is more useful to HDL
transfer protein (CETP)
● HDL will gain triglycerides; VLDLand 2. Apolipoprotein B100
LDL will gain the cholesteryl esters ● 100% BAD - LDL
● VLDL will become LDL and its role is ● Primarily synthesized in the liver along with the synthesis
to transport the transferred cholesteryl of VLDL
ester towards the liver by binding to ● Will not be incorporated in chylomicron
LDL-receptors ● Important in the assembly of VLDL and cellular uptake of
● The uptake of apoB-rich particles via LDL
hepatic LDL receptors enables the ● Present in VLDL, IDL, and LDL
delivery of cholesterol to the liver
(approximately 50% of RCT)
Tangier Disease
● An inherited disorder characterized by significantly
reduced levels of high-density lipoprotein (HDL) in the
blood
● Mutations to chromosome 9q31 lead to a defective
ABCA1 transporter. These mutations prevent the
ABCA1 protein from effectively transporting cholesterol
and phospholipids out of cells for pickup by ApoA1 in the
12
VLDL 55 18 20 5 9
LDL 10 50 29 11 20
HDL 6 40 46 7 50
● Apo B100 will be used by the LDL to bind to the LDL

receptors present on the cell membrane of human cells
for the LDL to be metabolized and internalized
3. Apolipoprotein B48
● ApoB48 is identical to the amino-terminal 48% of
ApoB100
● Involved in the synthesis, assembly and secretion of
chylomicrons
● Stabilizes the chylomicron lipoprotein molecules PART 2
4. Apolipoprotein C-II Adult Reference Range for Lipids

● Activator of lipoprotein lipase Analyte Reference range
● Present in chylomicrons, VLDL, IDL, nad LDL Total Cholesterol 140 -200 mg/dL (3.6-5.2 mmol/L)
HDL-C 40 -75 mg/dL (1.0-2.0 mmol/L)
5. Apolipoprotein E
LDL-C 50 -130 mg/dL (1.3-3.4 mmol/L)
● Facilitates the binding of lipoprotein rEmnants to the liver
for elimination or metabolism Triglycerides 60 - 150 mg/dL (0.7-1.7 mmol/L)
● Present in chylomicrons and VLDL
Lipid and Lipoprotein Population Distribution
● Blood levels of cholesterol and triglycerides increase with
age
○ Circulating levels of total cholesterol, LDL, and
TAG in young children are lower than in adults
○ Can be affected by diet
● Women have higher HDL and lower total cholesterol than
men due to the hormone estrogen
○ The differences in HDL and total cholesterol
disappears after menopause
○ Men would have lower HDL and higher total
and put men at higher risk than women of
developing coronary heart disease
● Chylomicron → converted to chylomicron remnant →

metabolised by the liver using its Apolipoprotein E
● VLDL → uses its Apolipoprotein E for the liver to
catabolize VLDL remnant
Class Origin Major function Diameter Major Concentration

(nm) protein in interstitial
fluid
chylomicrons Ileum Transport 200-1000 apoB48 Absent
ingested fat
and fat-soluble
vitamins
VLDL Liver Transport 30-90 apoB100 Absent
synthesized
glyceride
LDL Lipolysis of Deliver 22-28 apoB100 ~9% that in
VLDL cholesterol to plasma ● Shows the differences in the cases of coronary heart
cells diseases between men and women of different age
HDL Liver and Reverse 7-11 apoA1 ~20% that in groups
Ileum cholesterol plasma ● In the age groups 20-54, men generally have higher
transport cases of coronary heart diseases primarily because men
have lower HDL and higher cholesterol levels at that
time
Average composition by weight (%) ● As soon as women will be in their menopausal stage,
Class they will also havee decreasing levels of estrogen and
TG CE PL Chol Protein
HDL levels of men and women will become equal and
chylomicrons 85 3 8 2 1 their total cholesterol level will be equal
13
● The risk to develop coronary heart disease will almost increasing awareness of CHD
be the same between men and women ● Stems from the deposition of cholesteryl esters in the
● HDL levels for boys and girls are comparable to that of arterial walls
adult women ○ The presence of cholesterol molecules will
○ However, a drop of 20% in HDL is seen in boys trigger the migration of monocytes from the
during puberty (same level as male adults) blood towards the wall of the artery
● Lower HDL cholesterol coupled with higher LDL ○ These monocytes will eventually differentiate to
cholesterol increases the risk of heart disease in men form the macrophages
● Genetic and lifestyle are two factors that increase the ○ These macrophages will try to engulf the
risk for heart disease cholesterol and cholesterol molecules in an
○ Genetic and lifestyle are factors that can alter attempt to clear them from the wall of the
the levels of lipids in the blood arteries
● Diet- people who tend to eat less animal fat and more ● Deposition results in the formation of fatty streaks
grains, fruits, and vegetables have lower LDL ○ Group of macrophages with fats in their
● National Cholesterol Education Program (NCEP) cytoplasm are called fatty streaks
developed a list of risk factors for heart diseases ○ Thin steaks of fats within macrophages within
the subendothelial spaces
Coronary Heart Disease Risk Factors Determined by

(Exclusive of LDL-C) NCEP ATP III
Positive Risk Factors:
● Age ≥ 45y for men, ≥ 55y for females, or premature
menopause
● Family history of premature CHD
● Current cigarette smoking
● Hypertension (BP ≥ 140/90 or taking antihypertensive
drugs)
● HDL-C Concentration < 40 mg/dL (< 1.0 mmol/L)
● Diabetes Mellitus = CHD rissk equivalent
● Metabolic syndrome (multiple metabolic factors)
Negative Risk Factors:
● HDL-C concentration ≥ 60 mg/dL (≥ 1.6 mmol/L); its ● Gross appearance of a fatty streak
presence removes one risk factor from the total count ● Expect that underneath this one, u have a group of
● Women would have a later onset of coronary heart macrophages that have ingested the deposited
diseases because they have high estrogen levels before cholesterol and cholesteryl ester molecules
the age of 55 ● The lipoprotein involved in the deposition of cholesterol
● The genetic makeup of the person will also be a factor in and cholesteryl molecules on the wall of the arteries is
determining whether the person will develop coronary LDL
heart disease or not ○ LDL functions to transport cholesterol towards
● Having low levels of HDL will also put you at risk to the cells or tissues
develop heart problems ○ If you have high amounts of LDL, the cells will
● DM and metabolic syndrome be saturated with cholesterol
○ Metabolic syndrome - there’s a combination of ○ The excess cholesterol will be deposited by
the other risk factors LDL on the walls of the arteries
■ E.g.: a person has diabetes and ○ The presence of cholesterol and cholesteryl
hypertension at the same time ester deposits will now trigger the migration of
the monocytes towards the walls of the arteries
Lipid Disorders: Diagnosis and Treatment and their differentiation into macrophages
● Collectively called dyslipidemia ○ These macrophages will try to engulf the
● Characterized by elevation of plasma cholesterol, molecules but the problem is, the engulfment of
Triglycerides (Gs), or both, or a low high-density this LDL molecule or cholesterol deposits will
lipoprotein cholesterol level that contributes to the trigger some genes inside the macrophages
development of atherosclerosis and other related ● LDL is believed to have a central role in initiating the
diseases formation of plaque on the walls of the arteries
● Can be caused by genetic abnormalities or ● LDL is oxidized and is then engulfed by various cells
environmental factors or lifestyle imbalances especially macrophages
● other occurs secondary to underlying disorders ○ This alters the gene expression of the
macrophages and they start synthesizing
Arteriosclerosis inflammatory mediators
● Occurs when arteries grow thick and stiff and restrict ■ Inflammatory mediators cause injury
blood flow to organs and tissues in the body to the area where the macrophages
○ Results to the occlusion of the artery are present
○ Imagine the coronary arteries of the heart, if ○ Referred to as foam cells
they will become occluded because of ■ The group of foam cells seen grossly
atherosclerosis, expect that the cardiac muscle is called the fatty streak
will no longer receive blood supply and will
undergo necrosis
● One of the causes of death and disability
○ If not treated could cause death
○ If treated, there would still be some level of
disability because cardia muscles can no longer
regenerate. So , the ability of the person to
perform strenuous activities will be reduced
● Mortality has decreased through time due to
advancements in diagnosis and treatment and
14
and this aggravates the plaque formation
● Some can burst and cause thrombosis
○ Cause thrombi or clots which could occlude
smaller blood vessels
● Final event = occlusion of blood flow
● Blood vessels with growing atherosclerotic plaque

● There is narrowing of lumen because of the plaque
● In the middle of the picture, there is the macrophage

recruited from blood monocytes. It will try to engulf the
excess LDL or the deposited cholesterol and cholesterol
ester molecules
● Macrophages will turn into foam cells
● The foam cells will produce inflammatory mediators
which causes injury to the walls of arteries
● If there is injury, there would be inflammation and ● B and C show a plaque that recently ruptured and there
damage to the wall of arteries and platelets will stick to is active bleeding and there is also a defect. This might
the wall of arteries causing the formation of plaque have thrown thrombi away which will be a problem if it
obstructs smaller capillaries
● Peripheral vascular disease - if the plaque develops in
the arteries f the arms or legs
● Coronary Artery Disease - heart
○ Acute myocardial infarction
● Cerebrovascular disease - brain
○ Stroke
● Lipid deposits are most of the time seen in cases of high
serum LDL concentration or decrease HDL concentration
● Goal: To lower LDL concentration
● For every 1% decrease in LDL concentration, there is 2%
reduction in the risk of developing arteriosclerosis
● For patients with a history of previous heart disease,
lowering the LDL below 100mg/dl (2.6mmol/L) is effective
in the stabilization and even in the regression of the
● A monocyte has differentiated to become a macrophage plaque
● Macrophages take up excess LDL and will become a ○ Problem: some of the plaques may rupture,
foam cell which can produce/elaborate a lot of cytokines may produce thrombi which may occlude
which can cause damages to the walls of the arteries smaller blood vessels or capillaries
○ We are contented with stabilizing the plaque
○ LDL must be maintained before 100mg/dl
● LDL should be lowered in patients:
○ Whose diets are rich in fats
○ Who lack daily exercise
○ Who are smoking and drinking alcohols
○ Have diabetes, hypertension and kidney/liver
diseases
○ Are obese
● Injury signals from the growing fatty streaks or plaque

trigger the expression of adhesion proteins which allow
the migration of more macrophages, platelets, and
lymphocytes
● The monocyte is usually found in the circulating blood
that’s in the lumen of the blood vessel
● Look at the orange-colored structure that the monocyte
will anchor itself to so it can go to the wall f the artery
and become the macrophage which becomes the foam
cell
● The elaboration of the cytokines by the foam cell will
increase the number of those orange-colored proteins ● Total cholesterol can be lowered by:
which are the adhesion proteins ○ Cholestyramine - bile acid sequestrants
● Chronic inflammation results from repeated cycles of ■ Bile acid is produced in the liver and
injury is temporarily stored in the gallbladder
● There would be the formation of plaque and the plaque ■ In the presence of fat in the diet, the
will increase its size gallbladder will contract releasing bile
● Increase in the plaque results in occlusion of blood flow ■ Bile will mix with cholesterol in the
● Narrowing of the blood vessels causes the blood to diet and absorbed in the blood
circulate in a nonlaminar manner under great pressure ■ In the presence of cholestyramine, it
15
will form a complex with the bile acid, ■ To prevent this from happening and to
bile acid cant interact with cholesterol only allow the HDL to bind to the liver
■ Cholesterol and bile acid will not through the SRB1 receptor and not
absorb in the blood transfer its cholesteryl ester to LDL,
then you have to inhibit CETP
○ Niacin-containing drugs - decreased hepatocyte
production of CETP
■ Can cause flushing
● Can cause discomfort, but
no harmful effects
● You have to monitor the liver
enzyme of the patient
■ Hepatotoxic
■ Niacin containing drugs will decrease
the production of CETP
● No CETP = HDL won't
transfer its cholesteryl ester
○ Ezetimibe - inhibiting the NPC1-L1 transporter to LDL
which is responsible for the absorption of ○ Fibrates - increases hepatic production of
cholesterol apaA-1
■ Bile acid will interact with cholesterol ■ Increase the production of
■ Cholesterol will be absorbed by the apolipoprotein A-1 by the liver =
intestinal cells via the NPC1-L1 higher number of discoid HDL =
receptor higher number of spherical or mature
■ If receptor is inhibit, the cholesterol HDL
will not be absorbed
○ Niacin Different diseases associated with abnormal levels of lipids in the
■ Increase blood HDL blood:
● High amount of HDL =
higher amounts of Familial hypercholesterolemia
cholesterol to be delivered ● Inherited
to the liver for excretion ● Increase cholesterol level in the blood (particularly LDL)
○ Statins - HMG-CoA reductase enzyme inhibitor ● Autosomal dominant
■ This enzyme is used by the cells to ○ You only need one abnormal gene to manifest
produce their own cholesterol the condition
■ If inhibited, the cell will not produce its ● Increased risk to heart diseases
own cholesterol, making it forced to ○ Increased cholesterol in the blood = increased
get cholesterol from the LDL chances that this will be deposited on the walls
lipoproteins in the blood arteries
○ Higher risk to develop stroke and coronary
heart diseases
● Caused by mutation in the gene for the LDL cholesterol
receptor
○ LDL receptors is important in the uptake of LDL
from the blood by the cells in the body
○ So if cells can't express LDL receptors, then
LDL in the blood will not be taken up by the
cells
■ Increased LDL levels in the blood =
atherosclerotic plaque formation
● Homozygous
● Cell that can produce its own cholesterol by ○ A person has 2 abnormal gene inherited from
metabolising HMG-CoA with the help of the both parents
enzyme HMG-CoA reductase ■ Absence of
● HMG-CoA will be converted to mevalonic acid, LDL receptors
which will be utilized by the cell or converted to ○ 1:1 million in population
cholesterol ○ Total cholesterol
● In people with hypercholesterolemia, you can 800-1000mg/dL
give them statins (Normal: 20-26mg/dL)
○ It will inhibit the HCG-CoA reductase ■ Normal value:
enzyme 140-200mg/dl
○ Cell cant produce its own cholesterol, ○ First heart attack in their
forcing it express LDL receptors on its teenage years
cell membrane to engulf or get the ■ Some would die young because of
LDL molecules from the blood thereby coronary heart disease
lowering the cholesterol level of the ● Heterozygous
blood ○ 1 normal gene from 1 of the parent
● HDL can be increase by drugs such as ■ Only reduction in the expression of
○ CETP inhibitors LDL receptors
■ CETP (Cholesteryl ester transfer ○ 1:500 in the population
protein) - causes HDL to transfer its ○ Total cholesterol: 300-600mg/dL
cholesteryl ester to LDL ○ Become symptomatic in their 20s to 50s
■ Problem: sometimes LDL will ○ Some may die of familial hypercholesterolemia
transport the transferred cholesteryl
ester to the tissues thereby increasing
the chance for arteriosclerosis
16
utilized by the cells in the body as source of
energy, so the body will have to look for other
potential sources of energy
■ So the fats stored in adipose cells will
be broken down to form glycerol and
fatty acids and will be secreted in the
blood
■ Blood will transport the fatty acids and
glycerol to the liver
So, how do we manage heterozygous and homozygous FH? ■ Liver will assemble the glycerol and
● Heterozygous FH patients (reduced expressions of LDL fatty acids into TAG, it will pack these
receptors) TAG into VLDL molecules
○ Treated with statins or HMG-CoA reductase ■ That’s why in diabetes, there is
enzyme inhibitors increase production of VLDL (rich in
■ Statin drugs - rosuvastatin and TAG)
atorvastatin ■ Subsequent increase in the blood
● Inhibit the HMG-CoA TAG level
reductase enzyme so that ■ Depresses the removal of the large
the cells in the body won't molecular constituents such as TAG
produce their own from the blood.
cholesterol, forcing to ● Can be caused by hormonal imbalance(epinephrine.
express more LDL receptors norepinephrine, growth hormone, ACTH, glucagon)
so that they will get the ○ Can activate the particular enzyme = hormone
LDLs from their blood sensitive lipase
● Results to the reduction on ■ Found in adipose cells.
the LDL levels of patients ■ Lipase
with heterozygous FH ● Breakdown the TAG stored
○ Medicines stimulate synthesis of additional LDL in adipose cells
receptors which will then cause the removal of ● Subsequent release of
the LDL in the blood glycerol and FA in the blood
● Homozygous similar to DM
○ Managed with LDL pheresis ● Glycerol and FA will be
■ Similar to hemodialysis transported to the liver→ will
● Blood of the patient goes form high amounts of TAG→
through a machine will be loaded in tbe VLDL
● In dialysis, the machine will molecules→ increase
remove all the waste hepatic production or
products, once blood is synthesis of VLDL-->
already filtered and waste Increase TAG level
product is already removed, ● Elevated triglyceride level is seen in coronary heart
then the blood will be disease
returned to the body of the ○ No direct relationship yet
patient ● Acute or recurrent pancreatitis
● In the case of LDL pheresis, ○ Pancreatitis
once the blood will go inside ■ Enzymes that is supposed to be
the machine, the machine released into the SI particularly the
will just remove all the LDL duodenum to digest the nutrients in
molecules and once its the food will digest the pancreas itself
removed, the blood is then ■ Activations of this enzymes are
returned to the body of the brought about by hypertriglyceridemia
patient ● Treatment
○ Statins wont work for patients with homozygous ○ Dietary modifications
FH, because even if you deprive cells from ○ Fibrates
producing their own cholesterol, they still cant ■ Known to lower TAG
express LDL receptors because they have ■ Knows to increase HDL level
missing genes for the expression of these
receptors Combined Hyperlipoproteinemia
● If both (hypercholesterolemia and hypertriglyceridemia)
Hypertriglyceridemia are increased at the same time.
● Increased level of TAG in the blood ● Increase LDL and VLDL
● Can be caused by genetic abnormality ● High risk of coronary heart disease.
○ Familial hypertriglyceridemia - mutation in the ● Increase hepatic production of APO B 100 containing
LPL enzyme gene or deficiency of ap CII lipoproteins
■ AP CII is the apolipoprotein involved ○ APO B 100
in the activation of LPL, consequence ■ LDL and VLDL
will the same with the absence of LPL ● abnormal pattern of human serum lipoproteins in which
○ LPL - Lipoprotein lipase levels of low-density lipoproteins (LDL) and
■ Involved in the metabolism of very-low-density lipoproteins (VLDL) are elevated.
chylomicrons and VLDL ● Diagnosis of the disorder in a particular patient requires a
■ If absent, chylomicrons and VLDL will family history of premature coronary artery disease
no longer be metabolized causing ● The pathophysiological mechanism underlying FCH is
build up in the blood, thee 2 believed to be hepatic overproduction of apoB-100
lipoproteins are rich in TAG containing lipoprotein particles (ie, VLDL and LDL),
● Can be caused by diabetes, renal failure resulting in increased plasma total cholesterol,
○ Not a genetic abnormality triglycerides, and apoB
○ Diabetes - glucose molecules are not properly
17
Lipoprotein Elevation Hypoalphalipoproteinemia
● Decrease in the circulating HDL
● <40mg/dL
○ Normal value of HDL
● Increased risks for Coronary heart disease
● A good example is Tangier's disease
○ Absence of ABCa1 receptor
■ ABCa1 receptor- Allows cholesterol
and phospholipids to leak out the cells
so they can become part of discoid
● Modified of lipoprotein HDL
Left: 1 LDL molecule ■ Will not allow the formation of
Right: lipoprotein A spherical ot mature HDL from
● LDL discoidal HDL
● Has an additional apolipoprotein ● Treated with niacin (risk for flushing and liver damage)
○ Apolipoprotein A
■ Different protein HOW DO WE TEST THIS LIPIDS IN THE LABORATORY?
■ Will convert LDL into lipoprotein A or
LPA Cholesterol
● Lp(a) was first identified and classified as a "low density
lipoprotein variant" over 40 years ago. Lieberman-Burchard test
● Lp(a) showed it to consist of a LDL covalently bound to a ● Cholesterol is treated with sulfuric acid, acetic anhydride,
unique protein called apo(a) encoded by Lpa gene and acetic acid elicits a green-blue color
● Non-specific reaction
○ Solution: do hexane extraction (modification)
○ Other cholesterol can produce the same type of
+ result.
○ If u want to know whether it contains
cholesterol or not, combine it with hexane first
■ Hexane
● Remove the cholesterol
● The cholesterol will be
separated from the other
chemicals in the sample
than can also produce the
same result with liebermann
● APO (a) portion of lipoprotein A is similar in terms of
test.
structure and appearance to plasminogen
● Principle
● Plasminogen
○ Cholesterol if treated with sulfuric acid, acetic
○ Protein in the blood that is converted to plasmin
anhydride, and acetic acid will produce green
and plasmin is capable of breaking down clots
blue color.
especially the tissue that has already repaired
○ (+) Green-blue color
itself, the clot has to be removed via plasmin. .
Enzymatic reaction
● In clin lab we are NOT using the Lieberman-Burchard
test to measure the cholesterol in the serum of the px
● Enzymes are specific
○ More superior
● No need to do extraction of cholesterol
● Reagents are not as dangerous as the acidic reagents
used in traditional methods
Problem:
● Has a similar appearance to that of plasminogen, it will
compete with plasminogen with binding site in the BV.
● Plasminogen cannot be converted to plasmin→ clots will
not be broken down
● If the clot is in the atherosclerotic plaque, the plaque can
grow bigger.
Lipoprotein (A) Elevation

● Apo(a) is a homologue of plasminogen
● Lp(a) has proven to be pathogenic in nature and involved
in the development of coronary heart disease (CHD)
○ Lp(a) has been shown to competitively inhibit Pic: Reaction of enzymatic test for total cholesterol level in the
the binding of plasminogen to its receptor on blood
endothelial cells as well as to its binding sites ● Starts with cholesteryl ester
on fibrinogen and fibrin. ○ Has cholesterol in its structure
○ Once oxidized, it is engulfed readily by ○ Will be acted upon by cholesteryl esterase
macrophages thus acceleration of formation of ■ Break the ester bonds linking the FA
fatty streaks to the cholesterol molecules in
■ Speed process of atherosclerotic cholesteryl ester
plaque. ● Catalyzed by cholesterol oxidase
○ Substrate: cholesterol and oxygen
○ Product: cholestenone and hydrogen peroxide.
■ hydrogen peroxide.
18
● known oxidizing agent concentration and we cannot determine specific
● In the chemical reagent in the cholesterol, there is that numbers.
incorporated dye ○ What should we do?
○ 4-aminoantipyrine ■ Put the cuvettes inside a
■ colorless spectrophotometer
■ In the presence of hydrogen peroxide,
the 4-aminoantipyrine will be Spectrophotometer
converted into a quinone pigment
(RED)
● ↑ no. of free cholesterol ↑ hydrogen peroxide generated
in the second reaction ↑ the more dye that will be
oxidized into quinone pigment by peroxidase, the darker
the color solution.
● Inside the spectrophotometer, the cuvette with the

colored product will be hit by a light coming from the light
source
● Colored solutions can absorb specific types of light
● The higher the concentration of cholesterol, the darker
the final color of the product is.
○ The darker the color of the final product, the
● Cholesterol reagent in determination of total cholesterol higher the amount of light absorbed
○ Reagent: ● The cuvette with the lightest color, it will only absorb a
■ Cholesterol or cholesteryl esters little amount of light. Majority of the light will pass through
■ Cholesterol oxidase it and will be read by the spectrophotometer
■ Dye ● The spectrophotometer will give us the numerical value
○ Transfer the reagent in test tube or cuvette in terms of the concentration of the sample
○ Pipette specific amount of the px serum to ● Inside the spectrophotometer, the sample will be hit by
measure the amount of cholesterol molecules the incident light coming from the light source. Some of
present in the sample the light will be absorbed and some will be transmitted
○ Dispense the serum of the px in the cholesterol ● The higher the amount of light absorbed, the higher the
reagent concentration of the analyte
○ So, the cholesteryl esters will be acted upon by
cholesterol esterase, liberating free cholesterol Cholesterol
○ The free cholesterol molecules will proceed to
● Enzymatic reaction
the second reaction which is catalyzed by
● Intensity of color is proportional to the concentration of
cholesterol oxidase
cholesterol
○ One of the products is hydrogen peroxide and
● Interference from reducing agents such as bilirubin and
this will oxidize the dye that is present in the
Vitamin C
reagent to produce a colored compound
○ The problem with this enzymatic reaction for
determination of total cholesterol is that, since
the last chemical reaction is an oxidation
reaction, the presence of reducing agents can
greatly affect the result bc they will act on the
oxidized dye, converting the dye to the
colorless form and will not show the exact value
of cholesterol = falsely low
How do we measure triglycerides in the lab?
● We also employ enzymatic reactions similar to total
cholesterol.
Triglycerides
Assume that these are samples from 5 different patients

● Left = lightest color
● Right = darkest intensity of color
● Cuvette 1 (left) = has the lowest concentration of
cholesterol bcs only few of the dyes were oxidized by
hydrogen peroxide
○ Low amounts of hydrogen peroxide would
correlate with low amount of free cholesterol
bcs hydrogen peroxide molecules were only
produced in the second reaction and the ● First reaction: involves the TAG and the enzyme lipase
second reaction will not proceed in the absence ○ This lipase enzyme will liberate the 3 fatty acids
of cholesterol in the TAG and the glycerol backbone
● Cuvette (right) = Has the highest concentrations bcs a lot ○ What will proceed to the second reaction is
of dyes were oxidized by the hydrogen peroxide. glycerol
Meaning, there are high amounts of cholesterol that ● Second reaction: involves glycerol and glycerokinase
proceeded to the second reaction which led to the ○ Kinase = there is the transfer of phosphate
production of high amounts of hydrogen peroxide. group in the enzymatic reaction
● We can only determine which has the highest or lowest
19
○ Thus, ATP will be paired with glycerol and sample
expect that one of the phosphate group in ATP ● So whatever will be the result of the reading in the
will be transferred to glycerol spectrophotometer will only reflect the levels of the free
○ That's why the products are Glycerol- endogenous glycerol, and it will not reflect the glycerol
3-Phosphate + ADP molecules that will be derived from the triglyceride of the
● Third reaction: involves Glycerol-3-Phosphate and patient
Glycerol-3-Phosphate Oxidase ● In both procedures, u need to prepare glycerol blank (no
○ Glycerol-3-Phosphate will be made to react with lipase)
oxygen ● Upon the addition of the serum of the px to the reagent
○ Product: Dihydroxyacetone-Phosphate + H2O2 that doesn't have the lipase, the endogenous free
(hydrogen peroxide) glycerol will all proceed to the 2nd, 3rd, and 4th reaction
● Fourth reaction: Oxidation of the dye ● But these glycerol blank will never measure the glycerol
○ That’s why u have aminoantipyrine molecules that will be derived from the triglycerides of the
○ This reaction will end up with the production of px bcs there is no lipase enzyme to break the TAG
the colored quinone pigment ● With this glycerol blank, you will now have an idea how
Triglycerides much free glycerol molecules are there in the serum of
● Enzymatic assay the patient
● In most specimens, the endogenous free glycerol
contributes 10-20mg/dL overestimation of triglycerides
○ One of the problems encountered in the
enzymatic assay for the determination of blood
triglyceride level is that, there is an interference
brought about by the presence of the
endogenous free glycerol in the blood
○ In normal individuals, these endogenous free
glycerol can increase the blood TAG level by
10-20mg/dL. Why?
■ Recall: The 2nd reaction in the
enzymatic assay, would involve the
use of glycerol
■ These glycerol molecules are
supposed to come from the 1st
reaction ● Aside from the glycerol blank, we also need to prepare
■ Problem: These free endogenous another cuvette or another test tube wherein the reagent
glycerol will also become part of the that we will use will contain the lipase enzyme
2nd reaction. Thus, there is over ● The triglycerides in the serum of the patient will be acted
estimation of the TAG level upon by the lipase and there would be generation of the
● About 20% of specimens will have high glycerol content glycerol molecules, which will proceed to the second
due to diabetes and liver disorder reaction
○ The problem will become worse in people w/ ● Whatever result that will come up with this sample
diabetes and liver disorder cuvette, you will just simply deduct the amount of the free
■ Diabetes - no utilization of glucose glycerol measured from the glycerol blank, then you will
molecules as source of energy; body now have the value that will only reflect the glycerol
will break down stored TAG in the molecules derived from the triglycerides of the patient
adipocytes and will release fatty acids that were broken down by the lipase in the first chemical
and glycerol. reaction
■ These glycerol molecules will proceed
to the 2nd reaction of the enzymatic Double Cuvet Blank Technique
assay for blood triglyceride level
which would falsely elevate the result
of the TAG
■ HOW TO SOLVE?
● Perform “double-cuvette”
blank or “single cuvette”
blank
How “double-cuvette” blank or “single cuvette” blank techniques
are performed?
● Both glycerol blank and sample test will be measured at

the same time
● The light coming from the light source will be first
directed towards the glycerol blank and whatever the
reading will reflect the level of endogenous free glycerol
● The light coming from the light source will also be
● Glycerol blank: Reagent has no lipase directed towards the sample test of the px which will
● There is no 1st reaction, which is catalyzed by lipase contain the lipase enzyme. Both glycerol molecules and
● In performing double-cuvette or single cuvette blank endogenous free glycerol are measured.
technique, use a reagent that doesn’t have lipase ● At the end of the procedure, deduct from the total
● So that the TAG in the serum of the px will not be broken glycerol measured in the sample test the amount of the
down into glycerol and fatty acid free glycerol measured in the glycerol blank
● What will only react to the reagents are the free ● Thus u will have the true value of TAG in the serum of
endogenous glycerol molecules originally present in the the px
20
Single Cuvet Blank Technique
● The glycerol blank and the sample are not read at the Proteins can change its charges based on the pH
same time
● After getting both readings, the MT will just do the
subtraction and whatever the result, it will reflect the
triglyceride level of the px
LIPOPROTEIN METHODS
1. Ultracentrifugation
● Amino acid on the left side:

○ Amino acid was placed in an acidic medium
○ In an acidic medium, amino acids through their
amino group will accept hydrogen ions = amino
acids will become positively charged
○ Proteins will become positively charged at an
acidic pH
● Amino acid on the right:
○ Amino acid was placed in an alkaline medium
○ Carboxylic group of that amino acid will release
● Chylomicrons, HDL, VLDL, and LDL have significant H
differences in terms of their density ○ Amino acids and proteins will become
● Samples will be spun at an exceptionally high speed = negatively charged at an alkaline pH
separation of components of serum
○ HDL has the highest density = bottom of the 2. Electrophoresis
test tube
○ Chylomicrons are lightest = superficial
● The denser, the higher the chance it will settle at the
bottom of the test tube and vice versa
● Ultracentrifugation is a specialized technique used to
spin samples at exceptionally high speeds
● Uses density differences or rate of floatation to
fractionate lipoprotein classes
● Negative electrode = cathode

● Positive electrode = anode
● Electrophoresis is used to separate proteins in the
laboratory
● Proteins will be placed on the sample wells
● Proteins will be exposed to an alkaline solution to bear
negative charges
● Negatively charged proteins will now be attracted to
anode
Electrophoresis
● Proteins are made to bear negative charges and are
made to migrate to anode
● One of the factors that can affect the rate of migration of
proteins in the electrophoresis gel is the:
1. Molecular weight/Mass
- The smaller the protein, the faster it
will migrate
- The faster that it will migrate, the
closer it will be to reach the anode
2. The number of negative charges that the
protein would bear
- The more negative charge, the more
it will be attracted to the anode
21
● Proteins are separated based on their sizes and based
on the number of negative charges
● But, even if these proteins are already separated in the
electrophoresis gel, there is no way for us to see them
because they are soluble substances
● Expect that HDL will migrate towards the alpha region

● Staining is done so proteins will be made visible because it contains APO lipoprotein A1
● Darkest stain = lightest and the highest amount of ● VLDL and LDL will migrate towards the beta region
negative charge because both of them contains APO B-100
● HDL will be closest to anode
○ Highest protein concentration
● Chylomicron stayed at the sample well because they are
primarily triglyceride
3. Chemical Precipitation
● Polyanions such as heparan and dextran sulfate together
with divalent cations such as Magnesium and
Manganese
● Apo-B is rich in positively charged amino acids that will
form complexes with polyanions and will aggregate and
become insoluble in the addition of cations
○ Thus VLDL and LDL are precipitated leaving
HDL in the solution
● Serum of the patient + polyanions and divalent cations
○ Polyanions will be attracted to APO-B
■ Polyanions are negatively charged
and APO-B has a lot of positively
charged ions
○ Will form complexes and the divalent cations
will precipitate them
○ In the serum of the px, all of the APO-B
containing lipoproteins will be precipitated
○ What will be left as soluble is HDL
● First arrow (from left) = protein 1

○ Able to migrate closest to the anode
○ Smallest
○ Darkest = most abundant
● 2nd arrow = protein 4
○ Lighter
Normal electrophoresis result:
● Serum of px + polyanions and divalent cations
○ VLDL, LDL, and Chylomicrons will be
precipitated if the px did not undergo fasting
○ If the px fasted, VLDL and LDL will be
precipitated bcs chylomicrons are formed from
diet-derived lipids
● Centrifuge
○ Sediments in pic C are precipitated VLDL and
LDL
○ Supernantent will contain HDL
● Pipette the supernatant and measure the cholesterol
content
● Results will reflect the HDL of the patient
● Chemical precipitation can measure HDL levels
● Albumin is the smallest among them, migrates closest,
the highest concentration 4. Immunochemical methods
● Gamma proteins are most of the time antibodies ● Make use of specific antibodies to the apolipoproteins
● Antibodies are immobilized in a column matrix or beds
and will capture specific lipoprotein
● Example: I want to separate HDL from the other
22
apolipoproteins, so I will make use of antibodies to Apo VLDL will cause an interference
A1, so that they will all bind to HDL. Separating now HDL ■ How?
from other apolipoproteins for they do not contain apo A1 ● If you are going to add the
similar to HDL. precipitation agents, they
will precipitate out VLDL and
chylomicrons
● Since they are from
chylomicrons and VLDL,
they are expected to have
low density
● So these precipitates will
actually float and
centrifugation will never
make these sediments settle
in the bottom
Example: ● That's why you can't
● These antibodies (red) are directed against Apo A1 separate the supernatant
which are designed to bind to HDL. and test for HDL
● That’s why, as soon as you add the sample of the patient ○ If VLDL and chylomicrons are present in large
in the setup, the HDL molecules (blue diamond amounts, the low density of aggregated
structures) will all be captured by the immobilized lipoproteins may prevent them sedimenting and
antibodies. While the other apolipoproteins (red boxes) may even cause floating during centrifugation
will remain suspended in the patient's sample. ○ The incomplete sedimentation (seen as turbid,
● To separate HDL, we will aspirate back the serum. cloudy or particulate matter) may cause falsely
● With the aspiration, we were able to separate HDL from high HDL values
the other apolipoproteins. ○ troubleshooting: ultrafiltration
■ Filtration will remove the precipitates,
High Density Lipoprotein Methods leaving behind the supernatant that
only contains the HDL
1. Chemical precipitation
○ Designed to measure HDL 2. Magnetic Method for Automated Machines
● Precipitation reagent is added to the serum which will ● Similar to chemical precipitation except that the
aggregate and precipitate non-HDL lipoproteins (will precipitation reagent is complexed with magnetic particle
include VLDL and LDL for fasting specimens; if the ● That’s why if these precipitation agents will cause the
patient has not fasted, it will include chylomicrons) VLDL and chylomicrons to precipitate, they will still settle
● Majority of the samples that we process for lipid profile at the bottom of the test tube because the machine will
are fasting samples. What will be precipitated are the use the magnet to attract them towards the bottom part
VLDL and LDL. of the container, leaving behind the HDL in the
● Centrifuged at approximately 1500 gravity for 10-30 mins precipitate-free supernatant
(10,000-15,000 g for 3 minutes) ● the sediments formed do not require centrifugation
● Supernatant is then separated and HDL is quantified as ● Allows the supernatant to be analyzed without the need
cholesterol to remove it from the sedimented complex
● For cholesterol determination:
○ Liebermann–Burchard assay CONCEPT
■ Long abandoned by a lot of labs ● Lipid profile - we need to determine the concentration of
■ Mostly performed in biochemistry labs the individual lipoproteins
but not in Clinical labs ○ The patients are instructed to undergo at least
○ Enzymatic reaction 10 hours of fasting. Expect that the patient will
■ Most commonly used by labs not have chylomicrons in the serum.
● In the laboratory, we process specimens for total ● Total cholesterol = VLDL + LDL + HDL
cholesterol and HDL cholesterol. Both samples are
processed using enzymatic reaction.
○ Total cholesterol
■ We will measure the cholesterol
molecules present in VLDL, LDL and
HDL, so we will not perform chemical
precipitation
■ After determining the TC, we will use
a separate serum and we will do the ● Add the cholesterol reagent for enzymatic reagent to the
chemical precipitation (precipitating serum of the patient. And all of the cholesterol from
VLDL and LDL) VLDL, LDL, and HDL will be measured.
■ So, what will be processed in the ● In the determination of HDL, we will do chemical
serum is purely HDL precipitation wherein we are going to precipitate out
● Chemical precipitants VLDL and LDL, leaving behind HDL in the supernatant.
○ Heparin plus manganese (manganese can ● Using the cholesterol reagent, we will be able to measure
inhibit enzyme) the cholesterol molecules in HDL alone.
○ Phosphotungstic acid plus magnesium (high ● Don't worry about VLDL because we can just simply
variability among results) measure the triglyceride level of the patient and divide it
○ Dextran sulfate plus magnesium by 5. Whatever will be the result is equivalent to the
● Problem: elevated triglyceride levels can cause VLDL concentration of the patient.
interference ● In the lab:
○ If the patient has hypertriglyceridemia, ○ 1 test tube - total cholesterol
secondary to high levels of chylomicrons and ○ 1 test tube - HDL determination via chemical
VLDL in the blood (like Familial Triglyceridemia precipitation
- absence of LPL, Apo CII) ○ 3rd setup - determination of the triglyceride
■ The high levels of chylomicrons and level = VLDL concentration in the patient's
23
serum
○ LDL concentration - no available method
■ Substract from total cholesterol the
VLDL and HDL
3. LDL Determination
● Dyslipidemia - Friedewald Equation
● Do not use if TG>400

○ if there is hypertriglyceridemia, there would be
precipitation of sediments that are not easily
separated from the supernatant = error
○ Can still be applied if performed via
ultrafiltration
● Friedewald equation
○ Total cholesterol = HDL + LDL + VLDL
○ VLDL = Tg (in mg/dL)/5
= Tg (in mmol/L)/2.2
24
LECTURE 4: AMINO ACIDS AND PROTEINS:

BIOCHEMISTRY AND CLINICAL SIGNIFICANCE
Prof. Fritdey Jad Doctolero, RMT
October 25, 2021
AMINO ACIDS
● Building blocks of more complex compounds
● Small biomolecules containing at least one amino group
and one carboxyl group bonded to the alpha-carbon
● Have three parts
○ Amino group
○ Carboxylic acid group
■ Named as such because of the
functional group that they are
○ Sidechain or R group
■ Hydrocarbon part of the amino acid
○ Hydrogen atom
○ Alpha Carbon
■ The carbon at the center
■ It is named such because it is the first
carbon atom attached to the
functional groupd and the side chain
● There are 22 amino acids in the book which includes
selenocysteine and pyrrolysine. (20 lang ang napakita
since yun ang established before)
● Remember the three-letter abbreviation and the

decriptions ● Amino acids can be classified according to the polarity of
Abbreviation Amino acid their side chains
○ Non-polar hydrophobic
Ala Alanine ■ Their R groups are mostly hydrogen
Arg Arginine and carbon
Asp Aspartic Acid ○ Polar and charged
Asn Asparagine ■ Their R groups are alcohol, phenol,
thiol, amide
Cys Cysteine
○ Polar acidic
Gln Glutamine ■ Their R group can’t be seen but
Glu Glutamic Acid supposedly, the oxygen has a
Gly Glycine negative charge
His Histidine ■ The negative charge of the oxygen
are one of the signs that describes
Ile Isoleucine the amino acid as polar acidic
Leu Leucine ○ Polar basic
Lys Lysine ■ Instead of having a negative charge,
Met Methionine a positive charge can be seen its side
chains
Phe Phenylalanine ● Can also be classified based on being essential and
Pro Proline non-essential or based on the its nutritional requirement
Ser Serine
Thr Threonine Essential Amino Acids
- It is required in the diet but cannot be synthesized in the body
Trp Tryptophan
- PVT TIM HALL
Tyr Tyrosine
Val Valine ● Phenylalanine
Sec Selenocysteine ● Valine
Pyl Pyrrolysine ● Threonine
● Tryptophan
● Isoleucine
● Methionine
1
● Histidines ● Helps manufacture RBC and WBC
● Arginine ● Protect the body from heavy metal toxicity
● Leucine
● Lysine
Non-Essential Amino Acids 3. Isoleucine

- Not required in the diet but can be synthesized by the body
● Alanine
● Asparagine
● Aspartic Acid
● Cysteine
● Glutamic Acid
● Cysteine
● Glutamic Acid
● Glutamine
● Glycine
● Proline
● Serine
● Tyrosine ● Needed for hemoglobin formation
● Helps to regulate blood and glucose levels and maintain
Amino Acids energy levels
1. Arginine 4. Leucine
● Boosts healing of muscle, skin, and bones

● Aids in recovery from surgery
● Lowers blood glucose levels
● Optimal growth of infants and for nitrogen balance in
● Plays a role in cell division adults
● Healing of wounds, stimulation of protein synthesis
● Immune function 5. Lysine
● Release of hormones
● Required for the generation of urea and synthesis of
creatin
○ Urea is the less toxic form produced from the
ammonia metabolized in the urea cycle
○ Creatin (creatine phosphate) is a compound
involved in the supply of energy for muscle
contraction
○ Creatine is synthesized with arginine
○ Creatine when broken down forms creatinine
2. Histidine
● Plays a role in the production of antibodies and

antibodies (immunoglobulins) and lowers triglyceride
levels
● Needed for proper growth and bone development in
children and to maintain a proper nitrogen balance in
adults
● Helps in the absorption of calcium and the formation of
collagen
● Direct precursor of histamine

● Repair body tissues
● Maintain myelin sheaths that protect the nerve cells
2
6. Methionine 9. Tryptophan
● Helps initiate translation of messenger RNA

● Precursor for serotonin (5-hydroxytryptamine) and
○ First amino acid encoded during translation
melatonin
○ If methionine is not encoded, translation won’t
○ Serotonin
happen
■ Stabilized the mood
○ The codon that encodes for methionine is the
■ Person feeling happy
start codon, AUG
○ Melatonin
● Source sulfur
■ Hormone secreted by pineal gland
○ Sulfur in its side chain
■ Sleep week cycle (highest during
○ Sulfur functions for the normal metabolism and
night time
growth of the body
● Natural relaxant
● Assist the breakdown of fats
● Alleviate insomnia by inducing sleep, soothes anxiety
● Helps to detoxify lead and other heavy metals
and reduces depression
● Helps diminish muscle weakness
● Used in treatment of migraine headaches
● Prevents brittle hair
● Aids in weight control by reducing appetite
● Helps control hyperactivity in children
7. Phenylalanine ● Heaviest among all the amino acids
10. Valine
● Promotes alertness and vitality, elevates mood,

decreases pain, aids memory and learning
● Used to treat arthritis and depression ● Needed for muscle metabolism and coordination, tissue
● Used by brain to produce norepinephrine repair and maintenance of nitrogen balance
● Uses active transport channel to cross the BBB ● Used by muscle tissue as an energy source
○ BBB: Blood-Brain Barrier ● Used in treatments for muscle, mental and emotional
● Interferes with the production of serotonin problems (insomnia, anxiety, liver and gallbladder
● Part of the composition of aspartame disease)
○ Common sweetener in (?) food as sugar
replacment 11. Alanine
8. Threonine
● Involved in the breakdown of glucose

● Product of the breakdown of DNA
● Transfer of nitrogen from the peripheral tissue to the liver
● Important in the component in the formation of protein, ● Helps in reducing the buildup of toxic substances that are
collagen, elastin, and tooth enamel released into muscle
● Helps maintain proper protein balance and aids in liver ● Strengthens the immune system through production of
function, metabolism, and assimilation antibodies
3
12. Asparagine ● Serves as a neurotransmitter and dysregulation has been
linked to epileptic seizures
● aids in transporting potassium to the spinal fluid
● responsible for the taste umami
● food additive/food enhancer(sodium salt, monosodium
glutamate (bitsin)
16. Glutamine
● First amino acid to be isolated

● From asparagus juice
● Converting one amino acid into another via amination
and transamination
○ Amination
■ Amine group is introduced to an
organic molecule
○ Transamination ● most abundant amino acid in the body
■ Amino acid is transferred to an alpha ● assists in maintaining the proper acid/alkaline balance in
keto acid (alpha ketoglutarate) the body (regulates pH)
○ Processes occur before urea cycle ● provides fuel for a healthy digestive tract
● Required by the nervous system and synthesis of ● supplement used for muscle growth in weightlifting and
ammonia bodybuilding
● transports ammonia to the liver
○ Ammonia is toxic to the body (increased levels
13. Aspartic acid (also known as aspartate)
is dangerous), may lead to comatose
○ Needs to be transported to the liver where
ammonia will be converted to urea (through the
urea cycle pathway)
17. Glycine
● A metabolite in the urea cycle and participates in

gluconeogenesis
○ Metabolic pathway wherein we produce
glucose (pyruvate) from non carbohydrate
sources
● Simplest amino acid
14. Cysteine
● One element in the side chain = no stereoisomer
● only amino acid not optically active because it has no
stereoisomers
● has a sweet taste and is used as a sweetener
● Inhibitory neurotransmitter in the cns
● helps in the synthesis of bile acids
○ Main bile acid in the body: folic acid and
chenodeoxycholic acid
● retards muscle degeneration, improves glycogen storage
and promotes healing
● Named after cystine (precursor)
18. Proline
○ Cystine will travel in your cells, and as it goes
inside the cells, it will be reduced to 2 cysteine ● precursor of hydroxyproline
molecules ● Technically not an amino acid, but an imino acid
● Also known as half-cystine residue ● role in wound healing and molecular recognition
● Production of flavors ● works with vit. c to promote healthy connective tissues
● Contains sulfur ● technically not a protein since it is an imino acid due to its
cyclic structure
15. Glutamic Acid (Glutamate)
4
19. Serine
PYRROLYSINE
● ENCODED BY UAG CODON
● USED BY ARCHAEA AND UNICELLULAR
ORGANISMS
● needed for proper metabolism of fats and fatty acids

● highly concentrated in all cell membranes
● Component of the protective myelin sheaths surrounding
nerve fibers
20. Tyrosine
How are amino acids metabolized?

● precursor of epinephrine, norepinephrine, dopamine, t3 ● From the food, ang makukuha pa natin ay complex na
and t4 version ng amino acid.
○ T3 and T4 are thyroid hormones ● Once u ingest the protein, it will be initially digested by
○ Thyroid hormones are not purely tyrosine, it trypsin and pepsin
also has -iodine (?) ● It will be degraded to amino acids
● stimulates metabolism ● This amino acids will now be absorbed in the intestine
● mood elevator, suppresses appetite and helps reduce and it will now be part of amino acid pool
body fat ○ Amino acid pool
● treatment of chronic fatigue, narcolepsy, anxiety, ■ Dito kinukuha ang mga amino acid na
depression, low sex drive, allergies and headaches gagamitin later sa translation.
● Once the body degrades the amino acid, the product of
TWO NEW AMINO ACIDS (established 2002) the degradation now will vary depending if the amino acid
● Selenocysteine is ketogenic or glucogenic
● Pyrrolysine ○ Ketogenic
■ By product = acetoacetyl CoA, acetyl
21. Selenocysteine (SEC) CoA
● Acetoacetyl CoA= Can be
(?) in the metabolic pathway
ketogenesis
● Acetyl CoA = can enter
ketogenesis or will be
allowed to enter the citric
acid cycle
○ Glucogenic
■ Can generate precursor of glucose:
Pyruvate→ gluconeogenesis or
create Acetyl CoA and enter citric
acid cycle
● encoded by uga codon ■ Another by product: citric acid cycle
● selenium analogue of cysteine intermediate→ will be part of the citric
○ Selenium analogue - a sulfur in cysteine is acid cycle.
replaced by selenium ● Oxidative deamination or Transamination
● found in some enzymes ○ Process of removing amino group
○ Formate dehydrogenase ○ Result:
○ Glycine reductase ■ keto acid : alpha keto glutamate
○ And some hydrogenases ● Will enter common
metabolic pathway
22. Pyrrolysine ■ Ammonia
● Toxic to the body and should
be converted to less toxic
form = UREA
AMINOACIDOPATHIES
● Rare, inherited disorders of aminoacid metabolism
● Abnormalities in activity of a specific enzyme in the
metabolic pathway
● Encoded by UAG codon
● Abnormalities in the membrane transport system for
● Used by archaea and unicellular organisms
amino acids
5
● Brought about by genetic defect.
mg/dL, phenylalanine counteracts the
PHENYLKETONURIA (PKU) antagonist and bacterial growth
occurs
● autosomal recessive trait ○ Positive = if may bacterial
○ Must inherit 2 mutated gene one from each growth around the disc
parent ○ Negative = if walang growth
● Total absence of activity of PHENYLALANINE
HYDROXYLASE Microfluorometric ● Filter paper w/ specimen must be
○ Catalyzes the conversion of phenylalanine to assay pre-treated with trichloroacetic acid
tyrosine ● Allow the extract to react with the
● In the absence of phenylalanine hydroxylase, microtiter plate which contains
phenylalanine will not converted to tyrosine leading to ninhydrin, succinate, leucylalanine,
accumulation or build up of phenylalanine and and copper tartrate
metabolites of phenylalanine. ● direct measurement of phenylalanine
● if there's accumulation in the blood = in dried blood filter disks
hyperphenylalaninemia ● Based on the fluorescence of a
● If the phenylalanine is present in the urine = complex formed of
phenylketonuria phenylalanine-ninhydrin-copper in
the presence of a dipeptide
(Lleucyl-L-alanine)
● Excitation/emission wavelengths of
360 nm and 530 nm respectively
Guthrie test for PKU

● Bacterial plate with newborn blood samples
● Positive blood test results: bacterial halo = PKU

● Negative controls: no bacterial growth
● Positive controls: increasing phenylalanine
COMPLICATIONS: concentrations give bacterial halos
Children ● Negative blood test results: no bacterial growth = healthy
● Retarded mental development babies
● Occurs as a result of the toxic effects of the ● Why does Bacillus subtilis grow if the px has PKU?
brain of phenylpyruvate or one of its metabolic ○ Phenylalanine present in the px will neutralize
by-products the inhibitor (beta-2- thienylalanine). Thus,
○ Expect microcephaly there will be growth of Bacillus subtilis.
● 2nd or 3rd week of life
● Metabolites High-Performance Liquid Chromatography (HPLC)
○ Phenylpyruvate – deamination of
● Reference method/standard method
phenylalanine
● Newborn: 1.2 – 3.4 mg/dL (70-200 umol/L)
○ Phenylacetic acid – decarboxylation and
○ PKU is one of the diseases that is tested in
oxidation of phenylpyruvate
newborn screening
○ Phenylacetylglutamine – glutamine conjugate
of phenylacetate
Urine Test
● If the mother has phenylketonuria and the baby kay wala,
maagapan mo na ma experience ng baby ang ● monitoring
phenylketonuria ● Reagent strip test
○ How? ○ Involves the reaction of ferric chloride with
■ Make sure that the mother will phenylpyruvic acid (possible analyte present)
undergo phenylalanine restricted diet in urine to produce a green color
○ *increased in PKU ■ “Possible” bcs metabolites present
may vary
METHODS OF DETERMINATION
Tyrosinemia
Guthrie Bacterial ● Spores of Bacillus subtilis are ● Characterized by excretion of tyrosine and tyrosine
Inhibition incorporated into an agar plate that catabolites in urine
Assay contains beta-2- thienylalanine ● Deficiency in:
(antagonist) ○ Fumarylacetoacetate (FAA) hydrolase (type I)
● Filter paper disk impregnated with ■ Most severe
blood from the infant is placed on the ■ Possible symptoms: failure to thrive
agar ○ 4-hydroxy-phenylpyruvic acid oxidase (type II)
● If blood phenylalanine exceeds 3-4 ○ tyrosine aminotransferase (type III)
● COMPLICATIONS: Leads to liver damage (Cirrhosis and
6
liver cancer) fluorescence: excitation wavelength of 360 nm
○ Cirrhosis = scarring of liver tissue (4) sensitivity = above 4 mg/dL
○ Liver cancer is possible ● COMPLICATIONS:
○ Lethargy, failure to thrive, Muscle rigidity,
Respiratory irregularities, Mental retardation,
Convulsions, Acidosis and hypoglycemia
Isovaleric Acidemia
● Deficiency of isovaleryl-CoA dehydrogenase in leucine
pathway
● “sweaty feet” odor
Alkaptonuria ● Perform chromatography
● Autosomal recessive disorder
● HGD gene Homocystinuria
● Familial inheritance ● Increased levels of homocysteine
● Lack of homogentisate oxidase ● Homocysteine
○ accumulates in connective tissue causing ○ Intermediate amino acid in the synthesis of
generalized pigmentation of these tissues cysteine from methionine
(Ochronosis), an arthritis-like degeneration ● Impaired activity of cystathionine beta-synthase
○ Characterized by darkening of urine (homocysteine to cysteine)
■ Accumulation of HGA -
homogentisate acid
○
● Cyanide-Nitroprusside Urine Spot Test

○ Cysteine and homocysteine are reduced by
sodium cyanide to free-thiol
○ Then, reacted to sodium nitroprusside to
produce a red-purple color
● Silver-nitroprusside Test
Maple Syrup Urine Disease ○ Sample: urine
● Also tested in newborn screen ○ Confirmation for homocysteine
● Autosomal recessive genetic disorder ■ Silver nitrate reduces homocysteine
● Characterized by burnt sugar odor of the urine, breath, to form reddish color
and skin ● COMPLICATIONS:
● Lack of branched-chain alpha-keto acid decarboxylase ○ Thromboembolism, Cardiovascular risk,
○ Blocking the normal metabolism of: Leu, Ile and Atherosclerotic disease, Low folate
Val concentrations, Vitamin B12 deficiency
● Modified Guthrie test: 4-azaleucine
● Microfluorometric assay: Leucine dehydrogenase above Citrullinemia
4 mg/dL is indicative of MSUD ● Results from inherited enzyme deficiencies in the urea
○ Difference between pku: (1) filter paper is pre cycle
treated with methanol acetone (2) extract is ● Type 1:
mixed with leucine dehydrogenase (3)
7
○ lack of the enzyme argininosuccinic acid Maple syrup Maple syrup urine disease
synthetase (ASS) Mousy Phenylketonuria
■ Found in the urea cycle
○ There is buildup of citrulline Rancid tyrosinemia
● Type 2: Sweaty feet Isovaleric acidemia
○ caused by a mutation of the gene that would Cabbage Methionine malabsorption
provide instructions for making the protein citrin Bleach Contamination
■ Citrin helps transport molecules inside
cells that used to produce simple
PROTEINS
sugars, proteins, nucleotides, and
molecules used in the urea cycle
● Covalently linked polymers of amino acids
○ inhibits the urea cycle and disrupts the
○ Carboxyl group of one amino acid combines
production of proteins and nucleotides
with the amino group of another amino acid
● COMPLICATIONS: Vomiting, high ammonia levels and
○ Water is removed- peptide bond
Mental retardation may lead to comatose
○ N-terminal end- amino group free
○ It is through the urea cycle that ammonia will be
■ Not bonded with other elements
converted to a less toxic form which is urea
○ C- terminal end- carboxyl group free
■ The group that is free is called the
Carboxyl group
● The bond between one amino acid that links to another
amino acid is called the peptide bond
● The peptide bond is seen between the carboxyl group of
one amino acid and the amino group of the other amino
acid
Argininosuccinic Aciduria
● Results from inherited enzyme deficiencies in the urea
cycle
● Deficiency in argininosuccinate lyase (ASL)
○ prevents the conversion of argininosuccinic
acid into arginine
● COMPLICATIONS:
○ Vomiting, high ammonia levels and Mental
retardation
● When peptide bonds are formed, water is released
Cystinuria
● Amino group is NH3. once the peptide bond is formed
● a defect in the amino acid transport system rather than a
nawala ang dalawang H that became H2 and hen form
metabolic enzyme deficiency
COO naging CO nalang ang isang O napunta kay H2
● Increased urinary excretion of cystine
● Yung dalawang H from NH from the amino group and
○ Thus, there is an inadequate re-absorption of
yung isang O from the carboxyl group ang naging water
cystine during the filtration process in the
kidneys
Structure of Proteins
○ Resulting from genetic defect in the renal
resorptive mechanism
○ Cystine
■ Insoluble
■ Tends to precipitate in the kidney
tubules
● Urinary calculi
○ Kidney stones
Possible Causes of Urine Odor
Odor Cause
Aromatic Normal
Bacterial decomposition, urinary tract
Foul, ammonia-like
infection
Ketones (diabetes mellitus, salvation,
Fruity, sweet
vomiting)
8
Classification of Proteins (Function)
● Enzymes
○ Catalyze chemical reactions
● Hormones
● Primary Structure ○ Control action of specific cells or organs
○ The different amino acids that compose a ● Transport proteins
specific protein in a linear way ○ Move substance inside the body
● Secondary Structure ● Immunoglobulins (antibodies)
○ The peptide chains are folded regularly which ○ Gives protection against foreign objects
forms ● Structural proteins
■ Alpha helix ○ Components of cells and tissues
■ Beta pleated sheets ● Storage proteins
● Tertiary structure ○ Acts as reserves
○ Folded secondary structure into a 3D form ● Energy source
○ Considered a polypeptide ● Osmotic force
● Quaternary Structure ○ Capable of affecting the distribution of water in
○ Combined tertiary polypeptide structure with the body
other polypeptide ● Blood coagulation
○ Clotting factors
■ Fibrinogen
Ponder: Are all proteins synthesized in the liver? If not, where are
they synthesized?
Protein Synthesis
FIVE FRACTIONS OF PLASMA PROTEINS
- Can be divided into 2 main groups: Albumin and
globulins
- It can also be divided into 5 fractions: (fractions is the
results of plasma being done in the electrophoresis --
observe bands)
- Basis: due to migration of proteins in the electrophoresis
- In your serum protein electrophoresis, this is the order of
the bands (in observing proteins in electrophoresis), but
there is this one part in electrophoresis that should also
be included, which is the PREALBUMIN (not in the list
because not considered as a main fraction)
● Albumin
● α1-Globulins
○ α1-fetoprotein
○ α-antitrypsin
○ HDL
● α2-Globulins
○ haptoglobin
○ ceruloplasmin
○ α2-macroglobulin
● Begins with transcription ● β-Globulins
○ The genes encoded in the DNA are used to ○ transferrin
produce pre-messenger RNA ○ C-reactive protein
○ Pre-messenger RNA undergoes some ● γ-Globulins
processes like splicing to get mRNA ○ Immunoglobulins
○ mRNA will go out of the nucleus and will
undergo translation PREALBUMIN
● Translation ● Also known as transthyretin
○ The mRNA is partnered with an anti-codon with ● Migrates ahead of albumin
the help of the ribosomal complex ● Transport protein of thyroid hormones
● Once the translation is done, the formed polypeptide will ○ Thyroxine (T4) and triiodothyronine (T3)
undergo folding and may bind with other polypeptides to ● Binds with retinol-binding protein to form a complex that
form a finished protein transports retinol (vitamin A)
● Rich in tryptophan
Classification of Proteins (Composition)
● Simple
○ Purely made up of amino acids
○ Contain peptide chains on hydrolysis yield only
amino acids
■ Albumin
○ If you’re going to hydrolyze the peptide chains
in your simple proteins, you will only acquire
amino acids
● Conjugated
○ Not entirely made up of amino acids only
○ Comprise a protein (apoprotein) and a
nonprotein moiety (prosthetic group)
■ Lipids (lipoprotein), CHO
(glycoprotein), porphyrins
(hemoglobin), metals (ceruloplasmin)
9
- Illustration on what is observed when you run
protein electrophoresis
- Prealbumin migrates ahead of albumin
- Arrow (movement of proteins)
- Prealbumin = quickest to degrade (followed by
albumin)
Clinical significance:
● Decrease prealbumin
○ Hepatic damage
○ Acute phase inflammatory response
○ Tissue necrosis
○ Sensitive marker of poor protein nutritional
status
■ If there is malnutrition (or simply di
kumain si patient), expect that
prealbumin results is quickly affected
■ Prealbumin in terms of half-life is only ● Anaalbuminemia
2 days ○ Bakante ang albumin (lower left)
■ Decreased food intake = prealbumin
will immediately decrease Bisalbuminemia
● Increased prealbumin ● Unusual molecular characteristics
○ Steroids, alcoholism and chronic renal failure ● Genetic in origin resulting from an autosomal recessive
trait
ALBUMIN ● condition of having two types of serum albumin that differ
● Present in highest concentration in mobility during electrophoresis
○ Colloid osmotic pressure
■ Albumin in the body can maintain the
appropriate fluid balance in our body
■ Regulating colloid osmotic pressure of
the intravascular fluid
○ Transcapillary escape rate
■ Rate of movement of albumin leaving
the blood circulation
○ Bind to various substances
■ Can bind to thyroid hormones
■ Similar to pre albumin
■ Can bind to conjugated bilirubin, iron,
calcium and magnesium
HYPOALBUMINEMIA
● ↓ levels of albumin
● Malnutrition
● Liver disease
○ Resulting in the inability of hepatocytes to
synthesize albumin
○ Albumin is synthesized in the liver
● Gastrointestinal loss
○ Inflammation and intestinal mucosal disease. ● Has 2 peaks
○ The interstitial fluid that contains albumin leaks ○ Differs in mobility
out in the intestine and will be easily excreted GLOBULINS
esp. The person is suffering from diarrhea ● Consists of alpha1, alpha2, beta, and gamma fractions in
● Loss in the urine in renal disease. electrophoresis
○ Kidney can't function properly and cannot filter
all proteins → proteins will go out from the ALPHA 1 - ANTITRYPSIN
blood circulation→ urine ● Acute-phase reactant
○ Concentration will change in response to
HYPERALBUMINEMIA inflammation
■ ↑ levels
● Not medically important ● Neutralize trypsin-like enzyme that can cause hydrolytic
● Seen in dehydration damage to structural protein
○ Relative increased ● Increased: inflammation, pregnancy, contraceptive use
■ Increased level is not truly increased ● Deficiency: Severe, degenerative, emphysematous
but rather it looks like that it is pulmonary disease
increased bcoz of decreased water in ● Among alpha 1 globulin, this one is the protein that
the blood circulation. majorly comprises the alpha 1 fraction (90%)
○ Fluid administration will decrease albumin ○ If ever there is deficiency in the alpha 1
levels back to normal antitrypsin, mapapansin sha agad in the
electrophoresis.
Analbuminemia
● Absence of albumin ALPHA 1 - FETOPROTEIN (AFP)
● Synthesized initially by the fetal yolk sac and then by the
parenchymal cells of the liver
○ Peak: 13 weeks’ gestation
○ Recede: 34 week’s gestation
■ Nag re recede sha after birth because
10
it has no known fxn in adult lab. Thus, there will be decreased
● Can pass across the placenta. haptoglobin.
○ An increase in this protein in the serum sample ■ Increased reticulocyte count,
of the mother can indicate that the fetus has a decreased RBC count, decreased
defect or a problem hemoglobin, decreased hematocrit
● ELEVATED AFP ● Tetramer
○ Spina bifida and neural tube defects ○ Contains 2 alpha & 2 beta chains
■ Incomplete closure of embryonic ● Acute phase reactant
neural tube ● Increased:
■ Incomplete spinal cord ○ Inflammation
○ Atresia of the gastrointestinal tract ○ Burns
■ Narrowing or absence of a portion of ○ Nephrotic syndrome
the intestine of the fetus ● There are times na hindi baba ang levels ng haptoglobin.
○ Fetal distress If this is the case, the site where there is hemolysis is
■ Not well; excessively fatigue happening in the spleen or liver
○ Ataxia-telangiectasia ○ Does not go into the blood circulation
■ Immunodeficiency disorder
○ Tyrosinosis CERULOPLASMIN
■ Increased level of tyrosine ● Conjugated group
○ Hemolytic disease of the newborn (HDN) ● Copper-containing, alpha2-glycoprotein
■ Anti-D of the mother attacks the RBC ● Acute phase reactant
with the D antigen of the fetus ● 90% of the total serum copper is bounded to
○ Tumor marker: hepatocellular carcinoma and ceruloplasmin, the rest is bound to albumin
gonadal tumors ● Elevated: inflammation, infection, tissue damage and
■ For adults pregnancy
● Decreased:
ALPHA 1 - ACID GLYCOPROTEIN ○ Wilson’s disease
● Also called as orosomucoid ■ Excess storage of copper
● Formation of certain membranes and fibers in ■ Increased copper levels of px
association with collagen ■
● Acute phase reactant ■ Hepatolenticular degeneration
● Increased: ■ Autosomal recessive inherited
○ Inflammation, stress, AMI disease
○ Cancer, Surgery ● Resulting to hepatic
○ Pneumonia cirrhosis and neurologic
○ RA damage
● Clinically significant if elevated ■ Kayser-Fleischer Ring
● Outline in the eyes
ALPHA 1 - ANTICHYMOTRYPSIN ○ Menke’s Syndrome / Kinky hair disease
● Serine proteinase inhibitor (serpin) ■ Curly hair
● Inhibits some enzymes ■ Problem with absorption of copper
○ Cathepsin G
○ Pancreatic elastase ALPHA 2 - MACROGLOBULIN
○ Mast cell kinase ● Found principally in the intravascular spaces
○ Chymotrypsin ● Inhibits proteases
● Acute phase reactant ● Slightly increased: pregnancy and contraceptive drugs
● Increased: ● Increased:
○ Inflammation ○ Nephrosis
● Deficiency: ■ Macroglobulin is large
○ associated with asthma and liver disease ○ Diabetes
○ Liver diseases
Gc - GLOBULIN ■ Many hepatocytes = many
● Group-specific Component/Vitamin D-binding protein synthesized proteins
○ High binding affinity for vitamin D, actin and etc
● Increased:
TRANSFERRIN
○ 3rd trimester of pregnancy
○ Patients taking estrogen oral contraceptives ● Also known as Siderophilin
● Decreased: ● transferrin is the major component of beta globulin
○ Liver diseases and protein-losing syndromes fraction
■ Gc-globulin is synthesized in the liver ● Transports iron to its storage sites, can carry it to cells
■ protein-losing syndromes = hindi na and prevents loss of iron through the kidney
ma filter ng kidney (excreted in the ● From transferrin, iron is transported to apoferritin then it
urine) will become ferritin
● The transferrin molecule can carry two ferric iron = 2
Fe3+
HAPTOGLOBIN
● Can cary cells to the bone marrow to synthesize
● An alpha2-glycoprotein hemoglobin
● Synthesized by the liver and in RES ● Increased:
● Bind free hemoglobin by its alpha-chain ○ Microcytic, hypochromic anemia
○ Helps in the detection of hemolytic anemia ■ Iron is decreased in level thus there
■ Haptoglobin is decreased bcs many would be less iron to bind in the
rbc are destroyed. Thus, there will be transferrin
many free hemoglobin ■ Madaming free transferrin
■ Haptoglobin will bind to these free ● Decreased:
hemoglobin and gamay nalang ○ Malnutrition, nephrotic syndrome, inflammation
mabilin ● Atransferrinemia
■ Free haptoglobin is measured in the
11
○ A condition wherein there is absence of ● Decreased:
transferrin due to autosomal recessive genetic ○ Malnutrition; SLE; DIC
defect ■ C3 is seen as decreased in
○ There would be anemia and hemosiderosis malnutrition and Systemic Lupus
(deposition of iron in the heart and liver) Erythematosus
■ C4 is seen as decreased in
HEMOPEXIN Disseminated Intravascular
● Removes circulating heme Coagulation
● Decreased: hemolytic anemia
● Ratio is 1:1 FIBRINOGEN
● Acute phase reactant ● Clotting factor (I)
● Increased: ● Form a fibrin clot when activated by thrombin (factor 2)
○ Diabetes mellitus ● Not seen in serum
○ Duchenne-type muscular dystrophy ● Acute-phase reactant
○ Administration of diphenylhydantoin ● Increased:
○ Inflammation ○ Pregnancy
○ Use of birth control pills
LIPOPROTEINS ● Decreased:
● Transport cholesterol, triglycerides and phospholipids ○ Extensive coagulation
● Can be run in electrophoresis
C - REACTIVE PROTEIN
● Appears in blood of patients with diverse inflammatory
diseases
● Rises:
○ Tissue necrosisPneumococcal infections
● One of the first acute-phase proteins to rise in response
to inflammation
IMMUNOGLOBULINS
● Synthesized in plasma cells
● IgG, IgM, IgA, IgE, IgD
● Synthesis is stimulated by an immune response to
● Chylomicrons foreign bodies
● VLDL – pre-beta globulin
● LDL – beta-globulin
● HDL – alpha-globulin
○ Based on the position and migration in the
electrophoresis
BETA 2 - MICROGLOBULIN
● Light chain component of the major histocompatibility
complex (HLA)
● Increased:
○ Impaired kidney clearance
○ Overproduction of acute-phase reactants
COMPLEMENT MYOGLOBIN
● A heme protein found in striated muscles
● Can reversibly bind oxygen but requires a very low
oxygen tension to release the bound oxygen
● Increased in AMI within 1-3 hours of onset and reaches
peak concentration in 5-12 hours
TROPONIN
● Bind to the thin filaments of striated muscles
○ Trop-T (TnT)
○ Trop-I (TnI)
○ Trop-C (TnC)
● Regulate muscle contraction
● Collective term for several proteins that participate in the

immune reaction
● Link to the inflammatory response
● Among the complements, C3 is the most abundant,
followed by C4 TOTAL PROTEIN ABNORMALITIES
● Increased: ● Hypoproteinemia
○ Inflammatory states ● Hyperproteinemia
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LECTURE 1: RENAL FUNCTION

Prof. Isaac Edron J. Orig, RMT
November 7, 2021
Introduction glomerulus
● Kidneys are important organs in the body because they ■ Efferent = Exiting the glomerulus
excrete the waste products of the body’s metabolism ● Efferent arteriole will go to
● When we don’t have our kidneys, the toxic substances in the tubules and then will
the body will accumulate and becomes deadly for us become capillaries
(Peritubular capillaries)
RENAL ANATOMY ● Leaves the nephron to
become the renal vein
Kidneys: General Characteristics ○ Bowman’s capsule and space
● Paired, bean-shaped organs found retroperitoneally in ● PCT (Proximal Convoluted Tubule)
either side of the spinal column ● Loop of Henle (LoH) - hairpin (u-turn)
○ Retro - at the back ○ Thin Descending LoH
○ Peritoneal cavity - abdomen ○ Thin Ascending LoH
● About the size of a fist (10-12 cm) ○ Thick Ascending LoH
● Between T12-L3 ● DCT (Distal Convoluted Tubule)
○ 12 thoracic vertebrae and the 3rd lumbar ● Collecting Duct
vertebra
● Urine will form sa kidneys, then will go to the ureters and
will go to the urinary bladder where it will be stored
● The bladder will contract and the urine will go throughout
the urethra and then will go to the penis/urethral orifice
Kidneys: Macroscopic Characteristics

● Renal cortex
○ Outside part of the kidney
○ Surrounded by a fibrous connective tissue that
supports the kidney
● Renal medulla
○ Heart-shaped
● Renal pelvis
○ The urine will go to the renal medulla and will
be collected in the renal pelvis and will go to the
ureters then bladder
RENAL PHYSIOLOGY
Kidney Functions
● Urine formation
○ Most important function
○ Filters unwanted substances in the body
● Fluid and electrolyte balance
○ Kidneys secretes and reabsorbs water
● Regulation of acid-base balance
○ Kidneys can also secrete acids (hydrogen) or
base (bicarbonate)
● Excretion of the waste products of protein metabolism
Kidneys: Microscopic Characteristics ● Excretion of drugs and toxins
● Kidneys are made up of nephrons ● Secretion of hormones
● Functional unit: nephrons ○ Renin
○ Can’t be seen in the naked eye ○ Erythropoietin
● Glomerulus - filter the substances needed to be filtered) ■ Hormone responsible for production
○ Made up of tuft of capillaries and covered by of RBC
the Bowman’s capsule and the space inside it ■ Kidney is also involved in production
is called Bowman’s space of RBC
○ Afferent arteriole→ tuft of capillaries → efferent ■ Renal function < anemia < decreased
arteriole EPO < bone marrow cant produce
○ Site of filtration of the substances RBC needed
○ Substances come from the blood vessels since ■ Kidney failure < rickets (osteomalacia)
the blood carries the substances that need to ● Active form of Vitamin D is
be filtered to the kidneys produced in the kidney
○ Fromm the different parts of the body, it will go ● Decreased Vit D = Rickets
to the renal artery from the heart and will ○ 1,25-Dihydroxy vitamin D3
eventually become the afferent and efferent ○ Prostaglandins
arteriole
■ Afferent = Approaching the BASIC RENAL PROCESSES
● Glomerular Filtration
1
○ Process of the substances from the glomerulus ● Bilirubin
to the Bowman’s space to the urine ○ Not that big but it is carried by albumin
○ Glomerulus filters unwanted substances CAN pass through the glomerulus:
● Tubular Absorption ● Water
○ Reabsorbs rom the tubules to the capillaries ● Electrolytes
back to the blood (back to the circulation) ● Glucose
● Tubular Secretion ● Amino acids
○ ● Urea
● Renal Blood Flow ● Creatinine
CANNOT pass through the glomerulus:
● Plasma proteins
○ Albumin, hemoglobin
● Cellular elements
○ RBC, WBC, PLT
● Protein-bound molecules (lipids, bilirubin)
Tubular Reabsorption
● Happens when the substances from the tubular lumen
are moved to the peritubular capillary plasma
○ Reabsorb from the tubules to the capillaries
● Happens mostly at the PROXIMAL CONVOLUTED
TUBULE (90%)
● 75% of the sodium, chloride, water
● 100% of the glucose
○ There should be no sugar found in the urine
Glomerular Filtration ● Almost all of the amino acids, vitamins, proteins
● Variable amounts of urea, uric acid, ions (Ca, Mg, K,
What are the factors that make the glomerulus the best site for
HCO3)
filtration?
● 98-100% of uric acid is reabsorbed, only to be secreted
● High pressure in the glomerulus - brought upon by the
at the DCT
position of the glomerular tuft of capillaries.
● There is tubular reabsorption because kailangan pa ng
○ Kasi galing sha sa renal artery
body yung mga na filter
■ Blood from the artery is mabilis ang
kanyang flow
■ That's why there is high pressure in
the glomerulus.
○ Bowman's space
■ Low pressure kasi walang laman
● Semi-permeability of the glomerulus - molecular cutoff
value of about 66,000 Da or 66 kDa
○ Cut off value
■ Dictates if the substance will be
filtered in the glomerulus.
■ Dapat below 66,000 Da ang isang Renal Threshold
substance for ot to be filtered . ● REMEMBER: If a substance’s concentration exceeds the
● Basement membrane is negatively-charged. renal threshold for tubular reabsorption, it will appear in
○ If the substance is negative and the basement the urine.
membrane is also negative = it will repel ● Example: Glucose 160-180 mg/ dL
○ Negative molecules will not be filtered in the ○ All of the glucose will be filtered and
glomerulus. reabsorbed by the
■ Ex: albumin is small (↓ 66K Da) PCT.
● Negatively charged. ○ What if the px has
● Cannot penetrate to the wall diabetes mellitus?
of the capillary. There will be a lot of
● Renal Blood Flow glucose in the blood.
○ 1,200 - 1,500 mL/min Hence, if it exceeds
● Glomerular Filtrate the renal threshold, it
○ 130 - 150 mL/min will be excreted in the
● GLOMERULAR FILTRATION RATE urine.
○ volume of blood filtered per minute.
○ Kapag ↑ ang glomerular filtration rate, marami
kang na fifilter sa glomerulus.
What can pass through the glomerulus?

Red = can pass through
● Water
● Sodium
● Glucose
○ Very small
● Glycine
○ Are also very small
● Urea
● Albumin
○ Small but negatively charged
● Red blood cells
○ Cellular elements: very big
2
Tubular Reabsorption ■ Sodium in urine = decreased
Substance Location ■ Sodium in blood = increased
● Addison’s Disease?
Active transport Glucose, amino Proximal convoluted ○ Deficiency of aldosterone
acids, salts tubule ○ Sodium will not be reabsorbed
Chloride Ascending loop of ■ Sodium in urine = increased
Henle ■ Sodium in blood = decreased
Sodium Proximal and distal ● Syndrome of Inappropriate ADH secretion (SIADH)?
convoluted tubules ○ Excess in ADH
Passive transport Water ● Proximal ○ Water will be reabsorbed from the tubule to the
convoluted tubule blood
● Descending loop ○ Water in the urine will be decreased
of Henle ○ Urine will be more concentrated
● Collecting duct ○ Water in the blood will increase
● Diabetes Insipidus?
Urea ● Proximal
○ Decrease in ADH
convoluted tubule
○ Water will be freely flowing and will not be
● Ascending loop of
reabsorbed
Henle
○ Water in the urine will increase
Sodium Ascending loop of ■ Urine will be more dilute
Henle ○ Water in blood will decrease
● Active transport
○ uses energy, transport mechanisms, carriers, Tubular Secretion
proteins to transport the substance from the ● Movement of the substances from the peritubular
tubules back to the capillaries capillary plasma to the tubular lumen
● Passive transport ○ From the blood to the tubules
○ Freely flowing ○ Baliktad ng tubular reabsorption
● Tubular cells secrete products of their own cellular
metabolism to the filtrate in the tubular lumen
● The tubules are made up of cells and these cells have
metabolic wastes
● Secrete waste products na hindi kaya ifliter ng
glomerulus
● Can contribute to acid base balance, fluid balance, and
electrolyte balance
● Substance A
○ Some of the solute is filtered and most are
*blue = passive transport; red = active transport secreted to the urine
● Proximal convoluted tubule ● Substance B
○ GAAs-WU ○ Substance is filtered but reabsorbed
■ Glucose ● Substance C
■ Amino acids ○ A lot of the substance is filtered in the
■ Salts glomerulus and all of the substance is
■ Water reabsorbed
■ Urea
● Descending loop of Henle RENAL FUNCTION TESTS
○ ONLY water is reabsorbed ● Tests that determine of the kidneys are functioning well
● Ascending loop of Henle
○ CloUrs 1. Glomerular Filtration Tests
■ Chloride 2. Tubular Reabsorption Tests
■ Urea 3. Tubular Secretion Tests
■ Sodium 4. Renal Blood Flow Tests
● Distal convoluted tubule
○ Sodium is reabsorbed but controlled by ● Why should we perform renal function tests?
aldosterone ● These rely on the measurement of the waste products in
■ When there is aldosterone, sodium the blood (usually urea and creatinine) which accumulate
will be reabsorbed in the DCT when the kidneys begin to fail
● Collecting duct ○ Urea and creatinine are waste products of the
○ ADH controlled H20 reabsorption blood
■ Water will only be reabsorbed when ○ Waste products should be excreted by the
there is antidiuretic hormone (ADH) kidney
■ kidney problems = increase in urea
What will happen to the Sodium and Water Balance? and creatinine in the bloof\d
● Conn Syndrome? ● There should be 20%-30% of the nephrons still
○ Excess of aldosterone functioning (advanced renal failure) before concentration
○ If there will be excess of aldosterone, sodium of these product begin to accumulate in the blood
will be reabsorbed. ○ 70%-80% of the nephrons ang masisira before
3
these product accumulate ○ Urinary Ammonia
○ Not sensitive markers to test for renal failure, ➢ These are not done in the present
but if it is vast, it can accumulate in the blood
and diagnosed as having kidney failure
Glomerular Filtration Tests
Clearance Tests
● Standard test used to measure the filtering capacity of
the glomeruli
● Measures the rate at which the kidneys are able to
remove a filterable substance from the blood
● Substances that can be filtered by the glomerulus:
○ Urea clearance test
○ Creatinine clearance test
○ Inulin clearance
○ Cystatin C
● To ensure accuracy of the test, substance to be analyzed
must:
1. Be neither reabsorbed nor secrete
○ Because it is the glomerular filtration NON-PROTEIN NITROGEN COMPOUNDS
that we want to test ● Urea
2. Be stable in the urine during a possible 24-hour ● Uric acid
collection period ● Creatinine
○ In testing GF, urine should be 24 ● Ammonia
hours old
3. Have a consistent plasma level UREA
4. Be available to the body (not toxic) ● NPN with the highest concentration in the blood
5. Be available for chemical analysis (can be ● Major excretory product of protein metabolism
tested) ○ When the proteins in the body are
○ There must be a test that is standard metabolized or broken down, it becomes urea
to measure the analyte ● BUN → Blood Urea Nitrogen
○ Obsolete term because we have to measure
Tubular Reabsorption Tests urea as a whole not just
the nitrogen in the urea
Concentration Tests ● BUN x 2.14 = Urea
● Often the first function to be affected in renal disease ● Has 2 amino groups and 1 carboxyl
● Tests to determine the ability of the tubules to reabsorb group
the essential salts and water that have been
non-selectively filtered by the glomerulus
● Sodium, chloride, & water will be filtered by the Urea Cycle
glomerulus but the body needs these, so the tubules will
reabsorbed those substances
● Osmolality and osmolarity - measures the “concentration”
of analytes in the urine
○ If there are a lot of analytes in the urine, it want
reabsorbed by the tubules = there is a defect
● Free water clearance - measures the amount of
solute-free water excreted in the kidney
● Obsolete tests:
○ Fishberg Test - 24 hours fluid deprivation
○ Mosential Test - Day vs Night concentration
function
● To ensure accuracy of the test, substance to be analyzed
must:
1. Be neither reabsorbed nor secreted
2. Be stable in the urine during possible 24 hour
collection period
3. Have a consistent plasma level
4. Be visible to the body
5. Be available for chemical analysis (can be
tested) ● Urea is formed in the liver from CO2 and ammonia (from
the deamination of proteins)
Tubular Secretion and Renal Blood Flow Tests ○ The major waste product of protein metabolism
● To measure the exact amount of blood flowing through ○ Exogenous or endogenous protein will undergo
the kidney, it is necessary to use a substance that is: proteolysis which is broken down into amino
○ How to measure the blood flowing to the acids and the amino acids are deaminated to
kidney? form ammonia
■ Use a substance that is completely ○ Ammonia will be combined with CO2 to form
removed from the blood (peritubular urea
capillaries) rather than being removed ○ Happens in the liver
when the blood reaches the ● Excreted by the kidneys
glomerulus ○ 90% excretion and appears in the urine
● To be secreted in the urine ● <10% are excreted in the GI tract and skin
○ PAH Test (para-aminohippuric test) ● Concentration of urea in the blood is affected by
○ Titratable Acidity ○ Protein content of the diet
4
■ Product of protein
metabolism/catabolism
○ Rate of protein metabolism
■ Increased levels of urea because of
protein metabolism from the muscles
○ Renal function and perfusion
■ When there is a failure of renal
function, the urea will not be excreted
from the urine and will accumulate in
the blood lading to increased levels
Urea Disorders: Pathophysiology

● Azotemia - elevation of urea in the blood
● Uremia - azotemia + renal failure
○ Elevation of urea with renal failure
● Causes of azotemia in the blood
○ Prerenal How do we differentiate the different types of causes of
■ Before the kidney azotemia?
○ Renal ● urea/creatinine ratio (normal - 10:1 to 20:1)
■ Kidney ● Prerenal azotemia - high urea/ creatinine ratio
○ Postrenal ○ Low creatinine
■ After kidney ● Renal azotemia/ uremia - normal urea/ creatinine ratio
● Postrenal azotemia - high urea/ creatinine ratio with high
CAUSES OF ABNORMAL PLASMA UREA CONCENTRATION creatinine
● Low urea/ creatinine ratio - low protein intake, severe
INCREASED CONCENTRATION liver disease
○ No urea due to lack of protein in the liver
Prerenal (causes of Congestive heart failure - heart can’t
azotemia) pump the blood to the kidneys; no
● Ex. A patient with azotemia
blood in the kidneys = can't filter blood
○ Remove urea in the blood through dialysis
(urea will stay in the circulation)
Failure of blood to be perfused to the
CREATININE
liver to be filtered
● Formed from creatine and creatine phosphate in the
Shock, hemorrhage - loss of blood muscle
(lower blood volume); lower perfusion ● Released to the plasma in proportion to muscle mass
in the kidneys (increased urea in the ○ If bigger muscle mass = more creatine and
blood) creatine phosphate = more creatinine
● Plasma creatinine is an indirect measure of GFR (MW =
Dehydration
113 Da)
Increased protein catabolism ○ GFR - Glomerular Filtration Rate
○ 113 Da is less than 60000 Da or 60 kDa (dapat
High-protein diet mafilter ng glomerulus ang creatinine)
Renal (causes of Acute and chronic renal failure - ○ Increased plasma creatinine = renal failure
azotemia) kidney can’t filter urea (urea will (glomerulus can’t filter the creatinine)
- Can be causes of accumulate in the blood) ● Diet?
uremia due to renal ○ Cannot affect the creatinine levels
Renal disease, including glomerular ○ Depends on the muscle mass
failure
nephritis and tubular necrosis
Postrenal Urinary tract obstruction - after the
kidney (ureter, urinary bladder)
Stones in the ureter may cause
backflow of urine to the kidneys =
kidneys can't filter (no storage) =
increase urea in the blood
DECREASED CONCENTRATION
Low protein intake
Severe vomiting and diarrhea
Liver disease - body cant produce
urea in the liver
Pregnancy - increase blood flow = ● Creatine is synthesized in the liver from arginine, glycine,
increase urea and methionine
● Becomes creatine phosphate(phosphocreatine) in the
muscle
○ creatine phosphate
■ High energy compound.
■ Ginagamit ng ating muscle
● Creatine phosphate and creatine become creatinine
○ Becomes creatinine if they are used by the
muscle as energy.
○ Creatine will become creatinine when it loses
water.
○ Creatine - water = creatinine
○ Phosphocreatine - phosphoric acid = creatinine
5
● Creatinine is released to the circulation in a relatively
constant rate in proportion to muscle mass.
○ Men have more creatine level than women
■ Men have more muscle mass.
● It is filtered by the glomerulus into the urine.
Clinical Application of Creatinine Measurement

● Determine the sufficiency of kidney function
● Determine the severity of kidney damage
● Monitor the progression of kidney disease.
○ Hypertension can cause kidney damage,so,
his/her creatinine levels will be measured from
time to time
● CREATININE CLEARANCE
○ amount of creatinine eliminated from the blood
by the kidneys (usually a 24-hour sample
● CrCI = UV/P x 1.73/A
● U = urine creatinine (mg/dL)
● V = volume of the 24-hour urine per minute = V/1440 =
ml/min
● P= plasma creatinine (mg/dL)
● A= body surface area (in m^2)
Creatinine Disorders
PATHOPHYSIOLOGY RNA or DNA → Purines → Hypoxanthine & Guanine → Xanthine
● When there is INCREASED plasma creatinine what does → (xanthine oxidase) → Uric acid → Excreted in the urine
it tell about the patient's renal function? ● Uric acid can form crystals in joints (Gout)
● It is an insensitive marker and is not measurably
increased until the renal function is decreased by 50% Biochemistry
○ 70-80% ● Purines (Guanine and Adenine) from the breakdown
● Muscle diseases? ingested of nucleic acids and tissue destruction are
○ If there are muscle diseases = ↑ in creatine converted to uric acid
■ Bcoz marelease niya yung creatine ● 98-100% of filtered uric acid is reabsorbed in the PCT
from the muscles. ● 70% excreted in the kidneys, others excreted in the GI
■ creatine → creatinine tract
○ Muscular dystrophy ● Most uric acid in the plasma is in the form of
○ Poliomyelitis monosodium urate
○ Trauma ○ Basic/neutral pH
○ Measurement of Creatine Kinase? ● At the pH of plasma (about ~7), urate is relatively
■ CK will convert creatine to creatine insoluble
phosphate ○ If urate or uric acid is increased, it will be
● Also found in the muscle deposited in the joints and tissue
■ ↑ creatine kinase =muscle destruction ● At concentrations >6.8 mg/dL, the plasma is saturated
forming urate crystals
● In acidic urine (pH <5.75), uric acid predominates and
URIC ACID is seen as uric acid crystals
GENERAL CHARACTERISTICS
● Product of the catabolism of purine nucleic acids
○ Urea = protein
○ creatinine = muscle
○ Uric acid = nucleic acid
■ Purine nucleic acid
● Guanin
● Adenine
● Relatively insoluble in plasma and, in high
concentrations, can be deposited in the joints and tissue
causing pain and inflammation
○ Gout - increased in uric acid Clinical Application of Uric Acid Measurement
● Diagnosis and monitoring of treatment of gout
● Prevent uric acid nephropathy during chemotherapeutic
treatment
○ In chemotherapy, there is increased destruction
of cells, RNA and DNA of the cells will be
released causing an increased uric acid
● Assess inherited disorders of purine metabolism
● Detect kidney function
● Assist in the diagnosis of renal calculi
○ Renal calculi - kidney stone
○ Uric acid stones
Pathophysiology
● Increased Uric Acid
○ Gout
○ Increased catabolism of nucleic acids
6
○ Renal disease novo purine synthesis)
■ Hindi kaya ifliter ng glomerulus ang ■ Konti and purine, konti ang uric acid
uric acid ○ Overtreatment with allopurinol
Gout ■ Drug used to lower uric acid
● Found primarily in men (30-50 years old) ■ Inhibits the enzyme xanthine oxidase
● Pain and inflammation of the joints (due to precipitation
of monosodium urates) Ammonia
● Plasma uric acid is usually greater than 6.0 mg/dL
● Can form renal calculi General Characteristics
● Postmenopausal women are prone to gout ● Produced from the deamination of amino acids during
● TOPHI formation protein metabolism
○ Precipitations in the join = tophi/tophus ● Converted to urea in the liver
○ Caused by increased uric acid ○ Protein → amino acid → AA will be deaminated
to ammonia = urea
● Free ammonia is toxic
Other causes of increased uric acid

● Increased metabolism of cell nuclei (chemotherapy)
● hemolytic/megaloblastic anemia
○ Increased destruction of red blood cells causing
release of rna and dna = increase in uric acid
● Renal disease
● Toxemia of pregnancy
● Lesch-Nyhan Syndrome
○ X-linked genetic disorder caused by the
complete deficiency of HGPRT (hypoxanthine
guanine phosphoribsyl transferase) ● Deamination of NH2= ammonia
○ Mostly boys are affected
○ No HGPRT Clinical Application of Ammonia Measurement
● Hepatic failure
○ The ammonia cannot be converted to urea
○ Increased in the ammonia, decreased in the
urea
● Reye’s syndrome
● Inherited defects in the urea cycle
○ Will stay as ammonia, thus it is increased and
is toxic
● From purines, it will become hypoxanthine and guanine

and will be salvaged by HGPRT to become purines and Reye’s Syndrome
gagawin ulit na RNA or DNA ● Serious and fatal disease
● HGPRT will decrease uric acid ● Occurs mostly in children
● Preceded by viral infection and administration of aspirin
Hyperuricemia ○ Damage the liver of the child
● Decrease in uric acid in the blood ● Acute metabolic disorder of the liver
● Causes
○ Liver disease Ammonia Neurotoxicity
○ Defective tubular reabsorption (Fanconi ● How can ammonia be toxic to the body?
Syndrome) ○ Ammonia will go to the brain and be added with
■ Defective tubular reabsorption of the glutamate to be glutamine by glutamine
uric acid synthase
○ azathioprine/6-mercaptopurine (inhibitors of de ○ Glutamine will cause:
7
■ Cerebral edema
■ Intracranial Hypertension
■ Neuronal dysfunction
➢ Causing hepatic
encephalopathy
Hepatic Encephalopathy
● The patient will just be sleeping, very confused, or in
coma (in severe cases)
8
LECTURE 2: LIVER FUNCTION TESTS

Prof. Francis Ian Salaver, RMT, MD
NOVEMBER 16, 2021
LIVER
● Largest internal organ of the body
● 1.2-1.5 kilogram in adults
● Located below the diaphragm
○ The diaphragm is the skeletal muscle we use
for respiration and it separate the organisms of
the thoracic cavity from the organs of the
abdominal cavity
● The liver is a soft organ and it must be protected by a

covering called the Glisson’s Capsule
● The Glisson’s capsule is composed of dense irregular
tissue
● Dense irregular tissue are designed to provide protection
to smooth organs
● Divided into right and left lobes by falciform ligament

○ (right lobe is 6x bigger than the left)
○ No known functional difference between the
two lobes
● Cross section of liver

● Appreciate the rows of hepatocytes within its stroma
● Look at the covering of the liver, the Glison’s capsule
● The Glisson’s capsule give rise to the connective tissue
septum
● This septum will invaginate the stroma of the liver and
will divide the liver into lobules
Septa divide the liver into classical lobules (hexagonal in

● Dissected cadaver
shape)
● Diaphragm is situated above the liver which is a skeletal
muscle that assists us during respiration
● It separates the thoracic cavity from the abdominal cavity
● As the connective tissue septa will penetrate the

stromma of the liver, they will divide the liver into lobules
● The blue colored structure are the connective tissue
septa
● Actual picture of a liver ● A lobule has a hexagonal shape meaning it has 6 sides
● The right and the left lobes are separated by the falciform
ligament
1
● Why is it important for the portal vein to carry the blood
coming from the stomach, and the small and large
intestine?
○ So that every time we absorb toxic chemicals
from the food we have ingested, these toxic
chemicals will first pass through the liver for the
chemicals to be detoxified so that they cannot
reach systemic circulation
● That’s the reason why oral administration of drugs is not
as effective as intravenous
○ Because in oral administration, the drugs have
to pass through the liver via the portal vein and
the liver might metabolize some of them. This is
called the First Pass Metabolism
● Has 6 sides and a central structure called the central vein

● Surrounding the central vein are rows of hepatocytes
● Underside of the liver is where you can find the portal

vein and the hepatic artery
● These two are accompanied by the green-colored bile
duct
● And the area where you can find the three is the porta
hepatis
● The liver is unique when it comes to its blood supply

because it receives blood from two blood vessels
● For a typical organ in the body, it receives blood via its
artery and he blood that will flow through will drain
through its vein
● The liver receive blood from two blood vessels, the larger
portal vein and the smaller hepatic artery
● Dual blood supply
○ Receiving well oxygenated blood from the
systemic circulation via the hepatic artery
(red-colored blood vessel arising from the
abdominal aorta) which a direct branch of aorta
(25%)
○ Larger volume (75%) of poorly oxygenated but
nutrient-rich blood coming from the intestinal
● Wherever the hepatic artery will go into the liver, it will be
tract via the portal vein
accompanied by the portal vein and the bile duct
● The always com as a group of three
● They are always referred to as the Portal Triads
Veins from GIT form portal veins
Portal triads around the lobules
● The portal vein is formed by the union of venous

drainages of the of the organs that belong to the GIT
● The very reason why the blood carried by the portal vein ● Recall that hepatic lobules have hexagonal shapes and
is nutrient rich they are formed by the invagination of the liver by the
● Since these are blood from the venous drainage of the connective tissue septa
organs of the GIT, expect that the blood is poorly ● Ther in the center is the central vein,
oxygenated ● The peripheral portion of the hepatic lobule is where you
2
can find the portal triads ● Expect that the blood that will reach the central vein is
● Blue= portal vein already poorly oxygenated and toxin free (even microbe
● Red = hepatic artery free)
● Green = bile duct ○ Because there are phagocytes found within the
sinusoids
● Sinusoids
○ Spaces between cords of hepatocytes’ where
the arterial and venous blood mix and drain
towards the central vein
● One hepatic lobule

● In the center, the central vein
● Found at te peripheral portions are the portal triads
● Center: central vein

○ Surrounded by cords of hepatocytes
○ Spaces between the cords of hepatocytes
(sinusoids)
■ Pointed by black arrow
● Higher magnification view of the portal triads

● Portal vein- presence of RBCs in the lumen
● Hepatic artery= RBCs in the lumen and is easily
identified by the presence of a thick layer of smooth
muscles on its wall
● Third arrow: bile duct
○ Identified by the simple cuboidal/columnar
lining epithelium
● Some sinusoids are filled with RBC
○ There is blood flowing through the sinusoids
coming from the portal triads
○ Drain towards the central vein
● Blue arrow = sinusoid

● Within the walls of the sinusoids, you have there the rows
or cords of hepatocytes
● First red arrow= hepatic artery
● 2nd red arrow= portal vein
● The blood coming from these two vessels will drain into ● BLUE: Portal vein
the sinusoids ● RED: Hepatic artery
● It is in the sinusoids that the blood will flow through so ● Follow the black arrows
that the blood can reach the central vein ○ Indicates that the blood coming from these 2
● S the blood flows through the sinusoids, the blood is blood vessels will drain and mix into the
acted upon by the hepatocytes on the walls of the sinusoids
sinusoids ○ From the sinusoids, the blood will reach the
● If there are toxic chemicals flowing through the sinusoids, central vein
they can be metabolized by the hepatocytes ● There is one particular cell that we can find in the
● These hepatocytes will also get oxygen from the blood sinusoids - Kupffer cell
flowing through the sinusoids ○ Kupffer cells are the phagocytes of the liver
3
○ Strategically found in the sinusoids, because ● Focus on the structure pointed by the arrows
they need to filter the microorganisms that ● If the hepatocytes are found adjacent to each other. They
could be brought by the blood from portal vein will form the bile canaliculus, or bile canaliculi
○ Once the blood reaches the central vein, it is ● The green-colored channel pointed by the 3 red arrows,
already poorly oxygenated, toxin free, and even is the bile canaliculus
microbe free ○ Drains into the large hepatic duct
(green-colored structure beside the portal vein)
Kupffer cells in sinusoids
● The cells pointed by the arrows are along the sinusoids,

therefore, these are the kupffer cells (phagocytes of the
liver)
● The space between cords of hepatocytes is called the
sinusoids
● The space between 2 adjacent hepatocytes is called the
bile canaliculus
○ Bile canaliculus is responsible for the transport
of bile and drainage of bile into the bile duct
(green-colored structure beside hepatic artery)
● Appreciate the association of the bile canaliculus to the
bile duct/ hepatic duct
● The blood that will fall through the sinusoids will

eventually drain through the central vein
● Central vein will fuse with other central veins to form the
larger interlobular veins
○ This interlobular veins will eventually drain into
the larger hepatic veins
● In the liver you have 2 hepatic ducts: left and right

● They will fuse together to form the common hepatic duct
● Duct of the gallbladder is called cystic duct
● The cystic duct and the common hepatic duct will form
the common bile duct
● The common bile duct and the pancreatic duct will drain
in towards the ampulla of vater of the duodenum
BIOCHEMICAL FUNCTIONS OF THE LIVER

● Synthesis of proteins, lipids and carbohydrates
● The hepatic vein will drain towards the inferior vena cava
● During this time, the blood from the GIT will be returned Synthesis of Proteins
back into the systemic circulation ● ALL proteins found in the blood are produced by the liver
● The toxins absorbed in the GIT were all detoxified by the EXCEPT Immunoglobulins and adult Hemoglobin
hepatocytes before the blood was allowed to go back to ○ Immunoglobulins - antibodies produced by
or enter the systemic circulation activated b cells or plasma cells
○ Adult hemoglobin - produced by the immature
precursors of red cells in the bone marrow
● During the fetal stage, liver plays an essential role in the
development of haemoglobin
○ Fetal hemoglobin was produced by the liver
● Albumin - most abundant protein the blood
● Acute phase reactants
○ Produced in high amounts during the early
stage or in the acute stage of infection
● Coagulation proteins
○ Responsible for the formation of fibrin clot
during the process of hemostasis
● Stores amino acids for production of proteins
4
Synthesis of Lipids
● Synthesizes 70% of the cholesterol in the body
○ 30% of lipids are derived from diet
○ People who have hypercholesterolemia to take
in statin drugs
■ Statin drugs - will not allow
hepatocytes of the liver to produce
cholesterol
■ Lower cholesterol level
● Synthesize VLDL in times of fasting and starvation
● Normal serum electrophoresis
● The fraction albumin, alpha 1, alpha 2, and beta are
produced in the liver
● Gamma contains the antibodies, antibodies are produced
by the plasma cells
● Electrophoresis of someone with liver cirrhosis

● Significant decrease on the level of proteins that belong
to the albumin, alpha 1, alpha 2 and beta fractions
● Since they are produced in the liver, so a person with
liver cirrhosis will have decrease levels of these proteins
● The decrease in the beta fraction is not obvious because ● Gathers free fatty acids from the diet and those that are
of the increase in the IgA levels of people with liver produced in the liver itself and converts them to acetyl
cirrhosis CoA→ will enter the krebs cycle→ participate in ETC→
● The increase in IgA will form the beta-gamma bridging production of ATP
● The decrease in the beta fraction is not directly
observable Synthesis of Carbohydrates
● All of the fractions are affected because they were ● Metabolism of the carbohydrates is one of the most
produced in the liver except for the gamma fraction important functions of the liver
○ The gamma fraction produce antibodies which 1. use glucose for its own cellular energy
are not produced in the liver requirement or allow glucose to reach the
● Coagulation proteins are the proteins responsible for the central vein so that it will reach the systemic
conversion of fibrinogen into fibrin, so that the fibrin can circulation and be used up by other cells and
form the meshwork of clot that will prevent further loss tissues.
during injury 2. circulate the glucose for use at the peripheral
○ Produced in the liver tissues
3. store glucose in the form of glycogen
Liver plays the role in maintaining stable glucose concentration

● Stores excess glucose to form glycogen (glycogenesis)
● Breaks down glycogen back to glucose in fasting states
(glycogenolysis)
● Create glucose from nonsugar carbon substrates
(gluconeogenesis)
BIOCHEMICAL FUNCTIONS OF THE LIVER

● Drug metabolism and detoxification
Drug metabolism and detoxification

● Every substance that is absorbed from the
gastrointestinal tract must first pass through the liver –
first pass effect
● Allows important substances to reach the systemic
circulation and serves as barrier to prevent toxic and
● 2 pathways of coagulation pathway: extrinsic and intrinsic
harmful substances from reaching the systemic
pathways
circulation
○ Both involve coagulation proteins represented
by roman numerals
○ But the point is, these proteins will eventually
end up activating or converting fibrinogen into
fibrin to form the clot during the process of
hemostasis
○ These proteins are produced in the liver
5
● The liver may detoxify substances in two ways:
○ Chemically modify the substance for excretion
(example is the conjugation of bilirubin) ● Hgb has four globin chains; 4 heme molecules; each
■ To make a chemical more excretable, heme has iron in its center.
the chemical must be water soluble.
● How? They will conjugate
the chemical with water
soluble compound
○ Bind the material with a chemical to inactivate it
Nitrogen Metabolism
● Embedded within the globin chains are the heme

molecules
● Product of protein catabolism = ammonia

○ Ammonia
■ Can be absorbed by the GIIT
■ Brought to portal vein into the liver
● Liver functions to convert ammonia→ urea
○ Ammonia is toxic; can kill neurons
○ Once urea enters the systemic circulation,
majority of urea will be excreted by kidney via
formation of urine
● Px with liver failure cannot anymore detoxify or convert
ammonia to urea→ encephalopathy
Ammonia
● Ammonia is the by product of protein metabolism Pic: heme molecule
● Ammonia is neurotoxic ● Central iron atom
● it is converted by the liver into urea and urea is then ○ Surrounded by protoporphyrin rings
excreted by the kidneys
● Liver disease = High ammonia levels in the blood which
can lead to coma
Excretory and Secretory Bile (Bilirubin)

● Processing and excretion of bile which contains bile
acids, bile pigments, cholesterol and other substances
extracted from the blood.
● Bilirubin is the major component of the bile pigment
○ Derived from the breakdown of haemoglobin in
red cells
● RBC only have 120 days life span - they lose their
deformability and flexibility. Thus they are trapped in the
spleen
6
● Splenic macrophages will engulf the trapped RBC and
destroy them. Upon the lysis of the RBC, hemoglobin is
released and is metabolized in the spleen.
● The hemoglobin is broken down into:
○ Globin
■ Is a protein thus if it is metabolized, it
is broken down into amino acids,
which will be re-utilized to form other
proteins or another globin molecule in
the bone marrow
○ Heme
■ Metabolized to form bilirubin
● Bilirubin formed from the heme is called unconjugated or

water insoluble bilirubin
● Once released into the blood, it cannot interact well with
the blood bcs it is 90+% water. Thus, it should form a
complex with albumin
● It is the albumin that will transport the unconjugated
bilirubin towards the liver for further metabolism
Bilirubin 1
● Water – insoluble – requires albumin for it to be
transported to the liver for excretion
● Unconjugated bilirubin
● The heme portion will be acted upon by heme
● Indirect bilirubin
oxygenase, which will remove the iron from the structure
● Cannot be removed from the body unless it is conjugated
of the heme
with glucuronic acid in the liver
● The remaining portion of the heme will be converted into
○ To be eliminated in the body, it must be
the green colored biliverdin, which will be acted upon by
converted by the hepatocytes to a water soluble
biliverdin reductase, converting it into the yellow colored
form.
unconjugated bilirubin
○ Bilirubin will react with glucuronic acid
● Biliverdin = oxidized form of bilirubin
converting it to a water soluble form (excretable
● Bilirubin = reduced form of biliverdin
form of bilirubin)
● Since albumin is a LARGE protein, bilirubin 1 which is
attached to it will never be filtered in the glomerulus, thus
its not expected to appear in the urine
RBC undergoes lysis and release hemoglobin

↓
Hemoglobin is broken down into heme and globin
● Take note: Bilirubin 1 is in complex with albumin as it is
↓
being transported in the blood. If it passes through the
Globin is broken down into amino acids which will be reused by the
glomerulus, it will not be filtered.
body for protein synthesis. Heme is acted upon by heme
● Unconjugated bilirubin will NEVER appear in the urine
oxygenase which will release the iron atoms
● Glomerulus is composed of the bowman’s capsule
↓
(yellow).
Iron atoms released from the heme may be stored in the form of
● Bowman’s capsule is surrounding tuft of capillaries
ferritin in the liver or transported to the BM for the production of
new hemoglobin molecules .
↓
Remaining portions of the heme will be converted to the green
colored biliverdin, which will be acted upon by biliverdin reductase
↓
Yellow colored bilirubin
↓
Bilirubin formed from biliverdin should be transported towards the
liver
7
● Once transported in the liver, the bilirubin will be picked
up by the protein ligandin
● Conjugation will involve the UDP-glucuronyl transferase
○ This will transfer glucuronic acid molecules
from UDP-glucuronate into B1
● Why is albumin and other proteins not easily filtered in ● Product is B2 = water soluble form of bilirubin
the glomerulus?
● 3 layers:
○ Fenestrated capillary
■ Has spores or fenestrae
○ Basement membrane
○ Processes of the podocyte
■ Participate in the process of filtration
● The fenestrated capillary is surrounded by the blue
colored basement membrane, and the basement
membrane is surrounded by the processes of the
podocyte.
Uridyldiphosphate glucoronyl transferase is the enzyme important

for the bilirubin conjugation
● Since B1 is in complex with albumin, it will never be

filtered in the glomerulus
● B2 will be excreted through the efflux transporter which

will drain it to the bile duct for elimination
Bilirubin 2
● Conjugated bilirubin
● Direct bilirubin
● Water soluble
● Secreted by the hepatocyte into the bile canaliculi then
● Albumin will transport the water insoluble B1 to the liver into the gallbladder or into the common bile duct
to undergo the process of conjugation
8
TYPES OF JAUNDICE
Pre hepatic Hepatic Post hepatic
Excessive amount Impaired cellular Impaired excretion

of bilirubin is uptake, defective due to mechanical
presented to the conjugation or obstruction to bile
liver due to abnormal secretion flow
excessive of bilirubin by the
hemolysis liver cell
Elevated Both conjugated Elevated

unconjugated and unconjugated conjugated bilirubin
bilitbrin in serum bilirubin may be in serum
elevated in serum
Heme will be converted to the green colored biliverdin ● Management of the case will depend on the type of
↓ jaundice
Biliverdin to unconjugated bilirubin ● Prehepatic
↓ ○ Liver is normal but is working at maximum
Albumin will transfer unconjugated bilirubin to the liver capacity
↓ ○ Occurs when the problem occurs prior to the
In the liver, B1 will be conjugated with glucuronic acid converting it liver metabolism
to B2 ○ Increased amount of bilirubin 1 being presented
↓ to the liver
B2 will be secreted in the bile duct ○ Increased production of indirect or
water-insoluble bilirubin
● Hepatic
○ Intrinsic liver defect
○ Transport problem
○ Hepatocellular injury
● Posthepatic
Prehepatic Jaundice
● Occurs when the problem occurs prior to the liver
metabolism
● The liver works at maximum capacity
● There is overload of unconjugated bilirubin into the liver
● Increase in the indirect or water-insoluble bilirubin is a
product of heme metabolism = increased in the
breakdown of heme = increased metabolism of
● Urobilinogen will be excreted into the feces in the form of hemoglobin = increased red c`ell destruction
urobilin or stercobilin = brown color of the feces ● Hemolytic anemia
● Metabolized by the intestinal bacteria into colorless ○ Destruction of red blood cells
urobilinogen ● Malaria
○ 80% is converted into the urobilin (stercobilin) ● Sickle cell anemia
which gives the feces the brown color
○ 20% reabsorbed back in the enterohepatic
circulation which will later be eliminated by the
liver (small amount are eliminated by the
kidney)
Jaundice
● From the French word jaune which means yellow
● Yellowish discoloration of the skin, eyes and mucous
membranes due to the retention of bilirubin
● Normal level of bilirubin is 1-1.5 mg/dL
● Jaundice will only become overt or noticeable to the
naked eye at above 3.0mg/dL
● Icterus – term used in the laboratory to refer to a serum
or plasma with a yellowish discoloration due to bile
● Destruction of RBCs by antibodies = hemoglobin is
released and metabolized by the body = production of
9
heme and globin which will be subsequently metabolized
to form the unconjugated bilirubin (presented to the liver)
● In hemolytic anemia, malaria, and sickle cell anemia,

there is an increase destruction of RBCs → increased
metabolism of hemoglobin → increased levels of
● Since there is an increase in the number of unconjugated
bilirubin molecules that the hepatocytes are working,
● The Plasmodium spp. which are the causative agents of they will work at maximum capacity → produce high
Malaria infect human RBCs and undergo schizogony amounts of conjugated bilirubin
● One protozoan = more than two protozoans ○ But, there will be no problem since it will be
● After the completion of schizogony, the parasite will immediately secreted into the bile duct
cause the lysis of the infected RBCs ● What will accumulate in the plasma or blood of patients
● In severe malaria = more destruction of RBCs → with prehepatic jaundice is only the unconjugated
increased production of unconjugated bilirubin bilirubin, because it is not processed immediately by the
liver because the hepatocytes are working at maximum
capacity
● B2 will be unaffected since it is directly secreted into the
bile duct
● The increase in the conjugated bilirubin will also cause
the increase in production of urobilinogen (derived from
conjugated bilirubin)
● RECALL: 80-90% urobilinogen is excreted through the
stool in the form of stercobilin or urobilin, while 10-20% is
reabsorbed back into the body via the enterohepatic
circulation
● The hepatocytes are working at maximum capacity, it is
also the liver that excrete the reabsorbed urobilinogen
● But in prehepatic jaundice, the hepatocytes cannot
anymore perform the function by eliminating the
reabsorbed urobilinogen → eliminated via the kidney
through the urine → there will be increased urobilinogen
level
● In sickle cell anemia, the mutations will cause the RBCs
to sickle at low oxygen environment
● Sickled RBCs are prone to destruction, so if destroyed,
they release hemoglobin and there will be increased
production of unconjugated bilirubin
COMPLETE THE TABLE
Total B1 B2 Urine Urine

bilirubin bilirubin urobilinogen
Malaria
Sickle cell
disease
Hemolytic
anemia
● Unconjugated bilirubin is known to bound to albumin
which has high molecular weight
● so, even if unconjugated bilirubin will elevate in the
blood, it will not be seen in the urine because it will not
be filtered due to its attachment to albumin
10
● The gene encodes the different proteins that we have in
our body
● DNA is double stranded nucleic acid so it’s composed of
two strands
● Even if the strands are complementary to each other,
they contain genes different from each other
● Bilirubin does not exceed 5 mg/dL because the liver can

still handle the overload
● Prehepatic jaundice is also referred to as unconjugated
hyperbilirubinemia
○ Because there is an increased unconjugated
bilirubin in the blood
○ “emia” = blood
● Normal value of bilirubin = 0.2-1.0 mg/dL
● Jaundice will only be overt or observable to the naked
● One of the strand is copied complementarily in the form
eye at the level of above 3.0 mg/dL
of mRNA. Because that portion of the DNA strand
● The hepatocytes wil work at maximum capacity just to
contains the gene that is needed to encode for a certain
convert Bilirubin 1 to Bilirubin 2
protein
● This mRNA will you out of the nucleus, will eventually
Hepatic Jaundice
interact with the ribosomes to initiate the production of
● Involves problems within the liver proteins
● Hepatocellular Injury (Animation)
○ Like in the case of Hepatitis, cells are dying and ● Two strands of the DNA have to be separated from each
the cells cannot effectively clear out bilirubin in other so that one that contains the gene can be copied
complementarily to form the mRNA
● The dark blue colored structure that is moving along the
strand that contains the gene is the RNA polymerase
● The large RNA polymerase is currently copying one of

the DNA strands complementarily to form the mRNA
● How will the RNA polymerase know where to start
transcribing the gene portion of that particular DNA
strand?
the blood thus, there is jaundice

● Problems with the process of conjugation
○ Bilirubin 1 is not converted to Bilirubin 2
● Transport problem
● Occurs when the primary problem causing the jaundice
resides in the liver (intrinsic liver defect of disease)
● Can be because of:
○ Diseases
○ Defects in the transport and metabolism of
bilirubin
■ Gilbert’s Disease ● The RNA polymerase will simply have to look food the
■ Crigler-Najar syndrome promoter region
■ Dub-Johnson Syndrome ● This promoter region will act with the RA polymerase to
■ Neonatal Physiological Jaundice of give the RNA polymerase the idea that it should start
the Newborn transcribing the gene portion of the DNA
● This promoter region is usually composed of the bases
thymine and adenine
● This promoter region can also be called TATA box
because u have there, thymine and adenine bases
● There is a small portion of the DNA referred to as the

gene Gilbert Syndrome
11
● In the gene that encodes for UDP-glucoronyl transferase
enzyme which is important for the conjugation of Bilirubin ● No problem with RBCs, therefore, expect that only the
1 to glucuronic acid to form Bilirubin 2, the promoter RBCs that have reached their lifespan are destroyed in
region is composed of the following sequences of the body
adenine and thymine bases you have A (TA) 6 TAA ● Expect that there is normal amount of hemoglobin
meaning you have there six repeating dinucleotides released from the RBC
thymine and adenine and the promoter region will end up ● There is also a normal amount of unconjugated bilirubin
with thymine and adenine bases that will be derived from the hemoglobin released from
● In the case of Gilbert’s Syndrome, there is insertion of old rbc
additional dinucelotide containing TNA into the promoter ○ Unconjugated bilirubin will be processed by the
region of the gene liver
● The resulting promoter region is A (TA) 7 TAA and you ● Problem: the person with gilbert’s syndrome doesn't have
have the last three bases TAA enough glucuronosyltransferase enzymes
● The problem is, the promoter region cannot efficiently ○ Consequence: not all of the unconjugated
activate the RNA polymerase to start the transcription of bilirubin will be converted to conjugated bilirubin
the gene or bilirubin glucuronide
● That will have an impact on the number of functional ○ Increase in unconjugated bilirubin of the px
enzymes that the person can produce ■ Hyperbilirubinemia
● Instead of producing 100 functional enzymes, the person ■ Failure in the process of conjugation
with Gilbert's Syndrome will only produce 20-30% ■ Produce less amount of conjugated
function enzyme and that will have an impact on the bilirubin
conjugation process of Bilirubin 1 to Bilirubin 2 ■ Less conjugate bilirubin that will reach
● Gilbert syndrome - caused by changes in the UGT1A1 the intestine
gene ■ Decrease in the production of
○ Encodes for UDP-glucoronyl transferase urobilinogen (few absorbed by the
○ There is the insertion of extra dinucleotide enterohepatic circulation)
containing thymine and adenine converting the
promoter region from A (TA)6 TAA to A (TA)7
TAA which will have an impact on the amount
of active and functional UDP-glucoronyl
transferase that the person can produce
○ Molecular defect in GS is the addition of an
extra dinucleotide TA to the promoter TATA box
of the conjugating enzyme UGT1A1
■ The resulting genotype is designated
A(TA)7TAA (instead of the normal Crigler-Najjar syndrome
A(TA)6TAA) ○ Mutations on the gene itself
■ This gene provides instructions for ○ Type 1
making the bilirubin uridine ■ SEVERE unconjugated
diphosphate glucuronosyltransferase hyperbilirubinemia at birth
(bilirubin-UGT) enzyme, which is ■ TOTAL absence of the
found primarily in liver cells and is UDP-glucoronyl transferase
necessary for the removal of bilirubin ● None of the B1 is
from the body. conjugated to form B2
■ 20-30% of the enzyme are still ■ Fatal because of kernicterus (bilirubin
functional is toxic to nerves)
○ Occurs during puberty ○ Type 2
■ Differentiates it from other disease ■ Partial absence of the
○ Has no morbidity or mortality and no clinical UDP-glucoronyl transferase
consequences ● Both Gilbert and Crigler-Najajr are problems involving the
■ Does not shorten life span of patient conjugation process of bilirubin
○ How to differentiate?
○ Gilbert syndrome occurs during puberty while
Crigler-Najjar will start affecting the patient as
early as childhood
12
● No increase lysis of RBC
○ Normal HGB
○ Normal unconjugated of bilirubin
○ ↓ production of urobilinogen
■ No secretion of conjugated bilirubin in
the small intestine
○ Problem: cannot secrete conjugated bilirubin
into the bile duct because of the absence of
MRP→ accumulate in the hepatocytes→
leaking into the blood→ ↑ B2 fraction
Bilirubin 1
↓
will be conjugated with glucuronic acid ( UDP glucuronyl
transferase enzyme encoded by UGTA1)
↓
B1→ converted to B2
↓
B2 secreted will be secreted by the hepatocytes by the bile duct
● The hepatocytes will use the multidrug resistance Px with Dubin Johnson Syndrome will have:
associated protein (MRP2) ● hyperbilirubinemia = because of B2 fraction
○ Dubin-Johnson Syndrome ○ B2 - conjugated bilirubin and is already water
■ Absence of MRP2 soluble; this will not bind with albumin to be
● Cannot secrete B2 into the transported in the blood → it can easily be
bile duct→B2 accumulation filtered in the urine→ urine bilirubin ↑
in the plasma or serum ● Since conjugated bilirubin will not be able to reach the
● Will have ↑ total bilirubin in small intestine→ no conversion to urobilinogen→ urine
the plasma or seri\um urobilinogen will be within normal range or low levels
Hepatic Jaundice
● Dubin Johnson Syndrome Rotor syndrome
○ Absence of the canalicular multidrug ● Similar to Dubin Johnson, except that pigmentation in the
resistance/multispecific organic anionic liver is not seen in biopsy
transporter protein (MDR2/cMOAT) which is ○ Similar bcoz of hyperbilirubinemia and ↑ B2
important in the excretion of the conjugated fraction
bilirubin into the bile duct ● The SLCO1B1 and SLCO1B3 genes are involved in
○ The CMOAT protein has also been called the Rotor syndrome.
multidrug resistance-associated ○ The SLCO1B1 and SLCO1B3 genes provide
○ protein 2 gene (MRP2) instructions for making similar proteins, called
○ Biopsy will reveal dark pigmentation of the liver organic anion transporting polypeptide 1B1
■ pigment accumulation was shown to (OATP1B1) and organic anion transporting
result from retention of anionic polypeptide 1B3 (OATP1B3), respectively.
metabolites of tyrosine, phenylalanine
and tryptophan, which polymerized to
form a similar melanin like pigment in
vivo
■ metabolites of tyrosine, phenylalanine
and tryptophan are secreted by
hepatocytes into the bile duct via
MRP2
13
○ Urobilinogen is produced by bacteria from
conjugated bilirubin
● Problem with these px: The hepatocytes fail to secrete
the conjugated bilirubin into the bile duct for it to be
strained into the small intestine
○ There is decreased production of urobilinogen
and urobilin
Physiologic jaundice of the newborn

● Physiologic bcs there is no underlying cause
● Last function of the liver to be activated after birth is the
synthesis of UDP-glucoronyl transferase
○ In some newborns, the liver is not yet capable
● Red star: sinusoids
of synthesizing this enzyme thus, the
● Blue: bile canaliculus
conjugation process will not occur immediately
○ Between them is hepatocyte
■ Hence, newborns will have
hyperbilirubinemia bcs of the B1
fraction
● Build of unconjugated bilirubin and deposition in the brain
○ Bilirubin is neurotoxic which can cause
significant damage to the brain
● Treated with ultraviolet radiation or in extreme cases,
exchange transfusion is done
○ Exposing the child to UV light, will oxidize
bilirubin back to biliverdin, which is not toxic to
the neurons
Red star: unconjugated bilirubin

↓
Will be conjugated with glucuronic acid to form B2
↓
B2 can be secreted into the sinusoid and can become part of the
blood
↓
B2 will be reabsorbed back by the (OATP1B1/1B3) ● Exchanged transfusion
↓ ● 5-10 ml of the blood of the baby is manually removed
B2 will be secreted in the bile canaliculi while at the same time, u transfuse 5-10 ml of donor’s
blood to eliminate the bilirubin in the blood of the
Px with rotor syndrome do not have (OATP1B1/1B3) newborn baby
● After B2is secreted in the blood, it remains in the blood
causing hyperbilirubinemia Posthepatic jaundice
● Results from biliary obstructive disease due to:
● In a normal liver, a majority of bilirubin is conjugated by ○ Gallstones
hepatocytes and secreted back into the blood. It is then ○ Tumors
reabsorbed in downstream hepatocytes by the OATP1B1 ■ Cholangiocarcinoma tumor - tumor of
and OATP1B3 proteins. bile duct
○ These proteins are absent in patients with rotor ● Bilirubin will regurgitate back
syndrome to the liver
■ Liver of this px are not darkly ● Fraction B2 will increase
pigmented bcs OATP1B1 and ○ Parasites
OATP1B3 are not responsible for ■ Hepatic flukes (clonorchis,,
eliminating the metabolites of opisthorchis, fasciola or some
tryptophan, phenylalanine, and nematodes esp. ascaris
tyrosine lumbricoides))
■ Thus, there is no dark pigmentation of How do we measure bilirubin in the lab?
the liver
Acholic stool due to lack of urobilin
● Px with dubin-johnson and rotor syndrome will have

● Make bilirubin react with diazotized sulfanilic acid reagent
acholic stool or clay colored stools
○ Diazotized sulfanilic acid = sulfanilic acid +
○ Stools are normally brown bcs of urobilin, which
hydrochloric acid + sodium nitrite
is derived from urobilinogen
14
○ Will split bilirubin into 2 isomers of:
■ Azobilirubin isomers
Analysis of Bilirubin
● Ehrlich (1883)
○ Used the diazo reaction in determining the
presence of bilirubin in urine samples
● Urine Bilirubin can react with diazotized sulfanilic acid to
form a red colored product (DIAZO reaction)
○ Product: Azobilirubin isomers
■ Red to purple color
● van den Bergh (1913) – found out that diazo reaction can
be applied to serum samples provided there is a use of
an accelerator but his method had a lot of errors
● Malloy and Evelyn (1937)
○ modified van den Bergh method by using 50% ● If the bilirubin in the serum is directly made with
methanol as accelerator/accentuator diazotized sulfanilic acid, out of the two fractions of
● Jendrassik and Grof (1938) bilirubin, it is only the conjugated bilirubin that will react
○ uses caffeine-benzoate-acetate as with the diazotized sulfanilic acid
accelerator ● If the solution turned purple, what only reacted with the
diazotized sulfanilic acid is the conjugated bilirubin
● It reacts directly to the diazotized sulfanilic acid
● Thus, conjugated bilirubin is also known as direct
bilirubin
● In the diazo reaction the bilirubin in the serum is made to

react with diazotized sulfanilic acid to produce the red to
purple colored azobilirubin isomers
● B1 fraction
● Prepare another serum and make it react with the
diazotized sulfanilic acid but with an accelerator
● Both b1 and b2 will react with the diazotized sulfanilic
acid = TOTAL BILIRUBIN ASSAY
○ Diazotized sulfanilic acid plus accelerator
● Add ascorbic acid to terminate the reaction

● After terminating, place the sample in a cuvette and
place in the spectrophotometer at 560 nm
● In the lab, we can measure the total bilirubin and the

conjugated bilirubin
● Level of the B1 fraction (unconjugated bilirubin)
○ Subtract from the total bilirubin level, the level
of conjugated bilirubin
● A certain color can absorb another color and they are

described to be complementary colors
○ Red - green
○ Purple - yellow
● The higher the bilirubin in the sample, the darker the
color is
● The problem with Diazo reaction, there are other
15
interfering substances that can produce the purple color ● Hemolysis can delay reaction of bilirubin with diazo
and they can add on to the amount of bilirubin in the reagent
sample and we don't want that ○ Can cause false decrease in the bilirubin level
● Thus, the Evelyn-Malloy and Jendrassik-Grof method ● Bilirubin is the reduced form of biliverdin, if bilirubin is
can be modified by adding alkaline tartrate solution oxidized, it will be reconverted back to biliverdin and u
○ alkaline tartrate solution can no longer measure it using the diazo reaction
■ Turn the purple color produced by the ○ Falsely low results
azobilirubin isomers into intense blue ● What causes the oxidation of bilirubin to biliverdin?
color ○ If exposed to light, bilirubin level may be
● other interfering substances decreased 30%-50% per hour
are not measured ● Store serum and plasma should be stored in dark. Serum
○ Put the cuvette in the spectrophotometer set at and plasma are stable for 2 days at room temperature, 1
600 nm week at 4 degree Celsius and indefinitely at -20 degree
■ Why? Celsius
● If the color of the solution is ● Evelyn Malloy should be done at pH 1.2 thus Jendrassik
blue, it will absorb the Grof is more preferred cause it is not affected by
complementary color orange changes in pH
(has a wavelength between
580-620 nm) Jendrassik-Grof Method (advantages)
● Not affected by pH changes
● Insensitive to a 50-fold variation in protein concentration
○ Methanol accelerator in Evelyn Malloy can
cause the precipitation of proteins in the plasma
- cause interference
● Higher sensitivity than Evelyn Malloy
○ sensitivity: the ability of the method to detect
even the smallest amount of the analyte in the
sample
● Is not affected by haemoglobin up to 750 mg/dL
Urine and Fecal Urobilinogen

● Colorless end product of bilirubin metabolism that is
Principle oxidized by intestinal bacteria to the brown pigment
called urobilin or stercobilin
● SERUM plus Diazotized sulfanilic acid = Direct bilirubin ● 10% is excreted in the feces while 80-90% is reabsorbed
● SERUM plus Accentuator + Diazotized sulfanilic acid = into the portal blood and returned to the liver
Direct and indirect bilirubin = Total bilirubin ● A small portion that is not taken up by the hepatocytes
● How to compute for the indirect bilirubin are excreted by the kidneys
○ Total bilirubin minus direct bilirubin ● Increased in haemolytic anemia and in defective liver-cell
function and other diseases characterized by increase
red cell destruction
○ Because if there is increased red cell
destruction, expected that there is an increase
in the metabolism of hemoglobin
○ That will cause an increase in the levels of
○ Since there is unconjugated bilirubin delivered
to the liver, then the liver will also produce
increased amounts of conjugated bilirubin
○ If the increased amount of conjugated bilirubin
will be secreted into the bile duct and into the
intestine, the bacteria will have a lot of
conjugated bilirubin to be converted into
urobilinogen
○ There’s an increase in the amount of
urobilinogen that will be absorbed via the
enterohepatic circulation
○ Since the hepatocytes of the liver are already
working at a maximum capacity just to remove
the unconjugated bilirubin from the blood, the
hepatocytes will no longer be able to secrete
the reabsorbed urobilinogen into the bile duct
○ Majority of this urobilinogen will be excreted
into the urine via the function of the kidney thus,
there is an increase urine urobilinogen
○ Fecal urobilinogen will also increase because of
Diazo Reaction the increase in the amount of the conjugated
● Serum and plasma may be used. bilirubin that is draining into the small intestine
○ Serum is more preferred for it has less proteins. form the bile duct
Plasma has fibrinogen which can precipitate ● Absence in the urine and stool is seen in in bile duct
with the methanol used in Evelyn Malloy obstruction
■ can cause interference in the assay ○ The conjugated bilirubin is not able to reach the
● Fasting sample is recommended in patients with small intestine
hyperlipidemia because fats can cause interference ○ Therefore, the intestinal bacteria cannot act on
○ Can cause false increase in the bilirubin level the conjugated bilirubin converting it into
16
urobilinogen CIRRHOSIS
○ Expect that urine and fecal urobilinogen will be
very low in cases of bile duct obstruction
● Urobilinogen is made to react with Ehrlich reagent
(paradimethyl aminobenzaldehyde pdab) to form a red
color compound
● This is the normal histology of the liver

● Left side, you have a test tube containing a urine sample ○ Septa forming the hepatic lobules
● Added to the sample is the PDAB or the Ehrlich’s reagent
● The Ehrllich’s reagent will react with the urine
urobilinogen producing now a characteristic red-colored
compound
○ The higher the amount of urine urobilinogen,
the darker is the final color
Determination of Urine Urobilinogen

● Urobilinogen reacts with
para-dimethylaminobenzaldehyde (Ehlich’s reagent) to
form red color
● Ascorbic acid is added to keep the urobilinogen in
reduced state (urobilinogen is oxidized to urobilin)
○ Because once it is oxidized, it will form the
urobilin which does not react with
para-dimethylaminobenzaldehyde ● Liver constantly exposed to injurious agents
○ If the urine urobilinogen is oxidized to the ○ Chronic inflammation
urobilin then you will have a falsely low result ● Normal tissues are replaced by fibers
● Saturated sodium acetate is used to stop the reaction ○ Referred to as liver cirrhosis
● Fresh 2 hour urine is collected
● Results are reported in Ehrlich units than in milligrams
because of interfering substances
○ Porphobilinogen
○ Sulfonamides
○ Procaine
○ 5-hydroxyindoleacetic acid
● Testing should be done without delay because
urobilinogen can be oxidized to urobilin and the final
color of the urobilinogen-aldehyde decrease in intensity
○ Read the absorbance immediately using
spectrophotometer
○ Why? The final color decreases in intensity
through time ● The scar formation will replace the normal tissue of the
● Normal value: 0.1-1.0 Ehrlich units for 2 hr urine liver
○ Expect that the liver wont function the same
Determination of Fecal Urobilinogen way it did when it was normal
● Visual inspection is usually sufficient to detect decreased
Cirrhosis
urobilinogen
● Clinical condition in which scar tissue replaces normal
○ If stool appears clay-colored, it indicates that
healthy liver tissue. The scar tissue is replacing the
the px has no urobilin or urobilinogen in his
normal liver tissue
stool sample
● Can be caused by non-alcoholic steatohepatitis (fatty
● Same principle as that of the urine urobilinogen
liver), chronic alcoholism, autoimmune hepatitis, hepatitis
determination
B, C and D,hemochromatosis, alpha-1-antitrypsin
● Alkaline ferrous hydroxide is added first to the feces to
deficiency, drugs and toxins
reduced urobilin to urobilinogen
○ D,hemochromatosis
○ Convert back urobilin to urobilinogen
■ Increase deposition of iron into the
● Normal value: 75-275 Ehrlich units per 100g of fresh
hepatocytes → can kill hepatocytes
feces or 75-400 units per 24 hour specimen
17
■ There's a chance that the px will
develop Reye's syndrome.
● Studies have shown that there is a strong epidemiologic
associated with ingestion of aspirin during a viral
syndrome
● Can lead to noninflammatory encephalopathy and
degeneration of the liver cells
○ Liver cells are already dying, they cannot
detoxify ammonia to urea
■ Ammonia = neurotoxic
Left: normal
Middle: fatty liver
● Yellow = deposition of fats
Right:accumulation of lipids within the stroma of the liver and
cytoplasm of hepatocytes
TUMORS
● Cancers of the liver can be classified as primary or
Left: normal metastatic
Right: brown = deposition of iron → can damage the liver→ ○ PRIMARY
cirrhosis ■ cancer that begins in the liver
○ METASTATIC
ALPHA-1-ANTITRYPSIN DEFICIENCY ■ cancer that occurs when tumors from
other parts of the body spread into the
liver
■ Much more common that primary liver
cancer (90-95%)
■ Metastatic tumor from colon, lungs
and breast.
● Cancers of the liver can be classified as benign and
malignant
○ BENIGN
■ Hepatocellular adenoma
■ Hemangiomas
○ MALIGNANT
■ Hepatocellular carcinoma or
hepatoma
HEPATOCELLULAR ADENOMA
● Is an uncommon solid, benign liver lesion that develops
● A neutrophil is a phagocyte in an otherwise normal-appearing liver
○ Problem: everytime that it is engulfing bacteria, ● Characterized by the presence of a single or solitary
some of its enzymes will leak out into the mass found within the normal appearing liver.
cytoplasm in its surrounding tissue= ● This mass doesn't cause significant damage to the liver
ELASTASE→ tissue destruction tissue. Hence, it is considered as BENIGN.
○ Alpha-1-antitrypsin ● Typically, hepatocellular adenomas are solitary and are
■ A protein that will neutralize the found in young women in association with use of
elastase enzyme estrogen-containing medications
■ In the absence of this protein, ○ Most of the time these women are
elastase is free to cause tissue ASYMPTOMATIC
destruction ○ If they develop pain and other associated
○ People who are born with Deficiency in symptoms, that's when these women could be
Alpha-1-antitrypsin will have organ damages subjected to surgical removal of the mass.
brought by elastase enzyme.
■ Lungs and liver HEMANGIOMAS
● Commonly affected organs
● Benign (non-cancerous) tumor in the liver that is made
REYE'S SYNDROME
up of clusters of blood- filled cavities.
● Occurs in children ● Most liver hemangiomas do not cause symptoms,
● Preceded by a viral syndrome (chicken pox, flu and although larger ones can cause poor appetite, nausea
gastroenteritis) and vomiting.
○ Patients were given aspirin ○ Most of the time, this mass does not cause any
18
symptoms and surgical removal is rarely LIVER ENZYMES
needed.
● Smaller hemangiomas do not need to be treated, but
larger hemangiomas may need surgery.
● The mass of the liver is dark red in color bcs there is a lot
of RBCs
● Hepatocyte and beside it is the sinusoid where you can
● The mass is basically composed of cavities that are filled
find the blood
up with blood.
● Yellow colored figures - hepatic enzymes
● Seen in the biopsy, you have cavities that are filled up
○ These enzymes should be found intracellularly
with red blood cells.
(within the cytoplasm of hepatocytes)
○ There are small amounts of these enzymes
HEPATOCELLULAR CARCINOMA
present in the blood.
● Malignant tumor of the liver
● HCC develops with liver cell damage that eventually
progress to cirrhosis
○ Caused by liver cell damage
○ If the liver cell damage becomes chronic, the
liver will develop cirrhosis.
○ If not treated immediately, that cirrhosis can
lead or progress to hepatocellular carcinoma.
● Causes:
○ Hepatitis B, C and D
■ Known to cause chronic hepatitis
■ With their chronicity, they can cause
cirrhosis, which can lead to the
occurrence of hepatocellular
carcinoma.
○ Aflatoxin of Aspergillus flavus ● If hepatocytes are exposed to injurious agents, these
○ Hemochromatosis cells will undergo lysis and the enzymes in their
■ Deposition of iron into the liver stroma cytoplasm will start leaking into the blood.
and parenchyma. ● This would signify cell damage
○ Alcohol
Aminotransferase
Aspergillus flavus ● Aspartate aminotransferase
○ Serum Glutamic Oxaloacetic Aminotransferase
○ Widely distributed in equal amounts in the
heart, skeletal muscle, and LIVER
● A fungus that is commonly found in peanuts and corn.

● Can produce an AFLATOXIN
○ Aflatoxin - potent carcinogen and can cause the Amino group of aspartate will be transferred to a-ketoglutarate
development of hepatocellular carcinoma. ↓
○ Thus, it is important for us to check the corn A-ketoglutarate will become glutamate
and the peanuts that we eat. ↓
○ green colored colonies Aspartate will be oxaloacetate
● Alanine aminotransferase
○ Serum Glutamic Pyruvic Aminotransferase
○ Mainly found in the liver, lesser amounts in
muscle and kidney
○ More liver specific than AST
19
Alanine and a-ketoglutarate and amino group of alanine will be
transferred to a-ketoglutarate
↓
A-ketoglutarate will become glutamate
↓
Alanine will become pyruvate
○ High levels are found in viral hepatitis, drug or

toxin-induced hepatitis and hepatic ischemia
○ Increased in ALT is greater than that of AST
○ Mildly elevated in bile duct obstruction
Alkaline phosphatase
● Can help us diagnose bile duct destruction
● In the liver, alkaline phosphatase is particularly found on
the cells lining the bile ducts
● Found in the liver, bone, intestine, kidney and placenta
○ HOPIK (Hepatic, Bone (Osteoblast), Placenta,
Intestine and Kidneys
● In the liver, it is found along the bile canaliculi and
therefore it is a good marker of extrahepatic biliary
obstruction
● More elevated in cases of biliary obstruction than in
hepatitis and cirrhosis
● Problem: widely distributed
● Why is alkaline phosphatase elevated in:
○ Pregnant women???
■ Because of the placenta
○ Children?
■ Because they are actively growing
and their osteoblast will deposit bone
matrix = prudence high amounts of
ALP
○ Bone disease?
■ If it is destroyed, the bone will try to
repair itself by activating the
osteoblast
5-Nucleotidase
● Level of this enzyme become significantly high in
hepatobiliary disease hence can be helpful in
differentiating ALP elevations from the liver or from other
sources
● Bile duct obstruction = elevated ALP and 5NT
● Bone disease = elevated ALP but normal 5NT
● Exclusively find in the bile duct
Gamma-glutamyl transferase
● Similar in importance as the 5NT
● GGT is also a microsomal enzyme; therefore ingestion of
alcohol can elevate GGT
○ microsomal enzyme
■ Enzymes in the liver that can be
produced in high amounts if the
hepatocytes are exposed to a
chemical
● Can be used to assess chronic alcoholism
20
LECTURE 3: BLOOD GASSES, pH, AND

BUFFER SYSTEMS
November 22, 2021
ACIDS AND BASES ● Consequently, expressing you acidity in terms of you Ka

● Acid value constant can be inconvenient and not very intuitive
○ Is a substance that can yield a hydrogen ion that’s why your pKa is introduced as an index to express
(H+) when dissolved in water the acidity of your weak acid
● Base ● pKa is preferred rather than the Ka value
○ Is a substance that can yield hydroxyl ions ● Strong Acids
(OH-) ○ have pK values of less than 3.0
○ raising the pH above the pK will cause it to
dissociate and yield a H+
○ pH: The measure of concentration of your
hydrogen ions in an aqueous solution
■ In exponential form raised to the
power of p or to the power of your
hydrogen ion
● Strong Bases
○ have pK values of greater than 9.0
○ lowering the pH below the pK will cause it to
release OH
● If pH is less than pKa then it is mainly protonated acid
● If pH is more than pKa, it is mainly deprotonated
● Acid can yield a hydrogen ion

● Base substance can yield a hydroxide ion
● An acid is a substance that can also donate your
hydrogen ion when dissolved in water
● Base substance is capable of accepting the hydrogen ion
● The relative strength of your acids and bases and their
ability to dissociate in water are described by their
dissociation constant
● Because there is what we call a strong acid, weak acid, ● Strong acids are assumed to dissociate completely when
strong base, weak base in an aqueous solution
● Dissociation Constant (K value) ● Weak acids dissociate only slightly in aqueous soultion.
○ aka ionization constant The majority of molecules remain undissociated
○ relative strengths of acids and bases ○ Some of its hydrogens is still attached
■ their ability to dissociate in water
● pKa
○ negative log of the ionization constant
○ Derived from dissociation constant
○ the pH in which the protonated and
unprotonated forms are present in equal
concentration
Dissociation Constant of Different Acids and their pKa Value
No. Acid Ka pKa

1 Hydroiodic acid (HI) 3.16x10^9 -9.5
2 Hydrobromic acid (HBr) 1.0x10^9 -9
3 Hydrochloric acid (HCl) 1.0x10^6 -6
4 Sulfuric acid (H2SO4) 1.0x10^3 -3
5 Hydronium ion (H3O+) 55 -1.74
6 Nitric acid (HNO3) 28.2 -1.45
7 Trifluoroacetic acid 5.62x10^-1 0.25 ● Strong acids and bases completely dissociate in water,
(CF3COOH) unlike your weak acid and base where you will still find its
8 Oxalic acid 5.37x10^-2 1.27 full contents
(HOOC-COOH) ● In strong acids naseseparate and protons from its
negative ions
9 Acetic acid (CH3C 1.75x10^-5 4.76
1
carbonic acid will neutralize forming bicarbonate ion and
water
○ Easily eliminated by the body
PARAMETERS OF BLOOD GASES
pCO2
● Partial Pressure of CO2 (pCO2)
● Pressure or tension exerted by CO2 gas dissolved in
blood (dCO2)
○ The concentration of CO2 in the blood is not
equal to the partial pressure
○ Partial pressure of CO2 - pressure or tension
BUFFER exerted
○ Total conc. - presence of CO2 in the blood
● An index of efficiency of gas exchange in the lungs
● Not a measure of CO2 concentration in the blood
● 35 to 45 mm Hg
● In plasma, a small amount of CO2 can be physically
dissolved or combined with other protein to form
carbamino compounds
Total CO2 Concentration

● Total CO2 content in the blood
● Consisting of ionized (HCO3-, CO3-, carbamino
● Combination of weak acid and weak base and its salt compound) and unionized fraction (H2CO3) and
● System that will resist changes in pH physically dissolved CO2
○ Tries to maintain normal pH of the body ● 23 to 27 mmol/L
○ If there will be changes in the blood pH, it will
become acidotic or alkalotic, and later the Bicarbonate Ion Concentration
different physiological processes of our body ● The bicarbonate ion concentration in the blood that has
● The effectiveness of the buffer depends upon the pk been equilibrated with CO2 at 40 mm Hg at 37 degrees
value of the buffering system and the pH environment in Celsius
which it is actually placed ● 22 to 26 mmol/L
○ Ex. plasma - bicarbonate carbonic acid
■ Pk value of 6.1, principal buffer PO2
● This shows that Pk and pH value are equal when they ● Partial Pressure of O2 (pO2)
are half of the acid that has dissociated ● The pressure or tension exerted by oxygen gas dissolved
○ If the ph of the solution is equal to the pka, then in arterial blood which reflects the availability of the gas
the acid is in equilibrium (halfy dissociated) in blood but not its content
○ If ph is less than pka then it is mainly ● 80 to 110 mmol/L
protonated acid ● Assess the pulmonary function; measures how well the
○ If ph is more than the pka value then it is lungs work
deprotonated
● Bicarbonate-Carbonic Acid System pH
● The pH of a solution is defined as the negative logarithm
of the hydrogen ion activity (pH = −log aH+).
● a measure of the concentration of hydrogen ions in an
aqueous solution
● average pH of blood (7.40)
○ Normal range: 7.35 - 7.45
1
● pK = 6.1 𝑝ℎ = 𝑙𝑜𝑔 𝑐𝐻+
=− 𝑙𝑜𝑔 𝑐𝐻 +
● Henderson-Hasselbalch Equation
𝐹𝑢𝑛𝑐𝑡𝑖𝑜𝑛 𝑜𝑓 𝑘𝑖𝑑𝑛𝑒𝑦𝑠 (𝑚𝑒𝑡𝑎𝑏𝑜𝑙𝑖𝑐)
𝑝𝐻 𝐹𝑢𝑛𝑐𝑡𝑖𝑜𝑛 𝑜𝑓 𝑙𝑢𝑛𝑔𝑠 (𝑟𝑒𝑠𝑝𝑖𝑟𝑎𝑡𝑜𝑟𝑦)
𝑐𝐻𝐶𝑂3−
pH= pK’a + log
0.0307.𝑝𝐶𝑂2
● A fall in HCO3- or rise in pCO2 will cause a fall in pH
○ ↓pH (<7.35) = Acidotic
● When any acidic substance enters the bloodstream, ■ Bumababa ang HCO3- and pCO2
bicarbonate carbonic acid system will function ■ CO2 = acidic
○ Compensates by providing more bicarbonate or ● A rise in HCO3- or fall in pCO2 will cause a rise in pH
it could terminate acidity ○ ↑ pH (>7.46) = alkalotic
○ Normalizing the blood pH
● If blood stream is to alkaline
○ The buffer system will eliminate bicarbonate, or
increase level of hydrogen (acidity)
● When any acidic substance enters the bloodstream,
bicarbonate ions will neutralize hydronium ions , forming
carbonic acid and water
● When a basic substance enters the bloodstream, the
2
○ NaH2PO4 - weak acid
● When Na2HPO4 comes in contact a strong acid → the
base will pick up your H+ ion and to form weak acid
BUFFER SYSTEM
● Solution or a substance that has the ability to maintain
pH
● Resist any changes in pH and brings back to its optimal
value by addition or removal of hydrogen ions.
● Phosphate buffer system
○ They will just attach to your different
substances (acid or base) and it will be
excreted from the body
PROTEIN BUFFER SYSTEM
● Nearly all of your proteins can function as buffers

○ Proteins are made up of amino acid, which
contains positively charged amino groups and
negatively charged carboxyl groups
○ These charged regions can bind to your
hydrogen and hydroxyl (?), which function as
buffer
● Can occur in intracellular and extracellular fluid ● In an alkaline medium, amino acid acts as an acid and
First line buffer system (chemical buffers) releases H+
● Phosphate buffer system ○ pH ↑ = alkalotic
○ Will play role in plasma and,RBC ○ if H+ are released, it lowers down the pH to
○ Involved in the exchange of sodium ion and maintain the normal level of the body.
urine in hydrogen filtrate ● In acidic medium, amino acid acts as a base and absorbs
● Protein buffer system H+
○ Almost all proteins have buffer capacity ○ pH decreases = acidic
● Carbonic acid-bicarbonate buffer system ○ Amino acid will try to absorb H+ to remove the
Buffer works as a combination of the different buffer systems acidity in the blood and maintain a normal pH
● Our body will try to make blood pH into normal level
PHOSPHATE BUFFER SYSTEM HEMOGLOBIN BUFFER SYSTEM
● Products are easily excreted thru urine etc.

● Bumababa ang H+ para ma maintain yung pH
● Still under protein buffer system bc Hb is a protein

● During the conversion of CO2 into HCO3- , the hydrogen
ion is liberated in the reaction by hemoglobin, which is
● Phosphate can be found in the blood in two forms reduced by the dissociation of oxygen
○ Na2HPO4 = weak base ● Helps to maintain normal pH
3
CO2 from tissue →goes in the RBC → CO2 & water forms
Bicarbonate & Hydrogen → Hydrogen will bind to Hb →
decreases hydrogen level
BICARBONATE-CARBONIC ACID BUFFER SYSTEM
● Most important buffer of plasma

● An acid-based homeostatic mechanism involving the
balance of carbonic acid, bicarbonate, and pH to
maintain pH to support your proper function
○ Catalyzed by carbonic anhydrase
● If acidic:
○ Carbonic acid could dissociate into bicarbonate
and hydrogen ion, which will compensate the
acidity
○ Carbonic acid, by the action of carbonic
anhydrase, it becomes water and CO2, which
can be eliminated thru exhalation
● H2CO3 dissociates into CO2 and water (CO2 eliminated
by the lungs thru expiration)
● Changes in CO2 modify the ventilation (respiration) rate
○ Ex. increased concentration of CO2 in the body,
our body changes its respiration rate
(hyperventilation and increased termination of
CO2)
● HCO3- concentration can be altered by the kidneys
○ Bicarbonate could be reabsorbed or excreted
by the kidney
● Second line
● Respiratory mechanism thru lungs or kidneys
RESPIRATORY MECHANISM IN THE REGULATION OF

ACID-BASE BALANCE
● Respiration
● Exchange of Gases in the Lungs and Peripheral Tissues
○ External respiration = exchange of gases in
the lungs between alveolar air and the blood.
○ Internal respiration = occurs at cellular level
○ If there is a change sin the blood pH, our lungs
will try to compensate by means of
hyperventilation or hypoventilation
■ Acidic blood pH = Hyperventilation/
increased respiration
● Respiratory Response to Acid-Base Perturbations
● Exchange of Gases in the Lungs and Peripheral Tissues
4
RENAL MECHANISMS IN THE REGULATION OF ● What is common in these scenarios?
ACID-BASE BALANCE ○ Bcoz of hypoventilation (retention of CO2)
● Na+-H+ Exchange ● COMPENSATION:
● Renal Production of Ammonia and Excretion of ○ Kidney retain HCO3 because of increased
Ammonium Ions pCO₂48
● Excretion of Hydrogen as Dihydrogen Phosphate ○ Reabsorb bicarbonate
● Reclamation of Filtered Bicarbonate
● Na+-H+ Exchange Respiratory Alkalosis
● Due to excessive CO2 loss (because of rapid breathing)
○ Results to Hyperventilation
● Anxiety, severe pain, aspirin over dosage, hepatic
cirrhosis and gram negative sepsis
● COMPENSATION:
○ Decreased reabsorption of HCO₃
Metabolic Acidosis
● Renal Tubular Acidosis: Impaired Hydrogen secretion
○ Hydrogen is being maintained in the body =
acidotic
● Diarrhea: Increased HCO3 excretion
● Vomiting: Increased HCO3 excretion
● Diabetic Ketoacidosis
● Renal Production of Ammonia and Excretion of ● Tissue hypoxia/ increased anaerobic respiration
Ammonium Ion ● COMPENSATION:
○ Hyperventilation
Metabolic Alkalosis
● Too much chloride is lost without replacement
● Sweating, Vomiting, Nasogastric suction
● Ingestion of alkaline drugs
● Too much amount of lactate, acetate and bicarbonate is
intravenously infused
● Aldosteronism: increased hydrogen excretion
● COMPENSATION:
○ Hypoventilation (lungs)
Acid-base Imbalances
● Respiratory Acidosis: excess CO2, caused by
hypoventilation
● Respiratory Alkalosis: deficit CO2, caused by
● Excretion of Hydrogen as Dihydrogen Phosphate
hyperventilation
● Metabolic Acidosis: deficit HCO3, common in cases of
kidney disease and diabetes
● Metabolic Alkalosis: excess HCO3, caused by diarrhea,
steroid or diuretic therapy.52
Acid-base Imbalances
pH pCO2 HCO3-
Respiratory
↓ ↑ Normal or ↑
Acidosis
Respiratory
↑ ↓ Normal or ↓
Alkalosis
● Reclamation of Filtered Bicarbonate
Metabolic Acidosis ↓ Normal or ↓ ↓
MetabolicAlkalosis ↑ Normal or ↑ ↑
Respiratory Acidosis
● Due to excessive carbon dioxide accumulation
● COPD, myasthenia gravis, CNS disease, drug overdose
(barbiturates, morphine and opiates) and pneumonia
5
6
CLINICAL CHEMISTRY LAB
LECTURE 1: GLASSWARES, PLASTICWARES

AND MEASURING VESSELS
Prof. Bea Angelli Laude, RMT
August 16, 2021
Clinical chemistry is the area wherein we are going to measure the GLASSWARE
different analytes that are present in the blood sample of the patient.
They are usually metabolites of the body which are quantified to Advantages
identify the disorders the patient is facing.
(Some) heating
LABORATORY GLASSWARE & PLASTICWARE Longer storage of some chemicals
● Equipments in the laboratory can either be made of glass
or plastic Regardless of the design, glasswares in the laboratory
○ If they are made up of glass, it is actually a must fall into 2 types:
specific type of glass that’s why they are ● Class A glasswares
correctly termed as glasswares ● Class B glasswares
○ Nowadays, the use of plastic equipments are These classes will be identified or labelled in glasswares
rampant, however plastics tend to have if they are able to satisfy certain tolerances of accuracy
limitations = they cant support heat
■ That's why we still continue to use Class A Glasswares
glasswares
Main Functions of Glasswares and Plasticwares
Why use glasswares/ plastic wares in the lab?
● Storage
○ In the laboratory, we tend to store both the
samples as well as the reagents
○ That’s why it's necessary that we make use of ● Found in professional laboratories
glasswares and plasticwares, that has a
specific quality such as: Class A Glasswares
■ Ability to withstand extreme ● Class B tends to have twice the tolerance limits of Class
temperature; A
■ And the corrosive effects of strong ● Class B are much more durable
acids and strong alkaline solutions ○ Often found in student lab
● Measurement
● Containment Cleaning of plastic/ glassware
● Those in direct contact with biohazard material is usually
PLASTICWARE disposable
- Since the early times, majority of the laboratory ○ Biohazard material such as samples, be in the
equipments are actually made up of glass form of stool, urine, or blood must be disposed
- Since the discovery of plastics, it slowly replaced the after use
glasswares that we often use in the lab ■ Regardless if it's made of plastic or
- However, nowadays we still tend to continue using glass
glasswares mainly because it is able to fill in the ○ Advantage of plastic = since it’s very cheap,
limitations of plastics they are often in disposable form
Advantages Disadvantages ○ Unlike glass, they tend to be much more
expensive = reusable form
Evaporation through breathing
● If not disposable, follow proper decontamination protocol
of plastic
○ Immediate rinsing + washing with powder/
Cheaper - Plasticwares are not often
liquid detergent
utilize as storage vessels
■ Wash intensely to remove the
- They tend to be porous
remaining detergent (which can be a
More Durable contaminant) if you are going to reuse
- Much efficient in resisting that particular lab equipment
the corrosive effects of ○ Pre-soaking in soapy water
strong acids and strong ■ May contain bleach (usually small
alkali solutions amount) mainly for contamination
Evaporation of dyes, stains, and
Preferred for some analyses purposes
proteins
- Example:Testing for heavy ○ (read furthermore Bishop Chapter 1)
- They will evaporate in
metals (NO glass) ○ For reusable lab equipments, it must also be
plasticwares
- Some heavy metals that we rinsed properly
- Essential to be stored in
need to analyze could also ■ Perform multiple rinsing to ensure that
glasswares
be present in glasswares, there are no remnants of detergent
and they could be considered left = Remnants may act as
as a contaminant contaminants
- These heavy metals are not ○ One way of determining proper rinsing of lab
present in your plasticwares equipment, you have to check the water used in
the last rinse
1
○ Check the pH twice before rinsing and after ● Characteristics:
rinsing ○ Flexible or rigid
○ If after rinsing and pH is higher, we can ○ Chemical-resistant
determine that the detergents were removed ○ Can be autoclaved
(Detergents make the solution more alkaline) ● Uses:
■ Higher pH of water after rinsing ○ For cryogenic procedures
compared to pH before it was rinsed, ○ Specially formulated to withstand temp down to
means that the detergents are -190°C
removed ● Several tube designs
○ specimen tubes and test tubes
PLASTICWARE ○ Microwavable containers and folders
Major types of resins used in Clin. Chem Lab: C. Polyethylene

(Details on Henry, chapter 4, p. 60) ● Disadvantage:
A. Polystyrene ○ NOT suitable when using picric acid, stains,
B. Polyethylene dyes and proteins
C. Polypropylene ● Uses:
D. Teflon ○ test tubes
E. Polycarbonate ○ Bottles
○ graduated tubes
The type of plastic you will use will depend on the procedure you ○ Stoppers
are going to perform ○ Plastic balls (toys)
○ Plastic wraps
● Indicated with arrows = types of resins that are incontact ○ Insulators
with our food:
○ PET or PETE (polyethylene terephthalate) D. Polycarbonate
○ HDPE (high density polyethylene) ● Characteristics:
○ LDPE (Low density polyethylene) ○ Very strong plastic but NOT chemically
○ PP (Polypropylene) resistant
● Others cannot be used for food because once ingested, ○ Autoclavable but with limitations
they may produce harm ● Usable temp. range: -100°C to +160°C
● Uses:
A. Polystyrene ○ tubes for centrifugation
● Characteristics: ○ graduated cylinders
○ Rigid ○ Flasks
○ Clear type ○ Roofs of waiting sheds
○ NOT to be autoclaved
E. Teflon
● Characteristics:
○ Almost chemically inert - unable to react to
chemicals
○ Chemical-resistant
● Suitable work temp. @: -270 ⁰C to 255 ⁰C
● Uses: stirring rods, tubing, cryogenic vials, bottle cap
liners
● Used for storage or mixing equipments
● Pans for cooking and Teflon tapes utilized in water lines
at home
GENERAL CATEGORIES OF GLASS

1. Borosilicate (Kimax/ Pyrex)
2. High silica Glass
3. Aluminosilicate (Corex)
4. Acid & Alkali resistant (Vycor)
5. Low actinic (amber-colored)
6. Flint glass (soda lime)
7. Disposable glassware
1. Borosilicate Glass (Kimax/ Pyrex)

■ Can be reused with other means of ● Most common type of glass encountered in volume
disinfection but NOT AUTOCLAVING measurements
● Disadvantage: ● Composition:
○ Not resistant to most hydrocarbons, ketones ○ 80% silica
and ROH ○ 13% boric oxide - contains BORON
■ Thus not utilized as storage ○ 4% sodium oxide
● Uses: capped graduated tubes and test tubes ○ 2–3% aluminium oxide
● Can be used as a container - short time ● Characteristics:
● Ex. Styrofoam ○ High degree of thermal resistance and low
coefficient of thermal expansion
B. Polypropylene ■ Object has the ability to change its
● Primary constituent of pipet tips shape at a given temperature
○ Pipets are utilized to transfer and measure ■ Change is minimal
samples as well as reagents ○ Low alkali content
○ Reagents may be strong acids because the ■ Resistant to alkali corrosion
pipet tips can withstand extreme pH ○ Free of heavy metals
2
■ Free from Mg-lime-zinc group of 4. Acid- Resistant & Alkali-Resistant Glass
elements, heavy metals, arsenic and ● Boron-free
antimony ● A.k.a “soft glass”
● Uses: ● Vycor® (No. 7900)
○ Heating ○ Made up of fused silica
■ Open flame or electric heating ○ 2 in 1 characteristic
elements such as hotplates ■ Heat-resistant (not as efficient as
● Precautions: borosilicate glass)
○ Storing concentrated alkali solutions will ■ Chemically inert (a substance not
etch/destroy the calibration chemically reactive)
○ Heavy-walled type of glass should not be ○ Unique characteristics:
heated with direct flame or hotplate ■ Stable to all acids except hydrofluoric
■ Only the thin-walled type of types
borosilicate glassware will be heated - Hydrofluoric solutions corrode
in direct flame or hotplate glasses with silica
○ Avoid heating beyond its strained point ■ Superior in resisting corrosion by
■ Strain point- the temperature where alkali than borosilicate glass
the glass softens or will transform ■ Unlikely contamination by contact
from solid to liquid with solutions
■ Heating beyond strained point could ■ Relatively inert to acids and neutral
lead to breakage salts
○ In the case of volumetric glassware, heating ■ Chlorine and acid gases do not affect
can destroy the calibration it at any temperature
● Popular Brands ○ Withstand high temperature (1200°C)
○ Pyrex® ■ Softening temp.: 1500 °C
■ Strain point: 515 ○ Withstand downshocks from this temperature to
°Celsius (Henry) ice water
○ KIMAX® ○ Use:
■ Strain point: 513 ■ ashing and ignition techniques
°C
2. High Silica Glass

● Silica fused to quartz
● More expensive than borosilicate glass
● Use: 5. Low Actinic Glass
○ Spectrophotometer cuvettes ● Contain materials that usually impart amber to red color
to the glass
○ Reduces the amount of light transmitted to the
substance in the glassware disallowing
3. Alumina-Silicate Glass oxidation
● A.k.a Aluminosilicate glass ● Common uses:
● With aluminum oxide ○ For light-sensitive substances
● Strengthened chemically rather than thermally ■ Bilirubin
○ Can withstand very strong acids, bases, ■ Vit. A
hydrocarbons, and concentrated alcohols ○ Store control materials and reagents
○ Greater chemical durability and can withstand
higher operating temperatures 6. Soda-Lime Glass
○ Compared to borosilicate, aluminosilicates are ● A.k.a flint glass
more difficult to fabricate ● Composition:
● Has a wide thermal range but not as wide ○ Silicon oxide
as borosilicate glass ■ Soda = sodiuum oxide
● When coated with an electrically ■ Lime = calcium oxide
conductive film, aluminosilicate glass is ● Most inexpensive glass
used as resistor for electronic ○ Most common type of glass found outside the
circuitry laboratory
● High impact, extremely strong glass ● Readily made into variety of types of glassware
○ Common use: manufacture of ● Has a high expansion coefficient and a high degree of
calibrated centrifuge tubes thermal resistance
● Corex® (Corning, N.Y.) ○ At given temperature, it will exhibit huge
○ Characteristics: structural changee
■ Radiation-resistant ● Mineral can be leached from the glass into the stored
■ 6X stronger than borosilicate (outlast solutions
conventional ● Common uses:
glassware by 10-fold) ○ Volumetric flasks
■ Resist clouding and ○ Stirring rods
scratching better ○ Single-use pipette or test tubes
○ Uses:
■ High-precision MEASURING VESSELS
analytical work a. Graduated cylinder
■ Optical reflectors and b. Burets
mirrors c. Volumetric flask
■ Not used as general d. Pipet
type of glassware in
the lab
3
A. GRADUATED CYLINDER A. Design = TC vs. TD (to contain or to deliver)
● Long, cylindrical tubes usually held upright by B. Drainage characteristics = Blow-out vs.
an octagonal or circular base with gradations Self-draining
along its length C. Type = Measuring vs. transfer
● Semi-accurate
● Extremely convenient for rapid measurement of
TABLE 1-4 PIPET CLASSIFICATION
liquid
● Should NEVER be heated especially if plastic
since it will destroy the calibration marks along
its body I.Design
A. To contain (TC)
B. BURETS B. To deliver (TD)
● Long cylindrical graduated laboratory glassware with
stopcock II. Drainage characteristics
● Type of buret used depends on the chemical A. Blowout
used B. Self-draining
○ Glass >> ACID
○ Rubber for >> ALKALI III. Туре
● Extremely accurate in dispensing aliquots of a A. Measuring or graduated
solution 1. Serologic
○ Mainly used to dispense known 2. Mohr
amounts of liquid reagents in 3. Bacteriologic
experiments where such precisions 4. Ball, Kolmer, or Kahn
are necessary 5. Micropipet
● Generally used for titration purposes only B.Transfer
1. Volumetric
C. VOLUMETRIC FLASK 2. Ostwald-Folin
● Round lower portion and a long, thin neck 3. Pasteur pipets
with an etched neck or calibration line 4. Automatic macropipets or micropipette
which is going to be the mark in
measuring a specific volume
A. Design
● Generally used for:
○ Preparation of standard solution 1.) To Contain (TC
○ Measuring liquid colume ● A.k.a. “Rinsed-out pipets”
accurately ● Able to hold a particular volume but is unable to dispense
the volume indicated.
● For u to dispense the exact volume, Must be refilled and
rinsed-out with the appropriate solvent after the initial
D. PIPETS liquid has been drained from the pipet
● Glass or plastic material in the lab used to transfer liquids ● Examples:
● Can either be reusable or disposable ○ Sahli-hemoglobin pipets
● Majority of pipette can hold only up to 20 mL of a specific ○ Long-Levy pipets
solution 2) To Deliver (TD
● Used to transfer measured volumes of liquid between ● Able to transfer the exact volume indicated in the pipette
containers ● Designed to drain by gravity
● Must be held vertically with the tip placed against the
side of the container and must NOT TOUCH the liquid in
it
● Vessel ang nakatilt
● Examples:
○ Mohr pipet
○ Serologic pipet
○ volumetric transfer pipet
NOTE:
● A TC pipet holds or contains a particular volume but does
not dispense that exact volume, whereas a TD pipet will
dispense the volume indicated.
B. Drainage Characteristics
1) Self-draining pipet
○ Example (pic above): you have 5 in 1/10 which

means that the capacity of the pipet in
measuring a particular volume is only up to 5
mL with 10 increments to the next demarcation
line
● Numbers are indicated at the top or lower portion of the
pipet which indicates the volume or total capacity for
measuring and the major divisions
● In order to properly measure a specific volume of a
particular solution, you have to look for the meniscus of
the aspirated fluid where it should be aligned to the
graduation line of your pipet ● with a single painted at the top
● Pipets are classified based on: (Bishop 7th ● Allow to drain by gravity
● edition, table 1-4 pg.13) ● No frost/ etched / double lines
4
2) Blown-out pipet Volumetric or Transfer
● Used to measure and transfer a predetermined volume of
liquid
● Kaya niya ma dispense or ma measure na volume of a
particular solution is only one volume
● If it is indicated that it is able to measure 10 mL, therefore
10 mL lang jud ang pede niya i measure kay isa lang
iyang etched line
● Dispense one volume without further subdivisions
○ Ostwald-Folin Pipet
○ Pasteur Pipet
● With double rings/ frost/ etched

● designed to be "blown out" by pushing a small amount of
air out of the pipet, completely emptying it
● For u to deliver the exact amount of the volume specified
by the pipette, it has to be blown out with the use of a
bulb.
Left: volumetric (transfer)

Right: oswald-folin
TOP portions of self-draining & blown-out pipet

Single colored line = self-draining
● Allow it to drain by gravity
Double ring or frosted/ etched ring= blown out pipet
● To deliver the exact amount of solution u have measured,
you have to introduce air to empty the pipet.
Pic above: illustrates the proper positioning of pipettes in aspirating

TIP portions of self-draining & blown-out pipet and dispensing solution.
Left: self-draining ● Top pic: held vertically upright with the tip slightly
- Graduation is much farther from the tip. submerged in the surface of your solution without
Right: Blown out pipet touching the side of the vessel.
- Graduation is up to the tip of the pipet. ● Bottom pic: dispensing solution
○ Tip is not supposed to touch the surface of the
C. Type solution contained in your vessel but rather the
1. Transfer pipet (or volumetric) tip is going to touch the side of the vessel for a
a. Volumetric smooth flow of the draining of your solutions.
b. Ostwald-Folin ● In both aspirating and in draining, the pipette must be in
c. Pasteur a vertical position.
d. Automatic macro- & micropipet ● Draining
2. Measuring or Graduated pipet ○ In order to maintain the vertical position of your
a. Serologic pipet and to allow the tip to touch the sides of
b. Mohr the receiving vessel, so u have to tilt the vessel
c. Micropipet to maintain its vertical position.
5
Positive Displacement Pipette
● Act or move like a syringe

● Each tip contains a disposable plunger like a syringe
○ Plunger is disposable but not the tip
● The plunger directly displaces the fluid with no error
● These are the most accurate type of pipette
● They are also the most expensive
https://www.youtube.com/watch?v=_YTwxakpY1U
The volumetric pipets are always SELF-DRAINING

Ostwald-Folin pipets are BLOWOUT PIPETS
● Volumetric – to transfer aquaeous solutions
● Ostwald-Folin – to transfer viscous fluid
DIFFERENCE VOLUMETRIC OSWALD-FOLIN
Drainage self-draining Blowout pipette

characteristics
Top portion Have single ring double/frosted/

etched ring
Measuring or Graduated
● Calibrated to distribute fractional quantity of liquid and
principally used for measurement of reagents
● The volume that it can measure is not fixed but rather 1. Air displacement method (left)
measure different volumes of a - It is going to rely on a piston for creating a
particular solution suction to draw the sample into a disposable tip
● Examples: that must be changed after each use
2.a. Mohr pipet - The piston does not come in contact with the
2.b. Serologic pipet liquid but there will be unprotected air space so
there could be a possible aerosol contamination
Mohr pipet of the sample or the reagents that is going to be
● No graduations to the tip aspirated
● Self-draining pipet - Can ensure that there will be no carry over
● Single ring since u are going to replace the tip
- However, it going to be less accurate compared
Serologic pipet to positive displacement pipet
● Has graduation marks to the tip 2. Positive displacement method (right)
● Generally a blowout pipet - Going to operate by moving a piston in the
● Designated by a frosted or double pipet tip or barrel
colored ring at the top - The piston is in the form of a plunger in which it
is going to be changed once u are going to
dispense one liquid after another
Micropipet - Advantage of piston:
● With a total holding volume of less than 1 mL - There will be protected air space
● It may be designed as either a Mohr or serologic pipet - No aerosol
● Comes in 2 forms: - Problem: Since you are not going to change the
○ Automatic and Semi-automatic pipet tip, there will be a carry over of samples or
■ Commonly used in the laboratory regents to another type of solution
2 Types of micropipet:
1. Air displacement
● Disposable, polypropylene tip
2. Positive displacement
● Use of capillary tip (siliconized, glass, plastic)
6
ANATOMY OF AIR DISPLACEMENT PIPET POSITIVE DISPLACEMENT PIPET
● No first stop or second stop

● Pull the plunger in order to aspirate your fluids
● In draining, simply push down your plunger and drain all
the contents
https://www.youtube.com/watch?v=NgosWmRjjAo
Semi-automatic & Automatic pipettes and

dispensers (Tietz)
● Push button
○ pushed in order to aspirate or dispense a
particular volume of sample or reagent
○ Not going push it but turn it bcs it is going to
● Semi-automatic pipettes
serve as a large volume adjustment knob
○ Although it allows us to have an easier
■ In order to adjust the volume to be
measurement of volumes of samples and
aspirated in much more larger
reagents, but the aspiration and dispensing of
amounts
such liquids can still be done manually
● Tip ejector button
● Automatic pipettes
○ Simply push this down to remove the pipette
○ It is attached to a machine and a tube is going
tips that is found at the bottom portion
to be submerged to a particular solution
● Thumbwheel (Fine volume adjustment ring)
○ The machine will be regulating the aspiration as
○ Used to adjust the volume to be aspirated in
well as the dispensing of a particular volume of
smaller amounts
a reagent ot sample
● Volumeter display
● Dispensers (Dilutors)
○ Where u are able to determine how much of the
○ Automatic pipettes that obtain the liquid from a
solution or what is the exact volume to be
common reservoir and dispense it repeatedly
aspirated, transferred or measured
○ May be bottle top, motorized, handheld, or
● Tip ejector
attached to a dilutor
○ Connected to the shaft
○ Simply push to move and lead to the removal of
your disposable tip
AIR DISPLACEMENT PIPET
● If you are going to push the push button, it has 2 stops:

○ First stop
○ Second stop: mainly for the draining of the
entire solution that was aspirated in the pipet tip
Prior to the submersion of your tip to the solution, it must be

pushed to the first stop
↓
Once it is submerged in the solution, you are going to release it.
By then you are able to aspirate a specific amount of the solution
↓
In transferring, push down the push button from the first stop all
the way to the second stop (mainly for the draining)
↓
Release
7
UNIT
% g/100mL
Valence eq/mole
MW g/mole
M Moles/L
N eq/L
UNIT FORMULAS

𝑥100

𝑥100
𝑣𝑜𝑙𝑢𝑚𝑒 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒

𝑥100
● %
PERCENT ● % w/v
CONCENTRATION ● % w/w 𝑥 (𝑔)
● %v/v 𝑔 = 100 𝑚𝐿
𝑥 (𝑚𝐿)
𝑚𝐿 = 100
𝑥 (𝑔)
𝑔 = 100𝑔
● M
MOLARITY ● mol/L 𝑀𝑜𝑙𝑎𝑟𝑖𝑡𝑦 = 𝑀𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝐿𝑖𝑡𝑒𝑟 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
● moles/Liter
● moles/kg
● mol/kg 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒
MOLALITY
● molal 𝑚𝑜𝑙𝑎𝑙𝑖𝑡𝑦 = 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝑘𝑖𝑙𝑜𝑔𝑟𝑎𝑚 𝑜𝑓 𝑠𝑜𝑙𝑣𝑒𝑛𝑡
● m

𝑁𝑜𝑟𝑚𝑎𝑙𝑖𝑡𝑦 = 𝐸𝑊 𝑥 𝐿 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
𝑀𝑊
𝐸𝑊 = 𝑉𝑎𝑙𝑒𝑛𝑐𝑒
NORMALITY ● N
or

𝑁 = 𝑀𝑊
( 𝑟𝑒𝑝𝑙𝑎𝑐𝑒𝑎𝑏𝑙𝑒 𝐻/𝑂𝐻 )(𝐿 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛)
M→N (𝑀) (𝑉𝑎𝑙𝑒𝑛𝑐𝑒)
N→M 𝑁/𝑉𝑎𝑙𝑒𝑛𝑐𝑒
LECTURE 3: ELECTROPHORESIS AND

ELECTROCHEMISTRY
Prof. Lyslie Jane Vasquez-Baylon, RMT
August 30, 2021
TERMINOLOGIES ○ Cerebrospinal fluid (CSF)

● Ampholyte ○ Erythrocytes and tissue and,
○ A molecule that contains both acidic and basic ○ other biologic body fluids
groups ○ Nucleic acid (DNA, RNA)
○ Helps establish a stable pH gradient and assist ● Using electrophoresis, we can separate particles, isolate
molecules such as proteins in migration them and observe the rate how these particles move or
● Electrophoretic Mobility travel through the electric field
○ The rate of migration of a charged solute in an ● Take note:
electric field, expressed per unit field strength ○ Electrophoresis is usually used for protein
○ Solute’s response to the applied electrical field determination
○ What is the response? ○ The proteins are separated on the basis of their
■ The solutes move towards a charged electric charge density
electrode
● Endosmosis
○ Preferential movement of water in one direction
through electrophoresis medium due to
selective binding of one type of charge on the
surface of the medium.
○ Movement of liquid such as water and buffer
ions
● Electrophoretogram
○ The migration of charged macromolecules.
○ Result of zoned electrophoresis and consists of
sharply separated zones of macromolecules
● Iontophoresis
○ The migration of small charge ions.
● Zone Electrophoresis Theory of Electrophoresis
○ The migration of charged macromolecules in a ● Electrodes:
porous supporting medium such as paper, ○ Cathode = negatively charged
cellulose acetate, and agarose gel film ○ Anode = positively charged
○ Most prevalent electrophoretic technique used ● Charged particles and electrophoresis migrate toward the
today oppositely charged electrode
○ It has different types like paper electrophoresis, ● Thus:
cellulose acetate electrophoresis, capillary ○ Cations (+) → Cathode (-)
electrophoresis and gel electrophoresis ○ Anions (-) → Anode (+)
ELECTROPHORESIS
● In electrophoresis, particles or solutes are charged and

are moving in an electrical field towards an electrode
● One electrode is positively charged and the other one is
negatively charged
● Principle:
○ The movement of electrically charged
compounds (proteins) in a medium resulting to
their separations based on their electrical
charges when an electric current is applied
○ Compounds or particles are separated and they
move through the medium ● Remember: Proteins are amphoteric and the during
● Macromolecule of interest: electrophoresis, these proteins are negatively charged
○ Proteins in serum anions and move towards the anode
○ Urine
1
● Using electrophoresis particles like proteins are
separated, detected and quantified.
Components of Electrophoresis
1. Driving force (electrical power)
● Supplies the constant current
2. Support medium
● Holds sample in place for migration
3. Buffer
● Carries applied electrical current and set the pH
of electrophoresis
4. Sample
● Body fluids containing our molecules of interest
5. Detecting system
● Biomolecules ● Visualizes our macromolecules of interest
○ Protein under the UV light, also uses stains
■ Contains an amino group, side chains ● Densitometer for quantitation
and, carboxylic group
■ The acidic and basic amino acids
determine the net charge of the
proteins
○ Lipoproteins
○ Nucleic Acid (DNA & RNA)
● These are the common molecules of interest separated
and isolated in electrophoresis
● Electrophoresis is a technique used in laboratories in
order to separate macromolecules based on size
Factors Affecting Rate of Migration

● Net electric charge of the molecule
● Size and shape of the molecule Driving force (Electrical Power)
● Strength of electrical field ● In electrophoresis, heat is produced when current flows
● Properties of Supporting Medium through a medium that has resistance, resulting in an
● Temperature of operation increase in thermal agitation of the dissolved solute
(ions) and leading to a decrease in resistance and an
increase in current
● Separation of molecules using electrophoresis often
require high voltages of about 50 to 200VDC
○ Power supply should supply a constant voltage
○ A constant rate is applied because it helps keep
● This equation shows how the rate of mobility of the the migration rate constant
molecule presented by mu is affected by the net charge
of the particle as well as the constant Supporting Medium
● The ionic radius of the particle and the viscosity of the
buffer ● Provides the matrix where charged molecules are added
● Based from this equation, the rate of migration is directly and separated
proportional to the net charge of the particle but inversely ● Common supporting media utilized in electrophoresis
proportional to its molecular size and viscosity
● Note: Net Charge of the compound depends on the ○ Cellulose acetate
solution of the ph ■ cellulose is acetylated to form
○ The greater the net charge of the molecule, the cellulose acetate by treating it with
faster it moves through the solution and acetic anhydride
towards the oppositely charged electrode ■ A dry, brittle film composed of about
80% air space (these air spaces are
Conventional Electrophoresis filled with electrolyte when cellulose
acetate is soaked in the buffer during
electrophoresis, then it becomes
pliable (flexible) and easily bent)
■ Homogenous medium with uniform
pore size and does not absorb the
protein
■ When electrophoresis is done, it can
be stained and made transparent for
quantitation
■ Can be stored for long periods
■ Advantages - provides good
background for staining glycoproteins,
gives sharp bonds and offer good
resolution
○ Agarose gel
■ Widely used supporting medium for
electrophoresis
● Power source ■ Highly purified uncharged
● Gel - supporting medium polysaccharide derived from agar
● Sample wells where we place our sample ■ Advantages: requires small amounts
● Sub cell - contain the buffer or electrolyte solution of sample (approx. 2ml), neutral --
2
does not produce electroendosmosis ■ Ionic strength
■ Once electroendosmosis happens, ● If buffer is more than the pI, it becomes positively
there will be movement of solvent charged and migrates toward the cathode]
such as water in the electric field ○ Binds more hydrogen ion
which affects the movement of ● If buffer is more basic than the pI, it becomes negatively
sampled macromolecules which may charged and migrates toward the anode
cause molecules to slow down, not ○ Loses hydrogen ions
move, or pushed farther (paspas ang ● Take note of the term isoelectric point = pI
mobility), ● Isoelectric point is the pH at which a particular molecule
■ Agarose gel doesn’t bind protein thus carries no net electric charge
migration is unaffected ● Take note that buffer solution should have carefully
controlled ionic strength
○ Polyacrylamide gel ● Ionic strength is the measure of the concentration of ions
■ Referred to as page in the solution
■ Layers of gel with different pore sizes ● Remember that during electrophoresis, ions cluster
are used around the migrating particle
■ Separates protein based on its charge ○ The higher the ionic strength/concentration, the
and molecular size, hence it uses bigger the size of the ionic cloud, making its
layers of prepared gel with different mobility slower
pore sizes ○ The lower the ionic strength/concentration, the
■ Unique about it is its ability to more the current is carried by the proteins
separate serum proteins into 20 or which causes faster mobility
more fractions rather than the usual ■ The longer the distance, the sharper
five fractions separated by cellulose the protein band separation
acetate or agarose Examples of buffers used in electrophoresis
■ Very useful and widely used in
studying individual proteins such as Buffers Used for
isoenzymes Barbitone Buffer (around 8.0 ● Serum protein separation
pH) ● Poor resolution, weak buffer
○ Starch
■ Separates proteins on the basis of Phosphate Buffer (around 7.0 ● Enzyme separation
surface charge and molecular size pH) ● Low buffering capacity, high
just like the polyacrylamide gel conductivity
■ Not widely used because preparation Tris-borate-EDTA buffer (TBE) ● Nucleic acid separation
is difficult -(pH around 8.)) ● Good resolution, high
buffering capacity, low
Agarose Polyacrylamide Gel conductivity
● Smaller DNA fragments
● Polysaccharide ● Cross-linked polymer
extracted from of acrylamide Tris-acetate-EDTA buffer (TAE) ● Nucleic acid separation
seaweed ○ Cross-linker (pH around 8.0) ● High resolution, high
● Gel casted horizontally such as buffering capacity, low
● Non-toxic bisacrylamide conductivity
● Separate large ● Gel casted vertically ● Used in agarose gel
molecules ● Potent neuro-toxic electrophoresis
● Commonly used for ● Separate small Tris-glycine buffer- (pH more ● Protein separation
DNA separations molecules than 8.0) ● High buffering capacity, low
● Staining can be done ● Used for DNA or conductivity
before or pouring the protein separations
gel ● Staining can be done
after pouring the gel Stains
● Gel casting ● Since the ions have already migrated to specific
○ Once supporting medium is already prepared, electrodes and electrophoresis is already completed, the
they are held in a casting tray supporting medium is treated with a dye or stain to help
○ The gel casted in trays provides a place where identify the separated fractions
we put our samples to test ● Help visualize the bands and macromolecules such as
proteins that have already migrated
Components of Electrophoresis ● Allows us to visualize the locations of the separated
particles
Buffer ● There are also methods where the sample is already
● Provides ions and mixed with a tracking dye
○ Allows current to be carried through the sample ● While the molecules migrate, we can visualize the
○ The buffer resists pH changes in the overall fractions or the separated molecules such as nucleic
solutions acids
○ Buffer maintains the pH at a relatively constant ● In agarose gel electrophoresis, Ethidium bromide is used
value as a dye and binds to the DNA
● AMPHOLYTE is a molecule whose net charge can be
either positive or negative Stains Used for
○ Ampholytes contains both acidic and basic Amido Black
groups Coomassie Proteins
○ These amphoteric molecules can either be Brilliant blue
positively or negatively charged Bromphenol Blue
○ Help establish a stable pH gradient and assist
Ethidium bromide Enzyme separation
molecules in migration
Sybr green or Sybr Gold DNA
○ Two properties affecting ampholyte
■ pH Sudan Black Lipoproteins
3
Ponceau red sample allowing the particles such as DNA to separate
Amido black Hemoglobins
Coomassie
Brilliant blue
● The photo shows how we did electrophoresis particularly

● Agarose gel is placed in the chamber and in this process,
the agarose gel electrophoresis
the gels are arranged in a way that the wells are closest
● Mateiaals are prepared
to the negative cathode indicated by the black color
● Includes
● Since the DNA is negatively charged, it will move from
○ Casting tray- supporting medium is placed
the negatively charged cathode to the positively charged
○ Comb- used to create wells where the sample
anode indicated by the red color
is placed
● Buffer is added to the reservoir of each chamber until the
○ Power supply- provide the constant electric
wells of the chambered are covered of at least 2
current required
millimeters
○ Sub-cell- containing the supporting medium
● Agarose gel that we need to prepare first is prepared by
mixing buffer to the desired concentration then we heat it
in a microwave oven until it has completely melted
● Once the agarose gel solution is melted it is allowed to
cool at about 60 degrees celsius
● Poured into the casting tray containing the sample comb
● Solution is allowed to solidify at room temp
● Once agarose gel solidifies, the comb is carefully
removed making sure the wells are intact
● Other materials:
○ Pipette
○ Graduated cylinder ● Samples are arranged in an order according to the lanes
○ PPE that they are assigned to be running as indicated by the
lab protocol
● Samples may contain proteins, DNA, etc.
● Use the pipette to obtain the sample such as the
extracted DNA in this experiment
● Once gel is prepared, pour the agarose solution in the

casting tray and the comb is pushed down into the gel to
from wells ● Observe proper use of a pipette
● Allow to solidify and remove the comb carefully ● Samples are loaded properly on the well
● Once ready, the DNA fragments are loaded to each well ● Pipette tip must be perpendicular to the row of wells and
sing micropipette the other parts of the gel will not be punctured
● Gel plate is immersed in charged buffer solution ● The tip of the pipette is lowered below the surface of the
buffer just above or inside the well
● This is a subcell that contains an electrode wire across

the bottom of each end of the subcell. With this, the
electric current will pass through the medium to the ● The subcell containing the samples bust not be bumped
4
or moved drastically so that the samples will not mix through the light beam at fixed rate
● Lid is placed
● Black to black and red to red NORMAL SERUM PROTEIN ELECTROPHORESIS
● Connect to power supply

● Timer is set as required by the protocol
● Once set, press the start button
● Bubbles will appear form the wires
● Samples will migrate from the wells through the gels
● If the electrophoresis is done at a very long time, the ● Albumin
proteins or particles may migrate off or beyond the gel ○ Most negative and will migrate farthest
into the buffer ● α1-Globulins
○ Α1-fetoprotein
AGAROSE GEL ELECTROPHORESIS ○ Α-antitrypsin
○ HDL
DENSITOMETRY ● α2-Globulin
● It measures the absorbance of stain-concentration of the ○ Haptoglobin
dye and protein fraction. ○ Ceruloplasmin
● It scans and quantitate electrophoretic pattern ○ Α2-macroglobulin
● β-Globulins
○ Transferrin
○ C-reactive protein
● γ-Globulins
○ immunoglobulins
Refractometry
● Can be used to measure protein concentrations, specific
gravity of urine, and column influence of high
performance liquid chromatography
● Determines the concentration of dissolved particles in a
Basic Components
specimen by measuring the refractive index
1. light source
2. Monochromator
3. movable carriage - will scan the medium over the entire
area
4. Photodetector - optical system
5. read-out device
● based on refractivity (the ability of the substance to light)

● when the light passes from one medium into another, the
path of the light beam changes direction at the boundary
surface if its speed in the second medium is different
from that in the first
● Refractive index
○ the ratio of the two speed of light
○ Comparison of the velocity of light in air and the
velocity of light in the solutions
● In densitometry, the supporting medium is moved
5
● Coulometry
○ is an electrochemical titration in which the
titrant is electrochemically generated and the
endpoint is detected by amperometry
● Amperometry
ELECTROCHEMISTRY
● involves the measurement of electrical signals
associated with chemical system that are incorporated
into an electrochemical cell
● the magnitude of a voltage or current signal originating
from an electrochemical cell is related to the activity or
concentration of a particular chemical species in the cell
ELECTROCHEMICAL CELL ● The change in voltage, indicates activity of each analyte

● Used to measure analyte concentration
● the measured potential is related to the molar
concentration by the Nernst equation
○ Describes the electromotive force generated
because of hydrogen ion at the glass tip
● How do we measure electrical potention?
○ ELECTRODES
a. Reference Electrode
■ Also known as reference
potential
■ Has a constant and known
voltage
■ Produces constant potential
● A galvanic cell converts chemical energy into electrical b. Indicator Electrode
energy ■ responds to changes in the
● Here the redox reaction is spontaneous and is activity of solution
responsible for the production of electrical energy ■ measuring electrode
● The two half-cells are set up in different containers, being ➢ The potential difference between
connected through the salt bridge or porous partition these two electrodes are measured
● Here the anode is negative and cathode is the positive ➢ Related to the molecular
electrode. The reaction at the anode is oxidation and concentration of ions in a solution
that at the cathode is reduction ● REFERENCE ELECTRODE
● The electrons are supplied by the species getting ➢ An ideal RE should conform to the Nernst equation,
oxidized. They move from anode to the cathode in the exhibit a potential that is constant with time, and
external circuit return to original potential after being subjected to
● Liquid junction - also known as a salt bridge are small currents
required to complete the circuit between the reference ➢ Insensitive to the changes in the solution
and without contaminating anything ➢ Exhibits little hysteresis such as lags with
○ Functions temperature cycling
■ It allows electrical contact between ➢ Standard Hydrogen Electrode (SHE) has those four
the two solutions characteristics, but not for practical means
■ It prevents the mixing of the electrode ○ Calomel Electrode
solutions ■ Reference electrode
■ It maintains the electrical neutrality in ■ mercury/mercurous chloride
each half cell as ions flow into and out ■ Composed of mercury in contact with
of the salt bridge a solution saturated with mercurous
○ Purpose: not to move electrons from the chloride plus potassium chloride
electrolyte, but rather to maintain charge ○ Silver/silver chloride
balance because the electrons are moving from ■ Reference electrode
one half cell to another ■ overall better and faster
■ Consists of silver electrode immersed
Electrochemical Techniques in a solution of potassium chloride
● that has been saturated with silver
○ the measurement of the electrical current at a chloride
single applied potential ○ Normal hydrogen electrode
● Voltammetry
○ based on the current potential relationship in an Ion-selective Electrode
electrochemical cell when the potential is ● indicator electrode that can respond to individual types of
applied anions or cations
● very sensitive and selective for the ion it measures
Potentiometry ● Composed of a membrane or barrier between the
● measurement of a potential or voltage difference reference solution and reference electrode
between two electrodes immersed in solution under the ● 2 types of ISE:
condition of essentially zero cuPotentiometry 1. Direct ISE
○ measurement of a cell potential (voltage) under ○ Does not have sample dilution
equilibrium conditions 2. Indirect ISE
○ has sample dilution
6
● Types of ISE
1. Glass electrodes
2. Liquid membrane electrodes
3. Precipitate impregnated membrane electrodes
4. Solid state electrodes
5. Gas electrodes
6. Enzyme electrodes
● Its ionic selectivity depends on the membrane/barrier
composition used:
a. glass aluminum silicate (Sodium)
b. valinomycin gel (Potassium)
c. organic liquid membrane ion exchangers
(Calcium & Lithium) Osmometry
d. gas and enzyme electrodes
● measurement of the osmolality of an aqueous solution
such as serum, plasma and urine; measurement of the
Uses:
concentration of dissolve solute particles in a solution
● pH electrode
● Osmolality of a solution is related to its colligative
○ selective for the detection of hydrogen ions
properties such as osmotic pressure, boiling point,
○ indicator electrode has a glass membrane
freezing point, and vapor pressure.
○ Indicator: Glass electrodes contain small bulbs
● based on measuring changes in the colligative properties
of glass which contains a chloride ion buffer
of solution that occur owing to variations in particle
solution
concentration that is related to the osmotic pressure of
○ internal reference electrode: Ag/AgCl
the solution
○ external reference electrode: Calomel electrode
● COLLIGATIVE PROPERTIES:
● pCO2 electrode
○ Depend on the concentration of solute
○ pH electrode contained within a plastic jacket.
molecules or ions.
○ sodium buffer and permeable membrane
○ Not affected by the mass, size, and type of
(Teflon or silicone)
molecule.
1. osmotic pressure
Voltammetry 2. boiling point
● The measurement of current after which a potential is 3. freezing point
applied to an electrochemical cell. 4. vapor pressure
● Anodic stripping for lead and iron Testing ● Osmotic particles
● Sensitive and uses minimal analyte. ○ Glucose
Three Electrodes: ○ Urea
A. WORKING ELECTRODE ○ Nitrogen
● makes contact with the analyte ○ Sodium
● facilitate the transfer of charge to and from the ■ If added into the sol’n osmolality
analyte increases and 4 colligative are
B. REFERENCE ELECTRODE affected.
● a half cell with a known reduction potential ● ↑Osmotic and boiling
C. AUXILIARY ELECTRODE ● ↓Freezing point and vapor
● to sustain electrolysis pressure
● Process by which electric current is passed
through a substance to effect a chemical Osmotic Pressure
change (substance loses or gains an electron)
Coulometry
● Measure of amount of electricity in coulombs .
● Involves the application of a constant current generate a
titrating agent
● is an electrochemical titration in which the titrant is
electrochemically generated and the endpoint is detected
by amperometry
● the time required to titrate a sample at a constant current
is measured and is related to the amount of analyte in a
sample by Faraday’s equation.
○ Faraday’s equation. - chloride testing in CSF,
serum, and sweat.
● Minimum pressure which needs to be applied to a
Amperometry
solution to prevent inward flow of pure solvent across a
● is the measurement of the current flow produced by an semi permeable membrane
oxidation–reduction reaction at a single applied potential
Vapor Pressure
● a measure of the cell current when the potential
difference between indicator and reference electrodes is
controlled ● Measure of the tendency material to change into a
● We use it to determine p02, glucose, Cl, and peroxidase. gaseous or vapor state.
○ pO2 gas-sensing electrode Electrical Impedance
○ GLucose electrode ● measurement is based on the change in electrical
resistance across an aperture when a particle in
conductive liquid passes through this aperture.
● Measure resistance produced for each particle.
● Used primarily in the hematology laboratory to count
leukocytes, erythrocytes and platelets.
7
BECKMAN-COULTER CHEMISTRY ANALYZER
● Counts size particles by measuring impedance or

resistance.
ABOT CELL DYN
● Size of pulses is directly proportional to cell size

● Number of pulses is directly proportional to cell count.
● 2-21 fL= platelets
● >30 fL = RB
● > 35 fL = leukocytes.
COULTER MACHINES
● Measure electrical impedance or resistance

● Uses coulter principle
○ Detects and measures electrical impedance
produced by a blood cell as it passes thru an
aperture
8
LECTURE 4: AUTOMATION
August 6, 2021
PART ONE
TERMINOLOGIES AUTOMATION
● Describe the process whereby an analytical instrument
Automation performs many tests with only minimal involvement of an
● The process whereby an analytical instrument analyst
performs many tests with only minimal involvement of ● Enable laboratories to process much larger workloads
an analyst; also defined as the controlled operation of without comparable increases in staff
an apparatus, process, or system by mechanical or ○ Somehow reduces the staff and lesser labor for
electronic devices without human intervention. the staff
Batch analysis ● Used for:
● Type of analysis in which many specimens are ○ Test performance
grouped in the same analytical session. ○ Processing and transport of specimens
Carry-over ○ Loading of specimens into automated analyzers
● The transport of a quantity of analyte or reagent from ○ Assessing the results of the tests performed
one specimen reaction into and contaminating a HISTORY
subsequent one. ● First automated analyzer
Continuous-flow analysis ○ “Autoanalyzer” by Technicon in 1957
● Type of analysis in which each specimen in a batch ○ continuous-flow, single-channel, sequential
passes through the same continuous stream at the batch analyzer
same rate and is subjected to the same analytical ○ Single test result on approximately 40 samples
reactions. per hour
Discrete analysis ● First commercial centrifugal analyzer: a spin-off
● Type of analysis in which the sample is aspirated into technology from NASA outer space research (1970)
the sample probe and then is delivered, often with ○ Was developed by Dr. Norman G. Anderson
reagent,through the same orifice into a reaction cup or ○ A prototype was created in the Oak Ridge
another container. National Laboratory
Multiple-channel analysis ● Automatic Clinical Analyzer (ACA) (DuPont, now
● Type of analysis in which each specimen is subjected Siemens) in 1970
to multiple analytical processes so that a set of test ○ First non-continuous flow, discrete analyzer
results is obtained on a single specimen; similar to ○ first instrument to have random-access
random-access analysis. capabilities
Parallel analysis ○ STAT specimens could be analyzed out of
● Type of analysis in which all specimens are subjected sequence on an as-needed basis
to a series of analytical processes at the same time ■ Different compartment where u can
and in a parallel fashion. able to place STAT samples
Random-access analysis ■ STAT = short turnaround time
● The most common configuration of an automated ● 1976: production of thin film analysis technology
analyser, in which analyses are performed on a ○ Uses a very thin film kung saan ilagay ang
collection of specimens sequentially and each sample and kung saan tanan processing na
specimen is analyzed for a different selection of tests. mahitabo and reading
Sequential analysis
● Type of analysis in which each specimen in a batch
enters the analytical process one after another, and
each result or set of results emerges in the same
order as the specimens are entered.
Single-channel analysis
● Type of analysis in which each specimen is subjected
to a single process so that only results for a single
analyte are produced; similar to batch analysis.
Throughput
● The number of specimens processed by an analyzer ● Kodak Ektachem (now VITROS) analyzer (now
during a given period of time, or the rate at which an ortho-clinical diagnosis) in 1978
analytical system processes specimens. ○ First to use microsample volumes and reagents
Workstation on slides for dry chemistry analysis
● A clinical laboratory workstation dedicated to a defined ○ First to incorporate computer technology
task and contains appropriate laboratory extensively into its design and use
instrumentation to carry out that task. ■ Comparing it to the autoanalyzer
where you can see the processing
(whats inside), this is built in or
covered, you can only see computer
and sample holder
1
○ Transcription of results
● Use of very small amount of reagents and samples
○ Allows less blood drawn from each patient
○ Small amounts of reagents decrease the cost
of consumables
Relevant Terms
● Dwell time
○ Minimum time from initial sampling to the
production of a result
● Throughput
○ The maximum number of test results that can
be produced by an analyzer in a given time
period (usually an hour)
● STAT Analysis
Automated System Designs

● Total Laboratory Designs
● Modular Integrated system
● Stand-alone systems
TOTAL LABORATORY AUTOMATION

● Employs an integrated track system that links all the
laboratory’s workstations together to create a continuous,
comprehensive network that automates almost all the
steps involved in laboratory testing
● Why do we need to process using automated machines?

○ Staff shortages
■ Manual processing of plenty patient
samples
○ Turnaround time demands
■ In manual, one patient may take an
hour
■ In automation, built-in and settings,
TAT will lessen
○ Specimen integrity
■ Samples that aren't processed ● Form the time you received the saamle and eveen ang
immediately may lose its integrity paaghataag sa inyong different sample, machine na
○ Safety ● No need for manual transfer of sample
■ Manual processing may expose you
to harmful chemicals Advantages
● Decrease in labeling errors
○ There are some automated machines that they
Advantages could also check if they are labeling errors
● Human factor is decreased during the mechanical and ● Reduced turn-around time
repetitive part of an assay ● Cost-savinng+reduction in labor
○ In manual processing, human factors are ● Reduction in full-time equivalents (FTEs)
common error ○ Are the hours worked on one employee on a
● Increase the number of tests performed by one full-time basis
laboratorian in a given period
● Minimize variation in results from one laboratorian to Major Limitations
another ● Needs substantial financial investment and increased
○ Big variation sa test in every result floor space (Wilson, 2003)
○ Coefficient of variation is lowered and ● Need for highly technical personnel to operate and
reproducibility is increased troubleshoot the system
● The accuracy is not dependent on the skill or workload of ● Personnel team building
a particular operator on a particular day ● Lesser interaction with the staff
○ Our role is to take care of the machines ● Infrastructure remodeling
○ Proper maintenance, calibrations, standards,
running of controls just to make sure na correct ROCHE DIAGNOSTICS SYSTEM
pud and gina release sa atong machine ● Modular pre-analytics (MPA) and platform C (i.e., the
● Eliminates the potential errors of manual analyses such chemistry analyzer
as ● Consists of an integrated tract device that connects all of
○ Volumetric pipetting stes the laboratory workstations, including front-end
○ Calculation of results processing, instrumentation, and archiving, to create a
2
continuous, inclusive network that serves to automate
nearly every step involved in the testing of each sample ADVANTAGES:
● Uniformity in the performance of tests
○ Each sample follows the same reaction path
DISADVANTAGES:
● significant carryover problems and wasteful use of
continuously flowing reagents
○ Major problem = carryover
● The machine does not allow test selection; all tests must
be performed even if not requested
○ Costly thus not being used
MODULAR INTEGRATED SYSTEMS
● link together multiple laboratory disciplines into a single
testing platform that is interconnected by a track
● Less capital investment than TLA
● Has a module
● For a specific section only
● Since taga sample, separated lang siya with air bubbles

(space bubbles to separate one sample from another)
● One reaction sa tanan
● SYSMEX
STAND-ALONE SYSTEMS
● to automate specific sections of the process that are still
manual operations.
○ Specimen processing
○ Sample archiving
● That's why carry over will be common with this type of

analyzer
● Observe the number of staff: ● Air bubbles in the tubings

○ Stand-alone = more staff ● Using only one needle to analyzes one sample to
○ By module = less staff another
● One tubing and reaction vessels in the machine
CLASSIFICATION OF AUTOMATED ANALYZERS
● Continuous flow analysis DISCRETE ANALYZER
● Discrete analysis
● The sample travel through the instrument in its own
● Centrifugal analysis
reaction vessel
● Sequential analysis
● Each test reaction takes place in a separate
● Batch analysis
compartment that is either cleaned out or disposed of
● Parallel analysis
after use
● Random access analysis
● Have the capability of running multiple tests one sample
at a time or multiple samples one test at a time
● Keeps sample and reaction carryover to a minimum but
CONTINUOUS FLOW ANALYZER
increases the cost per test due to disposable products
● the samples flow through a common reaction vessel or (like plastic cuvette)
pathway ● Most commonly used compared to CFA and most
● liquids (reagents, diluents, and samples) are pumped versatile type of analyzer
through a system of continuous tubing ● developed as a result of space-aged technology and
● assists the laboratory that needs to run many samples were one of the first types of discrete analyzers
requiring the same procedure ● Samples and reagents were mixed together, reacted, and
● An oil heating bath is used to promote color development flowed by centrifugal force into separate cuvettes in
or the completion of enzymatic reaction which spectrophotometric analysis could occur
● Colorimetric method; color development
3
● DISADVANTAGE: Only one test type can be performed CENTRIFUGAL ANALYZER
each time ● developed as a result of space-aged technology and
were one of the first
● Samples and reagents were mixed together, reacted, and
flowed by centrifugal force into separate cuvettes in
which spectrophotometric analysis could occur
ADVANTAGES
● Running multiples samples at a time (batch analysis
DISADVANTAGE:
● Only one test type can be performed each time.
Similar to discrete but differs in way of processing is thru
centrifugal force
Classification of Automated Analyzers

Skalar SEQUENTIAL
● Cuvette with optical path-length: 15 mm ● performing a set of test reactions in a particular order on
● 640 tests running without operator intervention each sample in the order in which it is received
● 100 sample positions BATCH
● 32 positions for reagents, (stock) standards and QC's ● all samples are loaded at the same time, and a single
● Possibility to add priority samples during a run test is conducted on each sample.
● Automatic preparations of working standards PARALLEL
● Sample pre- and post-dilution functions ● more than one test is analyzed concurrently on a given
● 8 optical filters ranging from 340 - 900 nm clinical system
● Disposable cuvette blocks RANDOM ACCESS
● Segregation of chemical waste ● any test can be performed on any sample in any
sequence
PART TWO
SPECIMEN PREPARATION AND IDENTIFICATION

SUMMARY OF CHEMISTRY ANALYZER OPERATIONS
IDENTIFICATION AND PREPARATION Technologies Used for Automatic Identification and Data
Collection
1. Sample This is usually done by
identification reading the bar code. This ● Bar coding
information can also ● Optical character recognition
be entered manually ● Magnetic stripe and magnetic ink character
recognition
● Voice identification
2. Determine test(s) The LIS communicates to the
● Radio frequency identification
to perform analyzer which test(s) have
● Touch screens
been ordered
● Light pens
● Hand print tablets
● Optical mark readers
CHEMICAL REACTION ● Smart cards
3. Reagent systems One or more reagents can be

and delivery dispensed into the reaction
cuvette
4. Specimen A small aliquot of the sample

measurement and is introduced into the reaction
delivery cuvette
5. Chemical reaction The sample and reagents are

phase mixed and incubated
DATA COLLECTION AND ANALYSIS
6. Measurement Optical readings may be

phase initiated before or after all ● Optical character recognition - uses a pen; i agri ra sa
reagents have been pen then ma recognize na
added ● Magnetic identification character recognition (MICR) -
very common sa mga check
7. Signal processing The analyte concentration is ● Optical marker recognition - ex. scantron
and data handling estimated from a calibration
curve that is EMERGING TECHNOLOGIES
stored in the analyzer ● Automated specimen inspection
○ Identify sample identification errors and sample
8. Send result(s) to The analyzer communicates integrity issues
LIS results for the ordered tests to ■ Ex. hemolyzed sample - it is able to
the LIS recognize
○ Could sort a random collection of different-sized
containers with different additives
4
● Assess each sample for proper labeling, adequate ■ Eliminates the need both to wait for
volume and interference from icterus, lipemia or the sample to clot and to aliquot the
hemolysis sample
● Radio-Frequency IDentification ○ Barcode-labeled tubes
○ Passive and without active human intervention ■ Scanning then transport
○ Advantages:
■ No line of sight reading SPECIMEN LOADING AND ASPIRATION
Ma scan gihapon maskin di ● Circular carousel o rectangular racks as specimen
nakatutok containers
■ Dynamic data storage ● Trays or racks are numbers to aid in sample
■ Not affected by cold storage identification and move automatically in one-position
○ Disadvantages: steps at preselected speeds
■ Yet to be commercialized on a large ○ Even trays have barcodes so when you set or
scale in the clinical laboratory encode samples in the computer, inyoha siyang
■ Greater cost iset-up in line kung unsa ang ga barcode sa
racks to sill have proper identification sa
SPECIMEN DELIVERY specimens
● Courier service ● The instrument can determine the slot number containing
○ Not common in sending your different samples the last sample and terminate the analysis after that
especially when it needs confirmatory testing sample
○ The hospital can deliver patient samples for ○ By the time it will reach the last specimen, the
confirmatory testing whole process stop
● Pneumatic tube systems ● The instrument’s microprocessor holds the number of
○ Mechanical problems in the switching process samples in memory and aspirates only in positions
have been known to cause misrouting of containing the samples
carriers
■ Misroute of samples
○ Prone to hemolysis (Avoid sudden acceleration
and deceleration and use of proper packing
material)
■ Can be damaged (affects specimen
integrity)
○ Rarely used because of the disadvantage
● Electric Track Vehicles
○ Larger carrying capacity than pneumatic tube
systems
○ Not associated with problems such as damage
to specimens caused by acceleration and/or
declaration forces
● Mobile robots
○ Delivery of specimens to lab benches by a ○ Lower left picture- circular carousel
mobile robot is usually more frequent than ○ Upper right picture- rectangular racks
human pickup and has been shown to be ○ Lower right picture- loading zone of auto
cost-effective analyzers
● Actual measurement off each aliquot for each test must
be very accurate and is generally done through
aspiration of the sample into a probe
○ There are some like tubes lanng ang
ginabutang sa rack, there are some machines
na dapat aang inyong serum kay up lang to a
certain level
● Tubes are typically decapped before sampling
○ Unicell Analyzer (Beckkman Coulter Inc., Brea,
Calif.) offer cap-piercing technology
○ Hematology analyzers
SPECIMEN PREPARATION AND IDENTIFICATION ○ There are machines na sila na ang ga mix
● Preparation of the sample for analysis has been and ● Wash Solution: deionized water (sometimes with
remains a manual process in most laboratories surfactant)
● Alternatives: ○ Important and dapat dili mabutangan ppa ug
○ Use of robotics or front-end automation othe reagents
○ Bypass specimen preparation by using whole ○ Since this is necessary kay every after mukua
blood for analysis ug sample ang probe, iyaha nanghugas before
■ It takes a lot of time to get a sample magkuha ug sample sa another patient
(clot of blood, centrifuge takes about ○ Common Problem: carryover
15-20 mins) ■ Carries one sample to the other,
■ To lessen the time, its best to use a which lessen the accuracy of results
whole blood analysis (has pros and ● Loading zone: area in which specimens are held inside
cons) the instrument before they are analyzed
■ There are substances in the body that
its levels are better in the serum Specimen Processing
■ There are autoanalyzers as of the ● Autoanalyzer should be capable of removing protein and
moment that use whole blood (ex. other interferents
ABG) ○ Dialysis
● Nakaset up na machines ■ Process of removing excess water,
○ Use of plasma separator tube and perform solutes, or toxins in the body
primary tube sampling with heparin plasma ○ Common chroatography
5
○ Filtration interfere with results
○ Protein is a very common interferent ● Reagent layer - reagent reacts with sample
○ One problem it poses is its size which is 3. Indicator layer - reacted sample collects for spectral
common to blocks other substances. analysis
○ Common causes of blockage on tubes, probes ● Support layer - optical interface; serves as exit slit
in the analyzer, especially if Dili mag clean first
thing in the morning Dry chemistry slide
○ Pre-treatment removes interferences ● The reagent layer contains; enzymes, dye precursors,
and buffers necessary for the analysis of a specific
REAGENT SYSTEMS AND DELIVERY component
● Open vs Closed system analyzer ● Sample, control, or standard is deposited on the
○ Open-system analyzer spreading layer
■ operator is able to change the ● Selected components are allowed to penetrate to the
parameters related to an analysis and reaction layer(s), which in turn activate the dehydrated
prepare “in-house” reagents or use reagents
reagents from a variety of suppliers ● Less laborious
■ Flexible and adapt readily to new
methods and analytes Between your liquid and dry reagent, it would always be best to
○ Closed-system analyzer have a dry reagent because:
■ requires the reagent to be in a unique ● Longer shelf life
container or format provided by the
manufacturer Techniques of preservation:
■ common ● Keep all reagents refrigerated until the moment of need
and then quickly preincubate them to reaction
● liquid reagents for open systems are less expensive than temperature or store them in a refrigerated compartment
the closed analyzers on the analyzer that feeds directly to the dispensing area
● closed systems contain a hidden cost advantage ● Common mistake:
because reconstitution or preparation of reagents for use ○ Do not immediately use your reagents after
does not require a technologist’s time getting it from the refrigerator
● Liquid reagent ● Provide reagents in a dried, tablet form and reconstitute
● Dry reagent them when the test is to be run
○ May be bottled as lyophilized powder ● Manufacture the reagent in two stable components that
■ Freeze-dried powder will be combined at the moment of reaction
○ Requires reconstitution with water or a buffer ○ Instead of combining immediately the reagents
○ Dry chemistry slide (another type of dry in one bottle, better to separate it first and mix
reagent)
■ have microscopically thin layers of dry Reagent delivery techniques:
reagents mounted on a plastic
support ● Sa machine na nimo on how it would get samples
■ slides are approximately the size and ● Syringes: driven by a stepping motor, pipet the reagents
thickness of a postage stamp into reaction containers
■ Example of thin film analysis ● Piston-driven pumps: connected by tubing, may also
dispense reagents
● Use of pressurized reagent bottles connected by
tubing to dispensing valves
○ The computer controls the opening and closing
of the valves
○ The fill volume of reagent into the reaction
container is determined by the precise amount
of time the valve remains open
CHEMICAL REACTION PHASE

● consists of:
○ Mixing - most difficult part
● Dry reagent ○ Forceful dispensing
● Each of these pads is already incorporated with reagents ○ Magnetic stirring
necessary to identify what needs to be identified ○ Vigorous lateral displacement
■ discrete analyzer - positive
displacement
■ Magnetic stirring
○ Rotating paddle
○ Ultrasonic energy
● Separation
○ In relation to removing interferences
○ Use a very high reagent-to-sample ratio
■ Use very diluted sample so that any
turbidity caused by precipitated
protein cannot be sensed by the
spectrum
○ Shorten reaction time to eliminate
slower-reacting interferents
● Incubation
3 layers of the dry slide technology: ○ Some testing process requires 37 C
1. Spreading layer -sample is distributed evenly ○ Heating bath
2. Central layer ■ maintains the required temperature of
● Scavenger layer - filters out substances that the reaction mixture
6
■ provides the delay necessary to allow
complete color development
■ Components: heat-transfer medium,
heating element and thermoregulator
● Reaction time
○ Completion of reaction
○ Rate at which the reaction is proceeding
○ One way of calculating the amount or level of
certain substances
MEASUREMENT PHASE
● UV light, fluorescent, flame photometry, ion-selective
electrodes, gamma counters and luminometers are
various methods for measuring product formed
● Most common methods are still visible and UV
spectrophotometer
● Analyzers that measure light need monochromators to
produce specific wavelength, classically filter wheels
have been used to separate light, which are usually
computer controller
Signal Processing and Data Handling

● Accurate calibration is essential to obtain reliable results
○ Assurance sa atoa na accurate ang
measurements
○ Calibration is done though comparing a known
substance (standard) to the measurement used
in the instrument
● This requires proper use of standards, which then reflect
the data on standard curve according to which the
sample results are interpreted
○ Standards: solutions containing precisely
known concentration of a substance
● After the calibration has been performed and the
chemical or electrical analysis of the specimen is either in
progress or completed, the instruments computer goes
into data acquisition and calculation mode
● This process may involve signal averaging which may
involve hundred of data pulses per second
Future trends in Automation

● Automation will continue to evolve.
● System integration and miniaturization with more
technologically advanced computer power will persist to
accommodate more portable analyzers for more precise
testing.
● Automated analyzers will have artificial intelligence
where by the computer will “think” or make decisions if
sufficiently programmed with infinite scenarios of data.
● Spectral mapping or multiple wavelength monitoring, with
high-resolution photometers and polychromators will
become standard.
7
LECTURE 1: GLASSWARES, PLASTICWARES

AND MEASURING VESSELS
Prof. Bea Angelli Laude, RMT
August 16, 2021
Clinical chemistry is the area wherein we are going to measure the GLASSWARE
different analytes that are present in the blood sample of the patient.
They are usually metabolites of the body which are quantified to Advantages
identify the disorders the patient is facing.
(Some) heating
LABORATORY GLASSWARE & PLASTICWARE Longer storage of some chemicals
● Equipments in the laboratory can either be made of glass
or plastic Regardless of the design, glasswares in the laboratory
○ If they are made up of glass, it is actually a must fall into 2 types:
specific type of glass that’s why they are ● Class A glasswares
correctly termed as glasswares ● Class B glasswares
○ Nowadays, the use of plastic equipments are These classes will be identified or labelled in glasswares
rampant, however plastics tend to have if they are able to satisfy certain tolerances of accuracy
limitations = they cant support heat
■ That's why we still continue to use Class A Glasswares
glasswares
Main Functions of Glasswares and Plasticwares
Why use glasswares/ plastic wares in the lab?
● Storage
○ In the laboratory, we tend to store both the
samples as well as the reagents
○ That’s why it's necessary that we make use of ● Found in professional laboratories
glasswares and plasticwares, that has a
specific quality such as: Class A Glasswares
■ Ability to withstand extreme ● Class B tends to have twice the tolerance limits of Class
temperature; A
■ And the corrosive effects of strong ● Class B are much more durable
acids and strong alkaline solutions ○ Often found in student lab
● Measurement
● Containment Cleaning of plastic/ glassware
● Those in direct contact with biohazard material is usually
PLASTICWARE disposable
- Since the early times, majority of the laboratory ○ Biohazard material such as samples, be in the
equipments are actually made up of glass form of stool, urine, or blood must be disposed
- Since the discovery of plastics, it slowly replaced the after use
glasswares that we often use in the lab ■ Regardless if it's made of plastic or
- However, nowadays we still tend to continue using glass
glasswares mainly because it is able to fill in the ○ Advantage of plastic = since it’s very cheap,
limitations of plastics they are often in disposable form
Advantages Disadvantages ○ Unlike glass, they tend to be much more
expensive = reusable form
Evaporation through breathing
● If not disposable, follow proper decontamination protocol
of plastic
○ Immediate rinsing + washing with powder/
Cheaper - Plasticwares are not often
liquid detergent
utilize as storage vessels
■ Wash intensely to remove the
- They tend to be porous
remaining detergent (which can be a
More Durable contaminant) if you are going to reuse
- Much efficient in resisting that particular lab equipment
the corrosive effects of ○ Pre-soaking in soapy water
strong acids and strong ■ May contain bleach (usually small
alkali solutions amount) mainly for contamination
Evaporation of dyes, stains, and
Preferred for some analyses purposes
proteins
- Example:Testing for heavy ○ (read furthermore Bishop Chapter 1)
- They will evaporate in
metals (NO glass) ○ For reusable lab equipments, it must also be
plasticwares
- Some heavy metals that we rinsed properly
- Essential to be stored in
need to analyze could also ■ Perform multiple rinsing to ensure that
glasswares
be present in glasswares, there are no remnants of detergent
and they could be considered left = Remnants may act as
as a contaminant contaminants
- These heavy metals are not ○ One way of determining proper rinsing of lab
present in your plasticwares equipment, you have to check the water used in
the last rinse
1
○ Check the pH twice before rinsing and after ● Characteristics:
rinsing ○ Flexible or rigid
○ If after rinsing and pH is higher, we can ○ Chemical-resistant
determine that the detergents were removed ○ Can be autoclaved
(Detergents make the solution more alkaline) ● Uses:
■ Higher pH of water after rinsing ○ For cryogenic procedures
compared to pH before it was rinsed, ○ Specially formulated to withstand temp down to
means that the detergents are -190°C
removed ● Several tube designs
○ specimen tubes and test tubes
PLASTICWARE ○ Microwavable containers and folders
Major types of resins used in Clin. Chem Lab: C. Polyethylene

(Details on Henry, chapter 4, p. 60) ● Disadvantage:
A. Polystyrene ○ NOT suitable when using picric acid, stains,
B. Polyethylene dyes and proteins
C. Polypropylene ● Uses:
D. Teflon ○ test tubes
E. Polycarbonate ○ Bottles
○ graduated tubes
The type of plastic you will use will depend on the procedure you ○ Stoppers
are going to perform ○ Plastic balls (toys)
○ Plastic wraps
● Indicated with arrows = types of resins that are incontact ○ Insulators
with our food:
○ PET or PETE (polyethylene terephthalate) D. Polycarbonate
○ HDPE (high density polyethylene) ● Characteristics:
○ LDPE (Low density polyethylene) ○ Very strong plastic but NOT chemically
○ PP (Polypropylene) resistant
● Others cannot be used for food because once ingested, ○ Autoclavable but with limitations
they may produce harm ● Usable temp. range: -100°C to +160°C
● Uses:
A. Polystyrene ○ tubes for centrifugation
● Characteristics: ○ graduated cylinders
○ Rigid ○ Flasks
○ Clear type ○ Roofs of waiting sheds
○ NOT to be autoclaved
E. Teflon
● Characteristics:
○ Almost chemically inert - unable to react to
chemicals
○ Chemical-resistant
● Suitable work temp. @: -270 ⁰C to 255 ⁰C
● Uses: stirring rods, tubing, cryogenic vials, bottle cap
liners
● Used for storage or mixing equipments
● Pans for cooking and Teflon tapes utilized in water lines
at home
GENERAL CATEGORIES OF GLASS

1. Borosilicate (Kimax/ Pyrex)
2. High silica Glass
3. Aluminosilicate (Corex)
4. Acid & Alkali resistant (Vycor)
5. Low actinic (amber-colored)
6. Flint glass (soda lime)
7. Disposable glassware
1. Borosilicate Glass (Kimax/ Pyrex)

■ Can be reused with other means of ● Most common type of glass encountered in volume
disinfection but NOT AUTOCLAVING measurements
● Disadvantage: ● Composition:
○ Not resistant to most hydrocarbons, ketones ○ 80% silica
and ROH ○ 13% boric oxide - contains BORON
■ Thus not utilized as storage ○ 4% sodium oxide
● Uses: capped graduated tubes and test tubes ○ 2–3% aluminium oxide
● Can be used as a container - short time ● Characteristics:
● Ex. Styrofoam ○ High degree of thermal resistance and low
coefficient of thermal expansion
B. Polypropylene ■ Object has the ability to change its
● Primary constituent of pipet tips shape at a given temperature
○ Pipets are utilized to transfer and measure ■ Change is minimal
samples as well as reagents ○ Low alkali content
○ Reagents may be strong acids because the ■ Resistant to alkali corrosion
pipet tips can withstand extreme pH ○ Free of heavy metals
2
■ Free from Mg-lime-zinc group of 4. Acid- Resistant & Alkali-Resistant Glass
elements, heavy metals, arsenic and ● Boron-free
antimony ● A.k.a “soft glass”
● Uses: ● Vycor® (No. 7900)
○ Heating ○ Made up of fused silica
■ Open flame or electric heating ○ 2 in 1 characteristic
elements such as hotplates ■ Heat-resistant (not as efficient as
● Precautions: borosilicate glass)
○ Storing concentrated alkali solutions will ■ Chemically inert (a substance not
etch/destroy the calibration chemically reactive)
○ Heavy-walled type of glass should not be ○ Unique characteristics:
heated with direct flame or hotplate ■ Stable to all acids except hydrofluoric
■ Only the thin-walled type of types
borosilicate glassware will be heated - Hydrofluoric solutions corrode
in direct flame or hotplate glasses with silica
○ Avoid heating beyond its strained point ■ Superior in resisting corrosion by
■ Strain point- the temperature where alkali than borosilicate glass
the glass softens or will transform ■ Unlikely contamination by contact
from solid to liquid with solutions
■ Heating beyond strained point could ■ Relatively inert to acids and neutral
lead to breakage salts
○ In the case of volumetric glassware, heating ■ Chlorine and acid gases do not affect
can destroy the calibration it at any temperature
● Popular Brands ○ Withstand high temperature (1200°C)
○ Pyrex® ■ Softening temp.: 1500 °C
■ Strain point: 515 ○ Withstand downshocks from this temperature to
°Celsius (Henry) ice water
○ KIMAX® ○ Use:
■ Strain point: 513 ■ ashing and ignition techniques
°C
2. High Silica Glass

● Silica fused to quartz
● More expensive than borosilicate glass
● Use: 5. Low Actinic Glass
○ Spectrophotometer cuvettes ● Contain materials that usually impart amber to red color
to the glass
○ Reduces the amount of light transmitted to the
substance in the glassware disallowing
3. Alumina-Silicate Glass oxidation
● A.k.a Aluminosilicate glass ● Common uses:
● With aluminum oxide ○ For light-sensitive substances
● Strengthened chemically rather than thermally ■ Bilirubin
○ Can withstand very strong acids, bases, ■ Vit. A
hydrocarbons, and concentrated alcohols ○ Store control materials and reagents
○ Greater chemical durability and can withstand
higher operating temperatures 6. Soda-Lime Glass
○ Compared to borosilicate, aluminosilicates are ● A.k.a flint glass
more difficult to fabricate ● Composition:
● Has a wide thermal range but not as wide ○ Silicon oxide
as borosilicate glass ■ Soda = sodiuum oxide
● When coated with an electrically ■ Lime = calcium oxide
conductive film, aluminosilicate glass is ● Most inexpensive glass
used as resistor for electronic ○ Most common type of glass found outside the
circuitry laboratory
● High impact, extremely strong glass ● Readily made into variety of types of glassware
○ Common use: manufacture of ● Has a high expansion coefficient and a high degree of
calibrated centrifuge tubes thermal resistance
● Corex® (Corning, N.Y.) ○ At given temperature, it will exhibit huge
○ Characteristics: structural changee
■ Radiation-resistant ● Mineral can be leached from the glass into the stored
■ 6X stronger than borosilicate (outlast solutions
conventional ● Common uses:
glassware by 10-fold) ○ Volumetric flasks
■ Resist clouding and ○ Stirring rods
scratching better ○ Single-use pipette or test tubes
○ Uses:
■ High-precision MEASURING VESSELS
analytical work a. Graduated cylinder
■ Optical reflectors and b. Burets
mirrors c. Volumetric flask
■ Not used as general d. Pipet
type of glassware in
the lab
3
A. GRADUATED CYLINDER A. Design = TC vs. TD (to contain or to deliver)
● Long, cylindrical tubes usually held upright by B. Drainage characteristics = Blow-out vs.
an octagonal or circular base with gradations Self-draining
along its length C. Type = Measuring vs. transfer
● Semi-accurate
● Extremely convenient for rapid measurement of
TABLE 1-4 PIPET CLASSIFICATION
liquid
● Should NEVER be heated especially if plastic
since it will destroy the calibration marks along
its body I.Design
A. To contain (TC)
B. BURETS B. To deliver (TD)
● Long cylindrical graduated laboratory glassware with
stopcock II. Drainage characteristics
● Type of buret used depends on the chemical A. Blowout
used B. Self-draining
○ Glass >> ACID
○ Rubber for >> ALKALI III. Туре
● Extremely accurate in dispensing aliquots of a A. Measuring or graduated
solution 1. Serologic
○ Mainly used to dispense known 2. Mohr
amounts of liquid reagents in 3. Bacteriologic
experiments where such precisions 4. Ball, Kolmer, or Kahn
are necessary 5. Micropipet
● Generally used for titration purposes only B.Transfer
1. Volumetric
C. VOLUMETRIC FLASK 2. Ostwald-Folin
● Round lower portion and a long, thin neck 3. Pasteur pipets
with an etched neck or calibration line 4. Automatic macropipets or micropipette
which is going to be the mark in
measuring a specific volume
A. Design
● Generally used for:
○ Preparation of standard solution 1.) To Contain (TC
○ Measuring liquid colume ● A.k.a. “Rinsed-out pipets”
accurately ● Able to hold a particular volume but is unable to dispense
the volume indicated.
● For u to dispense the exact volume, Must be refilled and
rinsed-out with the appropriate solvent after the initial
D. PIPETS liquid has been drained from the pipet
● Glass or plastic material in the lab used to transfer liquids ● Examples:
● Can either be reusable or disposable ○ Sahli-hemoglobin pipets
● Majority of pipette can hold only up to 20 mL of a specific ○ Long-Levy pipets
solution 2) To Deliver (TD
● Used to transfer measured volumes of liquid between ● Able to transfer the exact volume indicated in the pipette
containers ● Designed to drain by gravity
● Must be held vertically with the tip placed against the
side of the container and must NOT TOUCH the liquid in
it
● Vessel ang nakatilt
● Examples:
○ Mohr pipet
○ Serologic pipet
○ volumetric transfer pipet
NOTE:
● A TC pipet holds or contains a particular volume but does
not dispense that exact volume, whereas a TD pipet will
dispense the volume indicated.
B. Drainage Characteristics
1) Self-draining pipet
○ Example (pic above): you have 5 in 1/10 which

means that the capacity of the pipet in
measuring a particular volume is only up to 5
mL with 10 increments to the next demarcation
line
● Numbers are indicated at the top or lower portion of the
pipet which indicates the volume or total capacity for
measuring and the major divisions
● In order to properly measure a specific volume of a
particular solution, you have to look for the meniscus of
the aspirated fluid where it should be aligned to the
graduation line of your pipet ● with a single painted at the top
● Pipets are classified based on: (Bishop 7th ● Allow to drain by gravity
● edition, table 1-4 pg.13) ● No frost/ etched / double lines
4
2) Blown-out pipet Volumetric or Transfer
● Used to measure and transfer a predetermined volume of
liquid
● Kaya niya ma dispense or ma measure na volume of a
particular solution is only one volume
● If it is indicated that it is able to measure 10 mL, therefore
10 mL lang jud ang pede niya i measure kay isa lang
iyang etched line
● Dispense one volume without further subdivisions
○ Ostwald-Folin Pipet
○ Pasteur Pipet
● With double rings/ frost/ etched

● designed to be "blown out" by pushing a small amount of
air out of the pipet, completely emptying it
● For u to deliver the exact amount of the volume specified
by the pipette, it has to be blown out with the use of a
bulb.
Left: volumetric (transfer)

Right: oswald-folin
TOP portions of self-draining & blown-out pipet

Single colored line = self-draining
● Allow it to drain by gravity
Double ring or frosted/ etched ring= blown out pipet
● To deliver the exact amount of solution u have measured,
you have to introduce air to empty the pipet.
Pic above: illustrates the proper positioning of pipettes in aspirating

TIP portions of self-draining & blown-out pipet and dispensing solution.
Left: self-draining ● Top pic: held vertically upright with the tip slightly
- Graduation is much farther from the tip. submerged in the surface of your solution without
Right: Blown out pipet touching the side of the vessel.
- Graduation is up to the tip of the pipet. ● Bottom pic: dispensing solution
○ Tip is not supposed to touch the surface of the
C. Type solution contained in your vessel but rather the
1. Transfer pipet (or volumetric) tip is going to touch the side of the vessel for a
a. Volumetric smooth flow of the draining of your solutions.
b. Ostwald-Folin ● In both aspirating and in draining, the pipette must be in
c. Pasteur a vertical position.
d. Automatic macro- & micropipet ● Draining
2. Measuring or Graduated pipet ○ In order to maintain the vertical position of your
a. Serologic pipet and to allow the tip to touch the sides of
b. Mohr the receiving vessel, so u have to tilt the vessel
c. Micropipet to maintain its vertical position.
5
Positive Displacement Pipette
● Act or move like a syringe

● Each tip contains a disposable plunger like a syringe
○ Plunger is disposable but not the tip
● The plunger directly displaces the fluid with no error
● These are the most accurate type of pipette
● They are also the most expensive
https://www.youtube.com/watch?v=_YTwxakpY1U
The volumetric pipets are always SELF-DRAINING

Ostwald-Folin pipets are BLOWOUT PIPETS
● Volumetric – to transfer aquaeous solutions
● Ostwald-Folin – to transfer viscous fluid
DIFFERENCE VOLUMETRIC OSWALD-FOLIN
Drainage self-draining Blowout pipette

characteristics
Top portion Have single ring double/frosted/

etched ring
Measuring or Graduated
● Calibrated to distribute fractional quantity of liquid and
principally used for measurement of reagents
● The volume that it can measure is not fixed but rather 1. Air displacement method (left)
measure different volumes of a - It is going to rely on a piston for creating a
particular solution suction to draw the sample into a disposable tip
● Examples: that must be changed after each use
2.a. Mohr pipet - The piston does not come in contact with the
2.b. Serologic pipet liquid but there will be unprotected air space so
there could be a possible aerosol contamination
Mohr pipet of the sample or the reagents that is going to be
● No graduations to the tip aspirated
● Self-draining pipet - Can ensure that there will be no carry over
● Single ring since u are going to replace the tip
- However, it going to be less accurate compared
Serologic pipet to positive displacement pipet
● Has graduation marks to the tip 2. Positive displacement method (right)
● Generally a blowout pipet - Going to operate by moving a piston in the
● Designated by a frosted or double pipet tip or barrel
colored ring at the top - The piston is in the form of a plunger in which it
is going to be changed once u are going to
dispense one liquid after another
Micropipet - Advantage of piston:
● With a total holding volume of less than 1 mL - There will be protected air space
● It may be designed as either a Mohr or serologic pipet - No aerosol
● Comes in 2 forms: - Problem: Since you are not going to change the
○ Automatic and Semi-automatic pipet tip, there will be a carry over of samples or
■ Commonly used in the laboratory regents to another type of solution
2 Types of micropipet:
1. Air displacement
● Disposable, polypropylene tip
2. Positive displacement
● Use of capillary tip (siliconized, glass, plastic)
6
ANATOMY OF AIR DISPLACEMENT PIPET POSITIVE DISPLACEMENT PIPET
● No first stop or second stop

● Pull the plunger in order to aspirate your fluids
● In draining, simply push down your plunger and drain all
the contents
https://www.youtube.com/watch?v=NgosWmRjjAo
Semi-automatic & Automatic pipettes and

dispensers (Tietz)
● Push button
○ pushed in order to aspirate or dispense a
particular volume of sample or reagent
○ Not going push it but turn it bcs it is going to
● Semi-automatic pipettes
serve as a large volume adjustment knob
○ Although it allows us to have an easier
■ In order to adjust the volume to be
measurement of volumes of samples and
aspirated in much more larger
reagents, but the aspiration and dispensing of
amounts
such liquids can still be done manually
● Tip ejector button
● Automatic pipettes
○ Simply push this down to remove the pipette
○ It is attached to a machine and a tube is going
tips that is found at the bottom portion
to be submerged to a particular solution
● Thumbwheel (Fine volume adjustment ring)
○ The machine will be regulating the aspiration as
○ Used to adjust the volume to be aspirated in
well as the dispensing of a particular volume of
smaller amounts
a reagent ot sample
● Volumeter display
● Dispensers (Dilutors)
○ Where u are able to determine how much of the
○ Automatic pipettes that obtain the liquid from a
solution or what is the exact volume to be
common reservoir and dispense it repeatedly
aspirated, transferred or measured
○ May be bottle top, motorized, handheld, or
● Tip ejector
attached to a dilutor
○ Connected to the shaft
○ Simply push to move and lead to the removal of
your disposable tip
AIR DISPLACEMENT PIPET
● If you are going to push the push button, it has 2 stops:

○ First stop
○ Second stop: mainly for the draining of the
entire solution that was aspirated in the pipet tip
Prior to the submersion of your tip to the solution, it must be

pushed to the first stop
↓
Once it is submerged in the solution, you are going to release it.
By then you are able to aspirate a specific amount of the solution
↓
In transferring, push down the push button from the first stop all
the way to the second stop (mainly for the draining)
↓
Release
7
UNIT
% g/100mL
Valence eq/mole
MW g/mole
M Moles/L
N eq/L
UNIT FORMULAS

𝑥100

𝑥100

𝑥100
● %
PERCENT ● % w/v
● %v/v 𝑔 = 100 𝑚𝐿
𝑥 (𝑚𝐿)
𝑚𝐿 = 100
𝑥 (𝑔)
𝑔 = 100𝑔
● M
● moles/Liter
● moles/kg
MOLALITY
● m

𝑀𝑊
NORMALITY ● N
or

𝑁 = 𝑀𝑊
LECTURE 3: ELECTROPHORESIS AND

ELECTROCHEMISTRY
Prof. Lyslie Jane Vasquez-Baylon, RMT
August 30, 2021
TERMINOLOGIES ○ Cerebrospinal fluid (CSF)

● Ampholyte ○ Erythrocytes and tissue and,
○ A molecule that contains both acidic and basic ○ other biologic body fluids
groups ○ Nucleic acid (DNA, RNA)
○ Helps establish a stable pH gradient and assist ● Using electrophoresis, we can separate particles, isolate
molecules such as proteins in migration them and observe the rate how these particles move or
● Electrophoretic Mobility travel through the electric field
○ The rate of migration of a charged solute in an ● Take note:
electric field, expressed per unit field strength ○ Electrophoresis is usually used for protein
○ Solute’s response to the applied electrical field determination
○ What is the response? ○ The proteins are separated on the basis of their
■ The solutes move towards a charged electric charge density
electrode
● Endosmosis
○ Preferential movement of water in one direction
through electrophoresis medium due to
selective binding of one type of charge on the
surface of the medium.
○ Movement of liquid such as water and buffer
ions
● Electrophoretogram
○ The migration of charged macromolecules.
○ Result of zoned electrophoresis and consists of
sharply separated zones of macromolecules
● Iontophoresis
○ The migration of small charge ions.
● Zone Electrophoresis Theory of Electrophoresis
○ The migration of charged macromolecules in a ● Electrodes:
porous supporting medium such as paper, ○ Cathode = negatively charged
cellulose acetate, and agarose gel film ○ Anode = positively charged
○ Most prevalent electrophoretic technique used ● Charged particles and electrophoresis migrate toward the
today oppositely charged electrode
○ It has different types like paper electrophoresis, ● Thus:
cellulose acetate electrophoresis, capillary ○ Cations (+) → Cathode (-)
electrophoresis and gel electrophoresis ○ Anions (-) → Anode (+)
ELECTROPHORESIS
● In electrophoresis, particles or solutes are charged and

are moving in an electrical field towards an electrode
● One electrode is positively charged and the other one is
negatively charged
● Principle:
○ The movement of electrically charged
compounds (proteins) in a medium resulting to
their separations based on their electrical
charges when an electric current is applied
○ Compounds or particles are separated and they
move through the medium ● Remember: Proteins are amphoteric and the during
● Macromolecule of interest: electrophoresis, these proteins are negatively charged
○ Proteins in serum anions and move towards the anode
○ Urine
1
● Using electrophoresis particles like proteins are
separated, detected and quantified.
Components of Electrophoresis
1. Driving force (electrical power)
● Supplies the constant current
2. Support medium
● Holds sample in place for migration
3. Buffer
● Carries applied electrical current and set the pH
of electrophoresis
4. Sample
● Body fluids containing our molecules of interest
5. Detecting system
● Biomolecules ● Visualizes our macromolecules of interest
○ Protein under the UV light, also uses stains
■ Contains an amino group, side chains ● Densitometer for quantitation
and, carboxylic group
■ The acidic and basic amino acids
determine the net charge of the
proteins
○ Lipoproteins
○ Nucleic Acid (DNA & RNA)
● These are the common molecules of interest separated
and isolated in electrophoresis
● Electrophoresis is a technique used in laboratories in
order to separate macromolecules based on size
Factors Affecting Rate of Migration

● Net electric charge of the molecule
● Size and shape of the molecule Driving force (Electrical Power)
● Strength of electrical field ● In electrophoresis, heat is produced when current flows
● Properties of Supporting Medium through a medium that has resistance, resulting in an
● Temperature of operation increase in thermal agitation of the dissolved solute
(ions) and leading to a decrease in resistance and an
increase in current
● Separation of molecules using electrophoresis often
require high voltages of about 50 to 200VDC
○ Power supply should supply a constant voltage
○ A constant rate is applied because it helps keep
● This equation shows how the rate of mobility of the the migration rate constant
molecule presented by mu is affected by the net charge
of the particle as well as the constant Supporting Medium
● The ionic radius of the particle and the viscosity of the
buffer ● Provides the matrix where charged molecules are added
● Based from this equation, the rate of migration is directly and separated
proportional to the net charge of the particle but inversely ● Common supporting media utilized in electrophoresis
proportional to its molecular size and viscosity
● Note: Net Charge of the compound depends on the ○ Cellulose acetate
solution of the ph ■ cellulose is acetylated to form
○ The greater the net charge of the molecule, the cellulose acetate by treating it with
faster it moves through the solution and acetic anhydride
towards the oppositely charged electrode ■ A dry, brittle film composed of about
80% air space (these air spaces are
Conventional Electrophoresis filled with electrolyte when cellulose
acetate is soaked in the buffer during
electrophoresis, then it becomes
pliable (flexible) and easily bent)
■ Homogenous medium with uniform
pore size and does not absorb the
protein
■ When electrophoresis is done, it can
be stained and made transparent for
quantitation
■ Can be stored for long periods
■ Advantages - provides good
background for staining glycoproteins,
gives sharp bonds and offer good
resolution
○ Agarose gel
■ Widely used supporting medium for
electrophoresis
● Power source ■ Highly purified uncharged
● Gel - supporting medium polysaccharide derived from agar
● Sample wells where we place our sample ■ Advantages: requires small amounts
● Sub cell - contain the buffer or electrolyte solution of sample (approx. 2ml), neutral --
2
does not produce electroendosmosis ■ Ionic strength
■ Once electroendosmosis happens, ● If buffer is more than the pI, it becomes positively
there will be movement of solvent charged and migrates toward the cathode]
such as water in the electric field ○ Binds more hydrogen ion
which affects the movement of ● If buffer is more basic than the pI, it becomes negatively
sampled macromolecules which may charged and migrates toward the anode
cause molecules to slow down, not ○ Loses hydrogen ions
move, or pushed farther (paspas ang ● Take note of the term isoelectric point = pI
mobility), ● Isoelectric point is the pH at which a particular molecule
■ Agarose gel doesn’t bind protein thus carries no net electric charge
migration is unaffected ● Take note that buffer solution should have carefully
controlled ionic strength
○ Polyacrylamide gel ● Ionic strength is the measure of the concentration of ions
■ Referred to as page in the solution
■ Layers of gel with different pore sizes ● Remember that during electrophoresis, ions cluster
are used around the migrating particle
■ Separates protein based on its charge ○ The higher the ionic strength/concentration, the
and molecular size, hence it uses bigger the size of the ionic cloud, making its
layers of prepared gel with different mobility slower
pore sizes ○ The lower the ionic strength/concentration, the
■ Unique about it is its ability to more the current is carried by the proteins
separate serum proteins into 20 or which causes faster mobility
more fractions rather than the usual ■ The longer the distance, the sharper
five fractions separated by cellulose the protein band separation
acetate or agarose Examples of buffers used in electrophoresis
■ Very useful and widely used in
studying individual proteins such as Buffers Used for
isoenzymes Barbitone Buffer (around 8.0 ● Serum protein separation
pH) ● Poor resolution, weak buffer
○ Starch
■ Separates proteins on the basis of Phosphate Buffer (around 7.0 ● Enzyme separation
surface charge and molecular size pH) ● Low buffering capacity, high
just like the polyacrylamide gel conductivity
■ Not widely used because preparation Tris-borate-EDTA buffer (TBE) ● Nucleic acid separation
is difficult -(pH around 8.)) ● Good resolution, high
buffering capacity, low
Agarose Polyacrylamide Gel conductivity
● Smaller DNA fragments
● Polysaccharide ● Cross-linked polymer
extracted from of acrylamide Tris-acetate-EDTA buffer (TAE) ● Nucleic acid separation
seaweed ○ Cross-linker (pH around 8.0) ● High resolution, high
● Gel casted horizontally such as buffering capacity, low
● Non-toxic bisacrylamide conductivity
● Separate large ● Gel casted vertically ● Used in agarose gel
molecules ● Potent neuro-toxic electrophoresis
● Commonly used for ● Separate small Tris-glycine buffer- (pH more ● Protein separation
DNA separations molecules than 8.0) ● High buffering capacity, low
● Staining can be done ● Used for DNA or conductivity
before or pouring the protein separations
gel ● Staining can be done
after pouring the gel Stains
● Gel casting ● Since the ions have already migrated to specific
○ Once supporting medium is already prepared, electrodes and electrophoresis is already completed, the
they are held in a casting tray supporting medium is treated with a dye or stain to help
○ The gel casted in trays provides a place where identify the separated fractions
we put our samples to test ● Help visualize the bands and macromolecules such as
proteins that have already migrated
Components of Electrophoresis ● Allows us to visualize the locations of the separated
particles
Buffer ● There are also methods where the sample is already
● Provides ions and mixed with a tracking dye
○ Allows current to be carried through the sample ● While the molecules migrate, we can visualize the
○ The buffer resists pH changes in the overall fractions or the separated molecules such as nucleic
solutions acids
○ Buffer maintains the pH at a relatively constant ● In agarose gel electrophoresis, Ethidium bromide is used
value as a dye and binds to the DNA
● AMPHOLYTE is a molecule whose net charge can be
either positive or negative Stains Used for
○ Ampholytes contains both acidic and basic Amido Black
groups Coomassie Proteins
○ These amphoteric molecules can either be Brilliant blue
positively or negatively charged Bromphenol Blue
○ Help establish a stable pH gradient and assist
Ethidium bromide Enzyme separation
molecules in migration
Sybr green or Sybr Gold DNA
○ Two properties affecting ampholyte
■ pH Sudan Black Lipoproteins
3
Ponceau red sample allowing the particles such as DNA to separate
Amido black Hemoglobins
Coomassie
Brilliant blue
● The photo shows how we did electrophoresis particularly

● Agarose gel is placed in the chamber and in this process,
the agarose gel electrophoresis
the gels are arranged in a way that the wells are closest
● Mateiaals are prepared
to the negative cathode indicated by the black color
● Includes
● Since the DNA is negatively charged, it will move from
○ Casting tray- supporting medium is placed
the negatively charged cathode to the positively charged
○ Comb- used to create wells where the sample
anode indicated by the red color
is placed
● Buffer is added to the reservoir of each chamber until the
○ Power supply- provide the constant electric
wells of the chambered are covered of at least 2
current required
millimeters
○ Sub-cell- containing the supporting medium
● Agarose gel that we need to prepare first is prepared by
mixing buffer to the desired concentration then we heat it
in a microwave oven until it has completely melted
● Once the agarose gel solution is melted it is allowed to
cool at about 60 degrees celsius
● Poured into the casting tray containing the sample comb
● Solution is allowed to solidify at room temp
● Once agarose gel solidifies, the comb is carefully
removed making sure the wells are intact
● Other materials:
○ Pipette
○ Graduated cylinder ● Samples are arranged in an order according to the lanes
○ PPE that they are assigned to be running as indicated by the
lab protocol
● Samples may contain proteins, DNA, etc.
● Use the pipette to obtain the sample such as the
extracted DNA in this experiment
● Once gel is prepared, pour the agarose solution in the

casting tray and the comb is pushed down into the gel to
from wells ● Observe proper use of a pipette
● Allow to solidify and remove the comb carefully ● Samples are loaded properly on the well
● Once ready, the DNA fragments are loaded to each well ● Pipette tip must be perpendicular to the row of wells and
sing micropipette the other parts of the gel will not be punctured
● Gel plate is immersed in charged buffer solution ● The tip of the pipette is lowered below the surface of the
buffer just above or inside the well
● This is a subcell that contains an electrode wire across

the bottom of each end of the subcell. With this, the
electric current will pass through the medium to the ● The subcell containing the samples bust not be bumped
4
or moved drastically so that the samples will not mix through the light beam at fixed rate
● Lid is placed
● Black to black and red to red NORMAL SERUM PROTEIN ELECTROPHORESIS
● Connect to power supply

● Timer is set as required by the protocol
● Once set, press the start button
● Bubbles will appear form the wires
● Samples will migrate from the wells through the gels
● If the electrophoresis is done at a very long time, the ● Albumin
proteins or particles may migrate off or beyond the gel ○ Most negative and will migrate farthest
into the buffer ● α1-Globulins
○ Α1-fetoprotein
AGAROSE GEL ELECTROPHORESIS ○ Α-antitrypsin
○ HDL
DENSITOMETRY ● α2-Globulin
● It measures the absorbance of stain-concentration of the ○ Haptoglobin
dye and protein fraction. ○ Ceruloplasmin
● It scans and quantitate electrophoretic pattern ○ Α2-macroglobulin
● β-Globulins
○ Transferrin
○ C-reactive protein
● γ-Globulins
○ immunoglobulins
Refractometry
● Can be used to measure protein concentrations, specific
gravity of urine, and column influence of high
performance liquid chromatography
● Determines the concentration of dissolved particles in a
Basic Components
specimen by measuring the refractive index
1. light source
2. Monochromator
3. movable carriage - will scan the medium over the entire
area
4. Photodetector - optical system
5. read-out device
● based on refractivity (the ability of the substance to light)

● when the light passes from one medium into another, the
path of the light beam changes direction at the boundary
surface if its speed in the second medium is different
from that in the first
● Refractive index
○ the ratio of the two speed of light
○ Comparison of the velocity of light in air and the
velocity of light in the solutions
● In densitometry, the supporting medium is moved
5
● Coulometry
○ is an electrochemical titration in which the
titrant is electrochemically generated and the
endpoint is detected by amperometry
● Amperometry
ELECTROCHEMISTRY
● involves the measurement of electrical signals
associated with chemical system that are incorporated
into an electrochemical cell
● the magnitude of a voltage or current signal originating
from an electrochemical cell is related to the activity or
concentration of a particular chemical species in the cell
ELECTROCHEMICAL CELL ● The change in voltage, indicates activity of each analyte

● Used to measure analyte concentration
● the measured potential is related to the molar
concentration by the Nernst equation
○ Describes the electromotive force generated
because of hydrogen ion at the glass tip
● How do we measure electrical potention?
○ ELECTRODES
a. Reference Electrode
■ Also known as reference
potential
■ Has a constant and known
voltage
■ Produces constant potential
● A galvanic cell converts chemical energy into electrical b. Indicator Electrode
energy ■ responds to changes in the
● Here the redox reaction is spontaneous and is activity of solution
responsible for the production of electrical energy ■ measuring electrode
● The two half-cells are set up in different containers, being ➢ The potential difference between
connected through the salt bridge or porous partition these two electrodes are measured
● Here the anode is negative and cathode is the positive ➢ Related to the molecular
electrode. The reaction at the anode is oxidation and concentration of ions in a solution
that at the cathode is reduction ● REFERENCE ELECTRODE
● The electrons are supplied by the species getting ➢ An ideal RE should conform to the Nernst equation,
oxidized. They move from anode to the cathode in the exhibit a potential that is constant with time, and
external circuit return to original potential after being subjected to
● Liquid junction - also known as a salt bridge are small currents
required to complete the circuit between the reference ➢ Insensitive to the changes in the solution
and without contaminating anything ➢ Exhibits little hysteresis such as lags with
○ Functions temperature cycling
■ It allows electrical contact between ➢ Standard Hydrogen Electrode (SHE) has those four
the two solutions characteristics, but not for practical means
■ It prevents the mixing of the electrode ○ Calomel Electrode
solutions ■ Reference electrode
■ It maintains the electrical neutrality in ■ mercury/mercurous chloride
each half cell as ions flow into and out ■ Composed of mercury in contact with
of the salt bridge a solution saturated with mercurous
○ Purpose: not to move electrons from the chloride plus potassium chloride
electrolyte, but rather to maintain charge ○ Silver/silver chloride
balance because the electrons are moving from ■ Reference electrode
one half cell to another ■ overall better and faster
■ Consists of silver electrode immersed
Electrochemical Techniques in a solution of potassium chloride
● that has been saturated with silver
○ the measurement of the electrical current at a chloride
single applied potential ○ Normal hydrogen electrode
● Voltammetry
○ based on the current potential relationship in an Ion-selective Electrode
electrochemical cell when the potential is ● indicator electrode that can respond to individual types of
applied anions or cations
● very sensitive and selective for the ion it measures
Potentiometry ● Composed of a membrane or barrier between the
● measurement of a potential or voltage difference reference solution and reference electrode
between two electrodes immersed in solution under the ● 2 types of ISE:
condition of essentially zero cuPotentiometry 1. Direct ISE
○ measurement of a cell potential (voltage) under ○ Does not have sample dilution
equilibrium conditions 2. Indirect ISE
○ has sample dilution
6
● Types of ISE
1. Glass electrodes
2. Liquid membrane electrodes
3. Precipitate impregnated membrane electrodes
4. Solid state electrodes
5. Gas electrodes
6. Enzyme electrodes
● Its ionic selectivity depends on the membrane/barrier
composition used:
a. glass aluminum silicate (Sodium)
b. valinomycin gel (Potassium)
c. organic liquid membrane ion exchangers
(Calcium & Lithium) Osmometry
d. gas and enzyme electrodes
● measurement of the osmolality of an aqueous solution
such as serum, plasma and urine; measurement of the
Uses:
concentration of dissolve solute particles in a solution
● pH electrode
● Osmolality of a solution is related to its colligative
○ selective for the detection of hydrogen ions
properties such as osmotic pressure, boiling point,
○ indicator electrode has a glass membrane
freezing point, and vapor pressure.
○ Indicator: Glass electrodes contain small bulbs
● based on measuring changes in the colligative properties
of glass which contains a chloride ion buffer
of solution that occur owing to variations in particle
solution
concentration that is related to the osmotic pressure of
○ internal reference electrode: Ag/AgCl
the solution
○ external reference electrode: Calomel electrode
● COLLIGATIVE PROPERTIES:
● pCO2 electrode
○ Depend on the concentration of solute
○ pH electrode contained within a plastic jacket.
molecules or ions.
○ sodium buffer and permeable membrane
○ Not affected by the mass, size, and type of
(Teflon or silicone)
molecule.
1. osmotic pressure
Voltammetry 2. boiling point
● The measurement of current after which a potential is 3. freezing point
applied to an electrochemical cell. 4. vapor pressure
● Anodic stripping for lead and iron Testing ● Osmotic particles
● Sensitive and uses minimal analyte. ○ Glucose
Three Electrodes: ○ Urea
A. WORKING ELECTRODE ○ Nitrogen
● makes contact with the analyte ○ Sodium
● facilitate the transfer of charge to and from the ■ If added into the sol’n osmolality
analyte increases and 4 colligative are
B. REFERENCE ELECTRODE affected.
● a half cell with a known reduction potential ● ↑Osmotic and boiling
C. AUXILIARY ELECTRODE ● ↓Freezing point and vapor
● to sustain electrolysis pressure
● Process by which electric current is passed
through a substance to effect a chemical Osmotic Pressure
change (substance loses or gains an electron)
Coulometry
● Measure of amount of electricity in coulombs .
● Involves the application of a constant current generate a
titrating agent
● is an electrochemical titration in which the titrant is
electrochemically generated and the endpoint is detected
by amperometry
● the time required to titrate a sample at a constant current
is measured and is related to the amount of analyte in a
sample by Faraday’s equation.
○ Faraday’s equation. - chloride testing in CSF,
serum, and sweat.
● Minimum pressure which needs to be applied to a
Amperometry
solution to prevent inward flow of pure solvent across a
● is the measurement of the current flow produced by an semi permeable membrane
oxidation–reduction reaction at a single applied potential
Vapor Pressure
● a measure of the cell current when the potential
difference between indicator and reference electrodes is
controlled ● Measure of the tendency material to change into a
● We use it to determine p02, glucose, Cl, and peroxidase. gaseous or vapor state.
○ pO2 gas-sensing electrode Electrical Impedance
○ GLucose electrode ● measurement is based on the change in electrical
resistance across an aperture when a particle in
conductive liquid passes through this aperture.
● Measure resistance produced for each particle.
● Used primarily in the hematology laboratory to count
leukocytes, erythrocytes and platelets.
7
BECKMAN-COULTER CHEMISTRY ANALYZER
● Counts size particles by measuring impedance or

resistance.
ABOT CELL DYN
● Size of pulses is directly proportional to cell size

● Number of pulses is directly proportional to cell count.
● 2-21 fL= platelets
● >30 fL = RB
● > 35 fL = leukocytes.
COULTER MACHINES
● Measure electrical impedance or resistance

● Uses coulter principle
○ Detects and measures electrical impedance
produced by a blood cell as it passes thru an
aperture
8
LECTURE 4: AUTOMATION
August 6, 2021
PART ONE
TERMINOLOGIES AUTOMATION
● Describe the process whereby an analytical instrument
Automation performs many tests with only minimal involvement of an
● The process whereby an analytical instrument analyst
performs many tests with only minimal involvement of ● Enable laboratories to process much larger workloads
an analyst; also defined as the controlled operation of without comparable increases in staff
an apparatus, process, or system by mechanical or ○ Somehow reduces the staff and lesser labor for
electronic devices without human intervention. the staff
Batch analysis ● Used for:
● Type of analysis in which many specimens are ○ Test performance
grouped in the same analytical session. ○ Processing and transport of specimens
Carry-over ○ Loading of specimens into automated analyzers
● The transport of a quantity of analyte or reagent from ○ Assessing the results of the tests performed
one specimen reaction into and contaminating a HISTORY
subsequent one. ● First automated analyzer
Continuous-flow analysis ○ “Autoanalyzer” by Technicon in 1957
● Type of analysis in which each specimen in a batch ○ continuous-flow, single-channel, sequential
passes through the same continuous stream at the batch analyzer
same rate and is subjected to the same analytical ○ Single test result on approximately 40 samples
reactions. per hour
Discrete analysis ● First commercial centrifugal analyzer: a spin-off
● Type of analysis in which the sample is aspirated into technology from NASA outer space research (1970)
the sample probe and then is delivered, often with ○ Was developed by Dr. Norman G. Anderson
reagent,through the same orifice into a reaction cup or ○ A prototype was created in the Oak Ridge
another container. National Laboratory
Multiple-channel analysis ● Automatic Clinical Analyzer (ACA) (DuPont, now
● Type of analysis in which each specimen is subjected Siemens) in 1970
to multiple analytical processes so that a set of test ○ First non-continuous flow, discrete analyzer
results is obtained on a single specimen; similar to ○ first instrument to have random-access
random-access analysis. capabilities
Parallel analysis ○ STAT specimens could be analyzed out of
● Type of analysis in which all specimens are subjected sequence on an as-needed basis
to a series of analytical processes at the same time ■ Different compartment where u can
and in a parallel fashion. able to place STAT samples
Random-access analysis ■ STAT = short turnaround time
● The most common configuration of an automated ● 1976: production of thin film analysis technology
analyser, in which analyses are performed on a ○ Uses a very thin film kung saan ilagay ang
collection of specimens sequentially and each sample and kung saan tanan processing na
specimen is analyzed for a different selection of tests. mahitabo and reading
Sequential analysis
● Type of analysis in which each specimen in a batch
enters the analytical process one after another, and
each result or set of results emerges in the same
order as the specimens are entered.
Single-channel analysis
● Type of analysis in which each specimen is subjected
to a single process so that only results for a single
analyte are produced; similar to batch analysis.
Throughput
● The number of specimens processed by an analyzer ● Kodak Ektachem (now VITROS) analyzer (now
during a given period of time, or the rate at which an ortho-clinical diagnosis) in 1978
analytical system processes specimens. ○ First to use microsample volumes and reagents
Workstation on slides for dry chemistry analysis
● A clinical laboratory workstation dedicated to a defined ○ First to incorporate computer technology
task and contains appropriate laboratory extensively into its design and use
instrumentation to carry out that task. ■ Comparing it to the autoanalyzer
where you can see the processing
(whats inside), this is built in or
covered, you can only see computer
and sample holder
1
○ Transcription of results
● Use of very small amount of reagents and samples
○ Allows less blood drawn from each patient
○ Small amounts of reagents decrease the cost
of consumables
Relevant Terms
● Dwell time
○ Minimum time from initial sampling to the
production of a result
● Throughput
○ The maximum number of test results that can
be produced by an analyzer in a given time
period (usually an hour)
● STAT Analysis
Automated System Designs

● Total Laboratory Designs
● Modular Integrated system
● Stand-alone systems
TOTAL LABORATORY AUTOMATION

● Employs an integrated track system that links all the
laboratory’s workstations together to create a continuous,
comprehensive network that automates almost all the
steps involved in laboratory testing
● Why do we need to process using automated machines?

○ Staff shortages
■ Manual processing of plenty patient
samples
○ Turnaround time demands
■ In manual, one patient may take an
hour
■ In automation, built-in and settings,
TAT will lessen
○ Specimen integrity
■ Samples that aren't processed ● Form the time you received the saamle and eveen ang
immediately may lose its integrity paaghataag sa inyong different sample, machine na
○ Safety ● No need for manual transfer of sample
■ Manual processing may expose you
to harmful chemicals Advantages
● Decrease in labeling errors
○ There are some automated machines that they
Advantages could also check if they are labeling errors
● Human factor is decreased during the mechanical and ● Reduced turn-around time
repetitive part of an assay ● Cost-savinng+reduction in labor
○ In manual processing, human factors are ● Reduction in full-time equivalents (FTEs)
common error ○ Are the hours worked on one employee on a
● Increase the number of tests performed by one full-time basis
laboratorian in a given period
● Minimize variation in results from one laboratorian to Major Limitations
another ● Needs substantial financial investment and increased
○ Big variation sa test in every result floor space (Wilson, 2003)
○ Coefficient of variation is lowered and ● Need for highly technical personnel to operate and
reproducibility is increased troubleshoot the system
● The accuracy is not dependent on the skill or workload of ● Personnel team building
a particular operator on a particular day ● Lesser interaction with the staff
○ Our role is to take care of the machines ● Infrastructure remodeling
○ Proper maintenance, calibrations, standards,
running of controls just to make sure na correct ROCHE DIAGNOSTICS SYSTEM
pud and gina release sa atong machine ● Modular pre-analytics (MPA) and platform C (i.e., the
● Eliminates the potential errors of manual analyses such chemistry analyzer
as ● Consists of an integrated tract device that connects all of
○ Volumetric pipetting stes the laboratory workstations, including front-end
○ Calculation of results processing, instrumentation, and archiving, to create a
2
continuous, inclusive network that serves to automate
nearly every step involved in the testing of each sample ADVANTAGES:
● Uniformity in the performance of tests
○ Each sample follows the same reaction path
DISADVANTAGES:
● significant carryover problems and wasteful use of
continuously flowing reagents
○ Major problem = carryover
● The machine does not allow test selection; all tests must
be performed even if not requested
○ Costly thus not being used
MODULAR INTEGRATED SYSTEMS
● link together multiple laboratory disciplines into a single
testing platform that is interconnected by a track
● Less capital investment than TLA
● Has a module
● For a specific section only
● Since taga sample, separated lang siya with air bubbles

(space bubbles to separate one sample from another)
● One reaction sa tanan
● SYSMEX
STAND-ALONE SYSTEMS
● to automate specific sections of the process that are still
manual operations.
○ Specimen processing
○ Sample archiving
● That's why carry over will be common with this type of

analyzer
● Observe the number of staff: ● Air bubbles in the tubings

○ Stand-alone = more staff ● Using only one needle to analyzes one sample to
○ By module = less staff another
● One tubing and reaction vessels in the machine
CLASSIFICATION OF AUTOMATED ANALYZERS
● Continuous flow analysis DISCRETE ANALYZER
● Discrete analysis
● The sample travel through the instrument in its own
● Centrifugal analysis
reaction vessel
● Sequential analysis
● Each test reaction takes place in a separate
● Batch analysis
compartment that is either cleaned out or disposed of
● Parallel analysis
after use
● Random access analysis
● Have the capability of running multiple tests one sample
at a time or multiple samples one test at a time
● Keeps sample and reaction carryover to a minimum but
CONTINUOUS FLOW ANALYZER
increases the cost per test due to disposable products
● the samples flow through a common reaction vessel or (like plastic cuvette)
pathway ● Most commonly used compared to CFA and most
● liquids (reagents, diluents, and samples) are pumped versatile type of analyzer
through a system of continuous tubing ● developed as a result of space-aged technology and
● assists the laboratory that needs to run many samples were one of the first types of discrete analyzers
requiring the same procedure ● Samples and reagents were mixed together, reacted, and
● An oil heating bath is used to promote color development flowed by centrifugal force into separate cuvettes in
or the completion of enzymatic reaction which spectrophotometric analysis could occur
● Colorimetric method; color development
3
● DISADVANTAGE: Only one test type can be performed CENTRIFUGAL ANALYZER
each time ● developed as a result of space-aged technology and
were one of the first
● Samples and reagents were mixed together, reacted, and
flowed by centrifugal force into separate cuvettes in
which spectrophotometric analysis could occur
ADVANTAGES
● Running multiples samples at a time (batch analysis
DISADVANTAGE:
● Only one test type can be performed each time.
Similar to discrete but differs in way of processing is thru
centrifugal force
Classification of Automated Analyzers

Skalar SEQUENTIAL
● Cuvette with optical path-length: 15 mm ● performing a set of test reactions in a particular order on
● 640 tests running without operator intervention each sample in the order in which it is received
● 100 sample positions BATCH
● 32 positions for reagents, (stock) standards and QC's ● all samples are loaded at the same time, and a single
● Possibility to add priority samples during a run test is conducted on each sample.
● Automatic preparations of working standards PARALLEL
● Sample pre- and post-dilution functions ● more than one test is analyzed concurrently on a given
● 8 optical filters ranging from 340 - 900 nm clinical system
● Disposable cuvette blocks RANDOM ACCESS
● Segregation of chemical waste ● any test can be performed on any sample in any
sequence
PART TWO
SPECIMEN PREPARATION AND IDENTIFICATION

SUMMARY OF CHEMISTRY ANALYZER OPERATIONS
IDENTIFICATION AND PREPARATION Technologies Used for Automatic Identification and Data
Collection
1. Sample This is usually done by
identification reading the bar code. This ● Bar coding
information can also ● Optical character recognition
be entered manually ● Magnetic stripe and magnetic ink character
recognition
● Voice identification
2. Determine test(s) The LIS communicates to the
● Radio frequency identification
to perform analyzer which test(s) have
● Touch screens
been ordered
● Light pens
● Hand print tablets
● Optical mark readers
CHEMICAL REACTION ● Smart cards
3. Reagent systems One or more reagents can be

and delivery dispensed into the reaction
cuvette
4. Specimen A small aliquot of the sample

measurement and is introduced into the reaction
delivery cuvette
5. Chemical reaction The sample and reagents are

phase mixed and incubated
DATA COLLECTION AND ANALYSIS
6. Measurement Optical readings may be

phase initiated before or after all ● Optical character recognition - uses a pen; i agri ra sa
reagents have been pen then ma recognize na
added ● Magnetic identification character recognition (MICR) -
very common sa mga check
7. Signal processing The analyte concentration is ● Optical marker recognition - ex. scantron
and data handling estimated from a calibration
curve that is EMERGING TECHNOLOGIES
stored in the analyzer ● Automated specimen inspection
○ Identify sample identification errors and sample
8. Send result(s) to The analyzer communicates integrity issues
LIS results for the ordered tests to ■ Ex. hemolyzed sample - it is able to
the LIS recognize
○ Could sort a random collection of different-sized
containers with different additives
4
● Assess each sample for proper labeling, adequate ■ Eliminates the need both to wait for
volume and interference from icterus, lipemia or the sample to clot and to aliquot the
hemolysis sample
● Radio-Frequency IDentification ○ Barcode-labeled tubes
○ Passive and without active human intervention ■ Scanning then transport
○ Advantages:
■ No line of sight reading SPECIMEN LOADING AND ASPIRATION
Ma scan gihapon maskin di ● Circular carousel o rectangular racks as specimen
nakatutok containers
■ Dynamic data storage ● Trays or racks are numbers to aid in sample
■ Not affected by cold storage identification and move automatically in one-position
○ Disadvantages: steps at preselected speeds
■ Yet to be commercialized on a large ○ Even trays have barcodes so when you set or
scale in the clinical laboratory encode samples in the computer, inyoha siyang
■ Greater cost iset-up in line kung unsa ang ga barcode sa
racks to sill have proper identification sa
SPECIMEN DELIVERY specimens
● Courier service ● The instrument can determine the slot number containing
○ Not common in sending your different samples the last sample and terminate the analysis after that
especially when it needs confirmatory testing sample
○ The hospital can deliver patient samples for ○ By the time it will reach the last specimen, the
confirmatory testing whole process stop
● Pneumatic tube systems ● The instrument’s microprocessor holds the number of
○ Mechanical problems in the switching process samples in memory and aspirates only in positions
have been known to cause misrouting of containing the samples
carriers
■ Misroute of samples
○ Prone to hemolysis (Avoid sudden acceleration
and deceleration and use of proper packing
material)
■ Can be damaged (affects specimen
integrity)
○ Rarely used because of the disadvantage
● Electric Track Vehicles
○ Larger carrying capacity than pneumatic tube
systems
○ Not associated with problems such as damage
to specimens caused by acceleration and/or
declaration forces
● Mobile robots
○ Delivery of specimens to lab benches by a ○ Lower left picture- circular carousel
mobile robot is usually more frequent than ○ Upper right picture- rectangular racks
human pickup and has been shown to be ○ Lower right picture- loading zone of auto
cost-effective analyzers
● Actual measurement off each aliquot for each test must
be very accurate and is generally done through
aspiration of the sample into a probe
○ There are some like tubes lanng ang
ginabutang sa rack, there are some machines
na dapat aang inyong serum kay up lang to a
certain level
● Tubes are typically decapped before sampling
○ Unicell Analyzer (Beckkman Coulter Inc., Brea,
Calif.) offer cap-piercing technology
○ Hematology analyzers
SPECIMEN PREPARATION AND IDENTIFICATION ○ There are machines na sila na ang ga mix
● Preparation of the sample for analysis has been and ● Wash Solution: deionized water (sometimes with
remains a manual process in most laboratories surfactant)
● Alternatives: ○ Important and dapat dili mabutangan ppa ug
○ Use of robotics or front-end automation othe reagents
○ Bypass specimen preparation by using whole ○ Since this is necessary kay every after mukua
blood for analysis ug sample ang probe, iyaha nanghugas before
■ It takes a lot of time to get a sample magkuha ug sample sa another patient
(clot of blood, centrifuge takes about ○ Common Problem: carryover
15-20 mins) ■ Carries one sample to the other,
■ To lessen the time, its best to use a which lessen the accuracy of results
whole blood analysis (has pros and ● Loading zone: area in which specimens are held inside
cons) the instrument before they are analyzed
■ There are substances in the body that
its levels are better in the serum Specimen Processing
■ There are autoanalyzers as of the ● Autoanalyzer should be capable of removing protein and
moment that use whole blood (ex. other interferents
ABG) ○ Dialysis
● Nakaset up na machines ■ Process of removing excess water,
○ Use of plasma separator tube and perform solutes, or toxins in the body
primary tube sampling with heparin plasma ○ Common chroatography
5
○ Filtration interfere with results
○ Protein is a very common interferent ● Reagent layer - reagent reacts with sample
○ One problem it poses is its size which is 3. Indicator layer - reacted sample collects for spectral
common to blocks other substances. analysis
○ Common causes of blockage on tubes, probes ● Support layer - optical interface; serves as exit slit
in the analyzer, especially if Dili mag clean first
thing in the morning Dry chemistry slide
○ Pre-treatment removes interferences ● The reagent layer contains; enzymes, dye precursors,
and buffers necessary for the analysis of a specific
REAGENT SYSTEMS AND DELIVERY component
● Open vs Closed system analyzer ● Sample, control, or standard is deposited on the
○ Open-system analyzer spreading layer
■ operator is able to change the ● Selected components are allowed to penetrate to the
parameters related to an analysis and reaction layer(s), which in turn activate the dehydrated
prepare “in-house” reagents or use reagents
reagents from a variety of suppliers ● Less laborious
■ Flexible and adapt readily to new
methods and analytes Between your liquid and dry reagent, it would always be best to
○ Closed-system analyzer have a dry reagent because:
■ requires the reagent to be in a unique ● Longer shelf life
container or format provided by the
manufacturer Techniques of preservation:
■ common ● Keep all reagents refrigerated until the moment of need
and then quickly preincubate them to reaction
● liquid reagents for open systems are less expensive than temperature or store them in a refrigerated compartment
the closed analyzers on the analyzer that feeds directly to the dispensing area
● closed systems contain a hidden cost advantage ● Common mistake:
because reconstitution or preparation of reagents for use ○ Do not immediately use your reagents after
does not require a technologist’s time getting it from the refrigerator
● Liquid reagent ● Provide reagents in a dried, tablet form and reconstitute
● Dry reagent them when the test is to be run
○ May be bottled as lyophilized powder ● Manufacture the reagent in two stable components that
■ Freeze-dried powder will be combined at the moment of reaction
○ Requires reconstitution with water or a buffer ○ Instead of combining immediately the reagents
○ Dry chemistry slide (another type of dry in one bottle, better to separate it first and mix
reagent)
■ have microscopically thin layers of dry Reagent delivery techniques:
reagents mounted on a plastic
support ● Sa machine na nimo on how it would get samples
■ slides are approximately the size and ● Syringes: driven by a stepping motor, pipet the reagents
thickness of a postage stamp into reaction containers
■ Example of thin film analysis ● Piston-driven pumps: connected by tubing, may also
dispense reagents
● Use of pressurized reagent bottles connected by
tubing to dispensing valves
○ The computer controls the opening and closing
of the valves
○ The fill volume of reagent into the reaction
container is determined by the precise amount
of time the valve remains open
CHEMICAL REACTION PHASE

● consists of:
○ Mixing - most difficult part
● Dry reagent ○ Forceful dispensing
● Each of these pads is already incorporated with reagents ○ Magnetic stirring
necessary to identify what needs to be identified ○ Vigorous lateral displacement
■ discrete analyzer - positive
displacement
■ Magnetic stirring
○ Rotating paddle
○ Ultrasonic energy
● Separation
○ In relation to removing interferences
○ Use a very high reagent-to-sample ratio
■ Use very diluted sample so that any
turbidity caused by precipitated
protein cannot be sensed by the
spectrum
○ Shorten reaction time to eliminate
slower-reacting interferents
● Incubation
3 layers of the dry slide technology: ○ Some testing process requires 37 C
1. Spreading layer -sample is distributed evenly ○ Heating bath
2. Central layer ■ maintains the required temperature of
● Scavenger layer - filters out substances that the reaction mixture
6
■ provides the delay necessary to allow
complete color development
■ Components: heat-transfer medium,
heating element and thermoregulator
● Reaction time
○ Completion of reaction
○ Rate at which the reaction is proceeding
○ One way of calculating the amount or level of
certain substances
MEASUREMENT PHASE
● UV light, fluorescent, flame photometry, ion-selective
electrodes, gamma counters and luminometers are
various methods for measuring product formed
● Most common methods are still visible and UV
spectrophotometer
● Analyzers that measure light need monochromators to
produce specific wavelength, classically filter wheels
have been used to separate light, which are usually
computer controller
Signal Processing and Data Handling

● Accurate calibration is essential to obtain reliable results
○ Assurance sa atoa na accurate ang
measurements
○ Calibration is done though comparing a known
substance (standard) to the measurement used
in the instrument
● This requires proper use of standards, which then reflect
the data on standard curve according to which the
sample results are interpreted
○ Standards: solutions containing precisely
known concentration of a substance
● After the calibration has been performed and the
chemical or electrical analysis of the specimen is either in
progress or completed, the instruments computer goes
into data acquisition and calculation mode
● This process may involve signal averaging which may
involve hundred of data pulses per second
Future trends in Automation

● Automation will continue to evolve.
● System integration and miniaturization with more
technologically advanced computer power will persist to
accommodate more portable analyzers for more precise
testing.
● Automated analyzers will have artificial intelligence
where by the computer will “think” or make decisions if
sufficiently programmed with infinite scenarios of data.
● Spectral mapping or multiple wavelength monitoring, with
high-resolution photometers and polychromators will
become standard.
7
UNIT
% g/100mL
Valence eq/mole
MW g/mole
M Moles/L
N eq/L
UNIT FORMULAS

𝑥100

𝑥100

𝑥100
● %
PERCENT ● % w/v
● %v/v 𝑔 = 100 𝑚𝐿
𝑥 (𝑚𝐿)
𝑚𝐿 = 100
𝑥 (𝑔)
𝑔 = 100𝑔
● M
● moles/Liter
● moles/kg
MOLALITY
● m

𝑀𝑊
NORMALITY ● N
or

𝑁 = 𝑀𝑊
LECTURE 1: QC/QA PART 2

Prof. Fritdey Jad Doctolero, RMT
September 27.2021
MEAN
● The mean (or average) is the laboratory’s best estimate LEVEY JENNINGS CHART
of the analyte’s true value for a specific level of control.
x=Σxi/n
● Where:
○ xi= each data point
○ n= the number of data points in the set
● Add the data values and the sum of it will be divided to
the number of data values
Glucose QC Values using Level 1 as control material:

Day 1 4.5
Day 2 4.7
Day 3 4.6
● Described as a graph of all the quality control results in a
Day 4 4.4 month
○ Why month?
Day 5 4.6 ■ In a clinical lab, dapat maka run ng
minimum of 20 controls
● Y-axis = concentrations
22.8 / 5 runs = 4.56 Mean ● Middle = mean
● Higher limit (above mean) = +1SD,+2SD, +3SD
STANDARD DEVIATION ● Lower limit (below the mean) = -1SD, -2SD, -3SD
● Standard deviation quantifies how close numerical values ● X-axis = day kung kelan gi run
are in relation to each other. ● Others dont use days to label the x-axis, some consider it
● Also used to set up limits as the number of run they did
● Ex. if u have acquired a low SD, then that implies that the ○ Ex, First run of quality control
data values/points are very close to the mean ○ Important to observe the levey jennings chart
● If u have a high SD, it implies that the data values are presented
spread out over a large range of values ○ Some uses days - specially on those
2 laboratories who run control materials/day
Σ(𝑋𝑛−𝑀𝑒𝑎𝑛) ○ Remember, theoretically hindi dapat per day
𝑆𝐷 = 𝑛−1 lang ginarun ang control, dapat at the beginning
of each shift
Limits
■ 3 shifts = 7-3, 3-11, 11-7
FORMULA: (MEAN) +/- (SD) (1) FOR 1SD ■ In one day, exactly 3 times irun ang
control
● LOWER LIMIT 4.56 - (0.1)(1) = 4.56 - 0.1 = 4.46 ■ In the real setting, once lang. Unless
● HIGHER LIMIT 4.56 + (0.1)(1) = 4.56 + 0.1 = 4.66 there are problems and it's in a big
hospital, run the control more than
FORMULA: (Mean) +/- (SD) (2) for 2SD once.
○ Aside from the mean, standard deviation, and
● LOWER LIMIT 4.56 - (0.1) (2) =4.56 - 0.2 = 4.36 solving of limits -- you also need to remember
● HIGHER LIMIT 4.56 + (0.1) (2) = 4.56 + 0.2 = 4.76 the concept of coefficient of variation
Coefficient of Variation
FORMULA: (Mean) +/- (SD) (3) for 3SD
● The coefficient of variation (CV) is a measure of
● LOWER LIMIT4.56 - (0.1) (3) = 4.56 - 0.3= 4.26 variability
● HIGHER LIMIT 4.56 + (0.1) (3) = 4.56 + 0.3 = 4.86 ● A method or instrument’s CV is expressed as a percent
● In the normal distribution of values, when we consider and is calculated as:
only 1SD, its is expected that 68.3% of the values will fall
within 1SD
○ 2SD = 95.5% CV (%) = ( 𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝐷𝑒𝑣𝑖𝑎𝑡𝑖𝑜𝑛
𝑀𝑒𝑎𝑛
)(100)
○ 3SD = 99.7%
0.1
Example: 4,56
(100) = 2. 19%
● Importance of CV
○ Coefficient of variation is useful for comparison
of precision for two different methods or
instruments..
1
○ It can also be used as a part of the Internal tinitingnan
Quality Control system when performing ● Better if sa consensus group na mean and standard
patients precision testing deviation
○ The lower the CV, the better. ● Since this is precision, we are just going to get the
○ Ex. You are assigned in hematology section inter-laboratory group CV
(you’re the section head) ● Ex. 10/5=2
■ You have 1 machine for CBC
■ A medrep approaches you
■ Gaano kaganda ang machine ng
medrep na yun?
■ To evaluate, use Coefficient of
Variation
■ Observe if the results of the machine Result Interpretation
is close to the results of the machine Less than 1 Acceptable performance; indicates that
in the lab precision is better than the peer group
■ If merong bago na machine, you can
use CV to compare the precision of 2 1 to 1.5 Acceptable to marginal performance; may
instruments (new and the one in the need to investigate test system imprecision
lab) 1.5 to 2.0 Marginal performance; may need to
○ You can use CV when you want to perform perform corrective action
patient’s precision testing
● Interpretations for the values of SDI and CVR s from
SDI (Standard Deviation Index) Bio-Rad
● A statistic that measures your lab’s bias relative to your
consensus group. An estimate of reliability Shift
● Is defined as abrupt changes in the control values
● Shifts in QC data represent a sudden and dramatic
𝑌𝑜𝑢𝑟 𝑚𝑒𝑎𝑛 − 𝐶𝑜𝑛𝑠𝑒𝑛𝑠𝑢𝑠 𝐺𝑟𝑜𝑢𝑝 𝑀𝑒𝑎𝑛 positive or negative charge in test system performance
SDI =
𝐶𝑜𝑛𝑠𝑒𝑛𝑠𝑢𝑠 𝐺𝑟𝑜𝑢𝑝 𝑆𝐷 ● What is the criteria for you to recognize na meron nang
100−98 shift?
Example:
2
= 1
○ If there are 6 or more QC results fall already on
one side of the mean, you can say that there is
● Reference method (consensus group)
already a shift
○ Usually, when calculating SDI, reference
○ If that happens, the recommendation of WHO is
method ang tinitingnan
to reject the results
○ Other references says that other from reference
method, you can also use Inter-laboratory
group
■ Results of different laboratories and
calculate for the mean and SD
○ Common - Reference method (standardized)
● SDI is useful in testing accuracy of a method
○ There may be modifications in the protocols or
SOPS, or you have own unique method in
performing a lab test
Result Interpretation
0 Ideal score; identical to your peer group
(+/-) 1 to 1.5 Acceptable to marginal performance;
may need to investigate test system
bias
(+/-) 1.5 to 2.0 Marginal performance; may need to
perform corrective action ● Malapit mo na masabi na shift siya because itong five
● 0 - in other words, this means that the data data vaues natin fall on the same side of the mean, nasa
values of the laboratory are in 100% agreement taas
with the consensus group/reference method. ● Pagdating sa Day 15, pag andito parin sa taas and QC
● You can say that the lab method you are doing result, then you can definitely say that there is already a
in the lab is accurate shift
● (+/-) 1.5 to 2.0 - this needs corrective action ● But if the 6th QC result falls sa baba, there is no need to
○ It implies that the method is already reject the results
giving inaccurate results ● Note: Regarding the example of a shift, I have said in the
○ Kay Bishop it’s 3 video that it lacks one data value for it to be a shift BUT
○ San nakuha ang 1.5-2? actually it isn't lacking. Day 8 should be counted as value
■ If i-search sa internet, may 1 since it's where data points are beginning to fall on one
isang company na mas strict side of the mean; then Day 9 as 2 and so on and so
ang kanila forth. I apologize if I have mislooked it. (Sir Fritdey, 2021)
CRV (Coefficient of Variation Ratio) Shifts may be caused by:

● A statistic that compares your lab’s precision to that of ● Sudden failure or change in the light source
other labs in a consensus group ● Change in reagent formulation
● Consensus group is still present but in this case, for your ● Change of reagent lot
CVR, only ginagamit is yung inter-laboratory group na ● Major instrument maintenance
CV hindi ang reference method ● Sudden change in incubation temperature (enzymes
○ Ang tinitingnan sa CVR is ang precision while only)
ang SDI is accuracy ng lab method ang ● Change in room temp. or humidity
● Failure in sampling reagent system and reagent dispense
2
system
● Inaccurate calibration/recalibration
● All of these affect the reading of results in some way
Trend
● indicates a gradual loss of reliability in the test system.
● Should be one side of the mean
● Trends are usually subtle.
● Example of trend
● This is a warning rule that is violated when a single
control observation is outside the ± 2s limits.
○ Not a reason for rejection
● This rule merely warns that random error or systematic
error may be present in the test system.]
22s Rule
● Violation indicates systematic error that occurs outside

● gradual the +/- 2SD
● Two consecutive controls exceed the mean by +/- 2SD
Trend may be caused by: ● There are laboratories who uses one chart where the two
● Deterioration of the instrument light source levels of control (abnormal and normal) can be seen, and
● Gradual accumulation of debris in sample/reagent tubing also those who separates the chart
● Gradual accumulation of debris on electrode surfaces
● Aging of reagents 41s Rule
● Gradual deterioration of control materials
● Gradual deterioration of incubator chamber temp.
● Gradual deterioration of light filter integrity
● Gradual deterioration of calibration
Trends and Shifts are indicative of a problem and these

should be investigated and corrected!
● This indicates systematic error
Error Detection ● 4 consecutive controls on one side of the mean exceeds
● Systematic Error +/-1SD
○ a trend or shift away from the laboratory mean
○ Not acceptable 10x Rule
○ Trend and shift
● Random Error
○ any random deviation away from the laboratory
mean
○ Shows no pattern
○ Noy likely to reoccur
○ Reject if the data value that shows the random
error exceeds ± 2SD
● This indicates systematic error
Number of Controls ● 10 consecutive controls on one side of the mean
● If one control:
○ accept results if control is within ± 2SD Sources of Systematic Error
○ The values are said to be in control if they fall ● Improper alignment of sample or reagent pipettes
within the ± 2SD resulting to change in reagent and calibrator volume.
■ If lampas, it is considered as out of ○ Calibrator, namali sa machine pag read kasi
control then reject the run and do nakulangan ng volume
corrective action ● Drift or shift in incubator chamber temperature,
● If 2 levels of controls deterioration of photometric light source
○ apply Westgard multirule system ○ Temperature can affect the rate of chemical
○ Usually one normal and one abnormal reactions
○ Light is a very important tool especially if the
Westgard Multirule System machine is using spectrophotometry
● a “multi-rule” system developed by Dr. James O. ● Change of Calibration /reagent lot
Westgard based on statistical concepts ● Deterioration of reagent/calibrator/control product while in
● a combination of decision criteria or rules to assess if a use, storage or shipment
system is in-control ● Incorrect handling of control product
● use when at least 2 levels of control are run with the ● Inadequate storage of reagent or calibrators/control
examination run products
● cannot use with only one control ● Change in test operator
●
12s Rule 13s Rule
3
● There is only one data value that occurs outside the ±
3SD
● This rule identifies random error
● Any QC result outside ± 3s violates this rule.
R4s Rule
● Data values are very far from each other.
IDEAL CONTROL RUNNING
● If there is at least a 4s difference between control values

within a single run, the rule is violate for random error
Sources of Random Error

● Power Supply
● Double pipetting of control sample
● Misplacement of Control sample within the run
● Random air bubbles in reagent or sample pipette system
○ Can affect the volume that can be seeped by
the machine ● Values are very close to the mean = values are
● Incorrect reconstitution of the control product accurate
● Inappropriate Storage of control ● Close to each other = precise
● Inadequately mixed reagents What will be our guide to determine if you are going to consider
● Individual operator variation in pipetting , timing , etc. the control values as still in control or out of control?
○ Dependent on the skills of the operator
BIAS/SDI RESULTS ACCURACY /RELIABILITY ARE

AFFECTED
If 1-2s lang na violate

● Picture above: tell us if there would be a bias in ● consider QC results as in control→ accept running
laboratory method, this is what u observe If nag violate sa 1-2s and 1-3s
○ They fall only on one side of the mean. ● Out of control → reject running or result of control
■ Violates the 10x (?) rule If QC is out of control
● STOP testing
PRECISION/REPEATABILITY/CV/ CVI ARE AFFECTED ● identify and correct problem
● repeat testing on patient samples and controls after
correction
● Do not report patient results until the problem is
solved and controls indicate proper performance
4
5
LECTURE 2: CARBOHYDRATE: LABORATORY

APPLICATIONS
Prof. Josephine Cuajotor, RMT
October 5.2021
SPECIMEN CONSIDERATIONS A. Oxidation-Reduction Method

1. Alkaline Copper Reduction Method
Possible samples: a. Folin Wu Method
● Whole blood b. Nelson Somogyi Method
○ 11% lower than serum or plasma c. Neocuproine Method
● Plasma d. Benedict’s Method
● Serum i. Modification of Folin Wu
○ Do not accept unhemolyzed venous 2. Alkaline Ferric Reduction Method
plasma/serum a. Hagedorn Jensen
● CSF (Cerebrospinal Fluid) B. Condensation Method
● Pleural fluid a. Dubowski Method
● Urine
● Most of the laboratory hospitals are using plasma and CHEMICAL METHOD
serum in glucose determination
● Hemolyzed samples are not accepted in the lab because A. Oxidation Reduction Method
it is mandated under the code of ethics to release 1. Alkaline Copper Reduction Method
accurate and precise results in accordance with the ● Principle: reduction of cupric ions to cuprous
quality assurance and quality control in the laboratory ions forming cuprous oxide in hot alkaline
solution by glucose
Separation of the liquid portion ○ Cupric ion - Cu2+
● Serum can be separated within 30 minutes ○ Cuprous ion - Cu1+
○ Must be separated within 30 minutes since
RBCs use glucose present in the sample,
glycolysis will happen to result to a false
decrease of glucose levels in the result a. Folin Wu Method
● Serum w/o bacterial contamination and without ● The glucose reduces cupric ions present in the
leukocytosis (increased WBCs) = up to 90 minutes delay alkaline copper reagent to cuprous ions or the
● Plasma must be separated from the cellular fraction cupric sulfate is converted to cuprous oxide
(even with Na fluoride) which reduces the phosphomolybdic acid to
○ Sodium fluoride is present in the gray tube phosphomolybdous acid
which is used for glucose determination ○ Which is blue in color when optical
○ Sodium fluoride inhibits the glycolytic enzyme density is measured at 420 nm
enolase which acts as an anticoagulant
○ There is also acetate which inhibits the
glycolytic enzyme, glyceraldehyde-3-phosphate
dehydrogenase
○ It is important to separate serum from the
sample upon receiving the blood sample
● Glycolysis decreases serum glucose by approximately
5% to 7% in 1 hour (5 to 10 mg/L) in normal
uncentrifuged coagulated blood at room temperature b. Nelson Somogyi Method
○ Pagkuha ng sample, allow it to stand for 5-1 ● Sugars with reducing properties are rising out
minutes and allow the sample to clot then of the presence of the potential aldehyde or
centrifuge then separate the serum or plasma keto group called the reducing sugars
from the (un)clotted blood at room temp ● Ex of reducing sugars
● In separated, non-hemolyzed sterile serum, stable as ○ Glucose, galactose, lactose, maltose
long as 8 hours at 25°C and up to 72 hours at 4°C. ● One of the classical and widely used methods
for the quantitative determination of the
Storage of Samples reducing sugars
● Refrigerated (2-8ºC)
○ Serum or Plasma: stable up to 48 hrs
○ Whole blood: 2mg Na fluoride per mL of whole
blood (48 hrs)
■ Still stable within 48 hrs at
refrigeration temperature
GLUCOSE METHODOLOGIES
● Different lab methods to determine glucose levels in the
● The reducing sugars when heated with the
sample
alkaline copper tartrate reduce copper from the
● 2 methods
cupric state and cuprous oxide is formed
○ Chemical method
● When cuprous oxide is heated with
○ Enzymatic method
arsenomolybdate, there will be reduction of
Chemical Method
1
molybdic acid to molybdenum which has the 2. Alkaline Ferric Reduction Method (Hagedorn Jensen)
color blue ● It involves reduction of yellow ferricyanide to a
● Blue colored is developed is compared to the colorless ferrocyanide by glucose (inverse
standards of colorimetry at 620 nm colorimetry)
c. Neocuproine Method
● (2,9 Dimethyl 1,10 Phenanthroline B. Condensation Method
Hydrochloride) ● Ortho-toluidine (Dubowski Method)
● The copper in oxidation state reacts with ○ Condensation of glucose with primary aromatic
neocuproine forming a complex, this complex is amine in glacial acetic acid, forming an
extracted into a chloroform methanol mixture equilibrium mixture of a glycosylamine and the
giving a yellow or yellow-orange solution corresponding Schiff base
Condensation Method
Ortho-toluidine (Dubowski Method)
Procedure:
● Glucose in a PFF (3% TCA) reacts to O-Toluidine in hot
acidic solution will yield a GREEN colored compound
with maximum absorbance at 630 nm
d. Benedict’s Method (Modification of Folin-Wu)
● Used for detection and quantitation of reducing ENZYMATIC METHODS
substances in body fluids like blood and urine.
● Acts on glucose but not on other sugars and reducing
● used citrate or tartrate as stabilizing agent
substances.
● Used to test for simple carbohydrates
○ GLUCOSE OXIDASE METHOD
● Identifies reducing sugars (ex.
■ Colorimetric Glucose Oxidase Method
Monosaccharides and some disaccharides
(Saifer Gerstenfield Method)
which have 3 ketone or aldehyde functional
■ Polarographic Glucose Oxidase
groups)
●
● Can be used for identification of glucose in the
○ HEXOKINASE METHOD
urine
○ GLUCOSE DEHYDROGENASE METHOD
● It has a principle that when the reducing sugars
○ DEXTROSTIX (cellular strip)
are mixed with the Benedict’s reagent and
○ INTERSTITIAL GLUCOSE MEASURING
heated, a reduction reaction causes the
DEVICE
benedict’s reagent to change color
○ ENZYMATIC METHODS
○ Color varies from green to brick red,
rusty brown = depending on the
amount of sugar GLUCOSE OXIDASE METHOD
● Fehling’s Test
○ Principle: Aldolases are easily Colorimetric glucose oxidase method (aka Saifer Gerstenfield
oxidized to yield carboxylic acid Method)
○ Cupric ion complex with tartrate ion is
reduced to cuprous oxide forming the
product of brick red color
● Difference of Fehling’s Test and Benedict’s ● Glucose oxidase is the most specific enzyme reacting
Test: only with B-D-glucose
○ Reducing sugars and aldehydes are ● Glucose oxidase converts B-D-glucose gluconic acid and
chemical compounds that can be the oxygen is consumed and H2O2 is being produced
oxidized by reducing other
components -- the concept can be
used to identify the presence of them ● Horseradish peroxidase is used to catalyze the second
in a compound measure reaction.
○ For the identification of this ● H2O2 is used to oxidize a dye compound to commonly
compound, the Benedict’s and chromogens are used in this type of method.
Fehling’s test can be used ● 3-methyl-2-benzothiazolinone hydrazone
■ It uses specific reagents ● N,N-dimethylaniline
such as Benedicts solution ● Shift in the absorbance can be monitored
and Fehlings solution spectrophotometrically and its proportional to the
○ Main difference: amount present in the specimen.
■ Benedict’s solution contains ● This couple reaction is known as the trinder reaction.
the Cu 2+ citrate
■ Fehling’s solution contains TAKE NOTE!
the Cu 2+ tartrate ● Peroxidase couple reaction used glucose oxidase
method is subject to + and - interference
● Increased levels of uric acid, bilirubin, and ascorbic acid
can cause falsely decreased values as a result of these
substances being oxidized by peroxidase, which then
prevents the oxidation and detection of the chromogen.
● Strong oxidizing substances, such as bleach, can cause
falsely increased values
Polarographic Glucose Oxidase

● Measures rate of oxygen consumption which is
proportional to glucose concentration.
● Glucose oxidase in the reagent catalyzes the oxidation of
2
glucose by oxygen under first order conditions, forming
hydrogen peroxide.
● Quantitated by the consumption of oxygen on an
oxygen-sensing electrode.
● Hydrogen peroxide is prevented from re-forming oxygen
by adding molybdate, iodide, catalase and ethanol.
DEXTROSTICS (cellular strip)

● Dextrostics is touched to a drop of blood and inserted
● Oxygen consumption can be used to perform the direct
into the meter which gives a digital reading of the blood
measurement of oxygen by polarographic technique.
sugar; gives the unit of mmol/L
● This oxygen depletion is measured and proportional to
● An enzyme-impregnated strip used with a small portable
the amount of glucose present.
electronic colour-measuring device for convenient
● This polarographic glucose analysers measure the rate
estimation of the blood sugar levels by diabetics.
of consumption because glucose is oxidized by the first
● One of the point of care (POC) device
ordered conditions using glucose oxidase reagent.
○ The whole blood capillary glucose values
● H2O2
obtained with POC devices are useful for the
○ Must be eliminated in the side reaction to
detection of hyperglycemia and hypoglycemia
prevent the reaction from reversing.
in individuals with diabetes
● Take note:
○ It should not be used to diagnose diabetes or
hypoglycemic disorders.
● The catalase in this type of reaction can be used to

catalyze oxidation of ethanol by the H2O2 forming the
acetaldehyde and water.
● OR using the molybdate to catalyze the oxidation of
iodide to iodine by the H2O2
INTERSTITIAL GLUCOSE MEASURING DEVICE
HEXOKINASE METHOD ● used for continuous monitoring of glucose levels in
● More accurate than glucose oxidase method people with diabetes
● MOST SPECIFIC GLUCOSE METHOD; REFERENCE ● uses electrochemical methods to automatically and
METHOD frequently measure glucose levels in the interstitial fluid
● Less interference of dermis or subcutaneous fat tissue.
● Plasma collected using heparin, EDTA, flouride, oxalate ● Result of this test provides an idea of the glucose
or citrate may be used for this test. patterns over hours or days
● Other samples; Urine, CSF and serous fluids
● Hexokinase in the presence of ATP converts glucose to
glucose 6 phosphate
○ glucose 6 phosphate and cofactor NADP are
converted into 6 phosphogluconate at 340
maximum absorbance measuring the rate of
appearance of NADPH and NADPH by glucose
6 phosphate dehydrogenase.
Laboratory Testing for GLUCOSE
1. Random plasma glucose (random blood sugar)
2. Fasting plasma glucose (FBS)
3. Tolerance test
● In the presence of ATP, it converts glucose to glucose 6 4. HbA1c (glycosylated hemoglobin test)
phosphate 5. Fructosamine
● This glucose 6 phosphate and the cofactor NADP are 6. Urine Microalbumin
converted to 6 phosphogluconate and NADPH by 7. Ketone testing
glucose 6 phosphate dehydrogenase
● This NADPH has a strong absorbance maximum at 340 Random Plasma Glucose
nanometer and the rate of appearance of NADPH can be ● Formerly random blood sugar (RBS)
monitored spectrophotometrically and is proportional to ● Specimens collected anytime of the day
the glucose present in the sample ● Usually done in a glucometer
● Gross hemolysis and extremely elevated bilirubin may ● NO NORMAL VALUES
cause a false decrease in results. ○ Bcs the glucose is measured randomly
GLUCOSE DEHYDROGENASE METHOD

● Glucose is reduced to produce a chromophore that is
measured spectrophotometrically or an electrical current
● NADH = glucose concentration
○ The amount of NADH generated is proportional
to the glucose concentration
● Close agreement with hexokinase procedures
● Mutarotose is added to shorten the time
3
Glycated/Glycosylated Hemoglobin
● HbA1c
○ Hemoglobin A is irreversibly glycosylated at
one or both N-terminal valines of the B-chains
of the tetrameric hemoglobin molecule
(International Federation of Chemistry Working
Group on HBA1c)
○ Is the largest subfraction of normal HBA
involved in diabetic and nondiabetic individuals
○ Represents the weighted average of glucose
levels with the youngest erythrocytes
contributing to a greater extent than the older
ones
● Principle:
Principle of glucometer
● Electrochemistry (Amperometry)
● Gluosse oxidase
● Works with the sensors or amperometry
● First step to measure the glucose in the blood is to
convert glucose concentration into voltage or amount or
the current signal
● This is possible with the special sensor strip for
amperometry
● The sensor uses the platinum or the silver electrode to
form part of the electric circuit where hydrogen peroxide
is electrolyzed
● The hydrogen peroxide is produced as a result of the
oxidation of glucose on the glucose oxide membrane and
the current flowing through the circuit provides the ● Now the preferred test to assess glycemic control
measurement of the concentration of hydrogen peroxide ● Widely used marker of chronic hyperglycemia (reflectin
hence giving the concentration of glucose average blood glucose levels over a 2-3 month period of
time)
Fasting Plasma Glucose
● Formerly fasting blood sugar (FBS) ● Methods
● Specimens collected after 80-10hrs fasting (new ○ Affinity Chromatography (common)
guideline) ○ HPLC
○ Nothing by mouth ○ Electrophoresis
○ Sometimes accept patients that have drank ○ Spectrophotometry (common)
small amount of water; physician must be ○ 2-step Non-enzymatic method
informed
○ >7.0mml/L or > 126mg/gL diabetes mellitus
(DM)
Oral Glucose Tolerance Test

● To determine how well your body metabolizes glucose
over a required period of time
● 3 samples in total
● Inform the patient of the procedure
Procedure:
1. Fasting sample + urine
2. 75 grams glucose load is orally taken within 15 minutes
3. 1hr sample + urine
4. 2hr sample + urine
Glucose Tolerance Test

● Diagnosis of GDM (for pregnant women)
○ Fasting: >5.1 mmol/L (>92 mg/dL) Specimen Req for HBA1c = Whole blood
○ 1h: >10.0 mmol/L (>180mg/dL) Anticoagulant of choice = EDTA
○ 2h: >153 mg/dL (>8.5 mmol/L)
4
Urine Microalbumin
● Test to detect very small levels of protein (albumin) in
urine
○ To detect early diabetic nephropathy (kidney
damage)
Ketone
● beta-HBA, acetoacetic acid, and acetone
○ Ratio: greatly increased in the Keto acidosis
due to the altered rate and elevated levels of
NADH in hepatic mitochondria
○ In serum acetone, it is indicative of defect in
carbohydrate metabolism when the same
acetone is increased
● Recommended when plasma glucose values reaches the
300 mg/dL
● To detect ketosis in DM type??
● Methods
○ Electrochemistry
○ Chromatography
○ Electrophoresis
○ Colorimetric method
● Methods
○ Gerhatt’s
■ Used of ferric chloride reacted with
acetoacetic acid to produce a red
color
○ Sodium Nitroprusside
■ Reacts with acetoacetic acid in an
alkaline pH to form a purple color
■ Urine reagent strip test and Acetest
tablets
○ 3-hydroxybutyrate dehydrogenase
■ To detect either 3-b-hydroxybutyric
acid or acetoacetic acid
2-hour Postprandial Glucose

● Any defect or abnormality in the RBC may affect your ● 2 hours following regular meal
HBA1c
2-Hour Postprandial Glucose (more standardized)
What if the Patient has Hemoglobinopathy? ● 75 grams glucose load
● It will cause the decreased value of your HBA1c ○ Blood collection after 2 hours
● Hemoglibonpathy ○ 200 mg/dL - DM
○ A genetic defect that results in abnormal
structure of one of the globin chains of the Diagnostic Criteria for Diabetes Melitus
hemoglobin molecule ● Random Plasma Glucose
○ Conditions associated with short and red blood ○ >200 mg/dL (11.1 mmol/L)
cell survival or lower mean red blood cell age ● Fasting Plasma Glucose
such as hemolysis recovery from acute blood ○ >126 mg/dL (7.0 mmol/L)
loss, transfusions, or splenectomy, will lower ● 2-hour Plasma Glucose
your HBA1c ○ >200 mg/dL (11.1 mmol/L)
● HbA1c
○ >6.5%
Laboratory Tests
Fructosamine
● Also known as the glycosylated albumin
● Most widely used to assess short-term (3-6 week)
glycemic control
● Most useful if the HBA1c is unreliable due to
hemoglobinopathy or hemolysis
● Not ideal: serum albumin level is <3 g/dL or when serum
albumin turnover is accelerated (cirrhosis)
5
PRODUCT INSERT concentration is normally maintained at a constant level.
Excessive glucose is stored as inactive glycogen mainly
in the liver and little in the muscles.
● Elevated blood glucose levels are found in diabetes
mellitus, hyperthyroidism, hyperadrenalism and certain
liver diseases.
● Decreased levels are found in insulinoma,
hypothyroidism, hypopituitarism
Principles
● Enzymatic colorimetric determination of glucose
according to the following reaction
Kit components
Reagent Storage and Stability

● The sealed reagents are stable up to the expiry date
stated on the label, when stored at 2-8 C and protected
from light
Open Vial Stability

● Once opened, the reagent is table up to 4 weeks at 2-8
C, if contamination is avoided,
Intended use
● This reagent is intended for in vitro quantitative
determination of Glucose in serum, plasma & CSF.
● GOD-PAP methodology
● Linear up to 600 mg/dL
Clinical Significance
● Glucose is a major carbohydrate present in the blood and
serves as a primary source of energy. It is usually
obtained from ingested starch and sugar. The glucose
6
LECTURE 2: LIPIDS: LABORATORY APPLICATIONS

Prof. Fritz Von Gella, RMT, MD
October 11, 2021
Blood Sampling and Storage resulting in inaccurate and variable results

● Protein aggregation occurs less frequently using Serum
1. Biological Variation ● Anticoagulant used:
● Age (↑ Chole) Large osmotic effects
○ Amn increase in age would mean an increase
in cholesterol levels
● Sex: women have lower level than men Alter electrophoretic mobiliy
● Seasonal (Chole ↑ in winter)
● Dietary intake (restricted diet for 2 weeks) *preferred
● Medications (oral contraceptives)
● Medical disorders ● Blue Tube: Citrate
● Lifestyle and other biological variation ○ Citrate will cause a large osmotic effect
○ Sedentary lifestyle ○ Cause falsely low results
○ Daily intake of fat increases cholesterol levels ○ Should be avoided
● Recent MI, stroke, cardiac catheterization ● Green: Heparin
● In different acute illnesses, it is recommended that ● Purple: EDTA
lipoprotein measurement should be made no sooner than ○ It inhibits certain kinds of oxidative and
8 weeks after any form of trauma, or acute bacterial or enzymatic alterations occurring in the
viral infection and 3-4 months after childbirth lipoprotein during storage
● Remember it depends on the reagent used
2. Fasting 5. Storage
● TC, TAG, HDL, Apolipoproteins – can be analyzed with
frozen samples
● Long-term (>2 mos) at -70C
● Short-term (1-2 mos.) at -20C
● Frozen samples are not appropriate for ultracentrifugal
analysis because your triglyceride-rich lipoprotein do not
withstand freezing
LABORATORY METHODS: CHOLESTEROL

● Ideal time: 12 hours
● NCEP: Adult treatment Panel III: at least 9 hours Chemical Methods
○ Enables fasting of 9 hours to accommodate the ● Liebermann-Burchard’s test
unable and unwilling to fast for 8 hours
3. Posture
● When standing patient reclines = ↓ 10% TC, LDL, HDL
○ When a standing patient reclines, extravascular
water transfers to the vascular system and
dilutes non-diffusable plasma constituent
● Position of the patient must be standardized for
venipuncture (sitting position)
● NCEP recommends: patient be seated for 5 minutes
before sampling
● Prolonged venous occlusion = hemoconcentration
(↑chole by 10-15%)
○ 2mg of dry extract was dissolved in acetic
4. Type of Plasma (Plasma or Serum)
anhydride, heated to boiling, cooled and then 1
● When applying Friedewald equation where LDL is
ml of concentrated sulphuric acid was added
calculated (Plasma/Serum)
along the sides of the test tube.
● Plasma: Ultracentrifugation & electrophoresis (LPP
○ Positive: Formation of green color indicates the
analysis)
presence of steroids
○ Plasma sample can be cold at 4ºC and retired
the changes occurring at room temperature
● Salkowski reaction
● Plasma should not remain in contact with the cells
overnight
○ Even in the presence of an anticoagulant,
protein aggregation can also occur in plasma
that is stored in the refrigerator for a few days
or frozen for longer periods
○ Can be difficult to obtain homogeneous aliquot
for analysis and can interfere with the flow of
the sample in the automated analyzers
1
○ Extraction of TAG by chloroform
○ Isolation of TAG by silicic acid chromatography
○ Release of glycerol by saponification
■ Saponification: alkaline hydrolysis of
triglycerides similar to van handful
and silversmith method
● Glycerol and Sodium periodate will be reacted to form

formaldehyde and formic acid
○ Formaldehyde is measured by sulfuric acid
○ 2mg of dry extract was shaken with chloroform, solution by chromotropic acid to produce the
to the chloroform later sulphuric acid was end color
added slowly by the sides of test tube ○ +H2SO4 = PINK END COLOR
○ Positive: Formation of red colour indicated the
presence of steroids. Enzymatic Methods
● Abell-Kendall
○ Another chemical method for cholesterol
○ Hydrolysis of CE using alcoholic KOH (potash)
to UE form
○ Extraction of UE form with petroleum ether
○ Measurement with Lieberm,ann-Burchard ● 2 first step that is common with all these methods
reagent ● Triglyceride will be acted by lipase forming Glycerol and
■ Sulfuric acid, acetic acid, and acetic Fatty acid
anhydride (SAA) ● Glycerol will be added with ATP (when you here kinase
○ Bluish Green Color has something to do with a transfer of phosphate group)
transfer one phosphate to glycerol forming
Enzymatic Methods glycerophosphate and then since kumuha ng phosphate
kay ATP naging ADP nalang
● Commonly used method

● Glycerophosphate is then oxidase to Dihydroxyacetone
and then the hydrogen peroxide in a glycerophosphate
oxidase will be catalyzed
● Hydrogen peroxide acted upon by these reagent (Phenol
+ 4-aminoantipyrine) by the action of peroxidase forming
● What is the advantage of using enzymatic? Quinoneimine dye (colored dye complex)
○ Use smaller sample volume, no preliminary
extraction step and it is rapid and precise vs
chemical method
● Cholesterol Ester with a combination of water will be
acted by the cholesteryl esterase forming cholesterol and
free fatty acid ● Glycerophosphate can be measured in a reduced form of
● Cholesterol with oxygen will be acted by cholesterol nicotinamide adenine dinucleotide (NADH) and is
oxidase forming Cholest-4-en-3-one + H2O2 measured by spectrophotometer set at 340 nm or in a
● Preferred si Hydrogen Peroxide (H2O2) is measured by diaphragm catalyzed reaction to form now a reaction
peroxidase catalase reaction that will now form colored product whose absorbance is measured at 500 nm
dye which is known Quinoneimine dye that will be ● Glycerophosphate will be acted by Glycerophosphate
detected by the device dehydrogenase with oxidized NAD forming reduced
○ Quinoneimine dye quantitates cholesterol level NADH
which is being utilized in spectrophotometer ● NADH will be acted by tetrazolium dye with the presence
● Interfering substances of Diaphorase. Therefore, we will be able to now
○ Plants sterol, measure the level of triglyceride
○ Ascorbic Acid
■ Consuming hydrogen peroxide
leading to false decrease of the result
○ Bilirubin
■ Consuming hydrogen peroxide
leading to false decrease of the result
○ Sample Turbidity ● ADP is now measured will be acted by Pyruvate Kinase
○ Presence of hemoglobin (high levels) with the presence of Phosphoenolpyruvate (kinase =
transfer phosphate) tinggalan ng phosphate ang
LABORATORY METHODS: TRIGLYCERIDES phosphoenol transferred to ADP kaya magiging ATP and
pyruvate
Chemical Methods ● Pyruvate with the action of Lactate dehydrogenase
● CDC - reference method ended up with the product Lactate + NAD which enables
○ Advantage: limits interfering substance; lower to detect triglyceride
interference of different substance using CDC -
reference method
2
Separated by chemical precipitation
● Earliest - heparin inc combination with manganese to
precipitate apo b- containing LPP
● Alternative - sodium phosphotungstate with magnesium
● More specific - dextran sulfate with magnesium
● Interfering factor: elevated TAG levels
● Clear supernatant - assay for cholesterol content
LABORATORY METHODS: HDL-CHOLESTEROL
Separated by chemical precipitation

- Precipitating Agents
● Earliest – heparin in combination with manganese to
precipitate apo B-containing LPP ● Std = standard
● Alternative – sodium phosphotungstate with magnesium ● Rgt = reagent
● More specific – Dextran sulfate with magnesium
● Interfering factor: elevated TAG levels
● Clear supernate – assay for cholesterol content PACKAGE INSERT CHOLESTEROL
● Enzymatic Colorimetric Test for Cholesterol with Lipid
Gold standard or Reference Method Clearing Factor (LCF)
● 3-Step Procedure
○ Ultracentrifugation – to remove VLDL
○ Heparin Manganese Precipitation – to remove
LDL
○ Analysis of supernatant cholesterol by the
Abell-Kendall Assay
LABORATORY METHODS: LDL-CHOLESTEROL
Reference Method
● Beta-Quantification
● Electrophoretic migration
● Ultracentrifugation + Chemical Precipitation
Routine Method
● Friedewald Calculation ● The cholesterol is determined after enzymatic hydrolysis
● Commonly used and oxidation. The indicator quinoneimine is formed from
● If u already have the levels of HDL, cholesterol, all u hydrogen peroxide and 4-aminophenazone in the
have to do is SUBSTRACT ur values to ur LDL presence of phenol and peroxidase
FRIEDEWALD EQUATION/CALCULATION
LDL = TC – HDL – (TAG/2.175) (mmol/L) - SI unit

LDL = TC – HDL – (TAG/5) (mg/dL)- conventional unit
- Divide first before subtracting
- Limitations
● Not appropriate in samples with TAG of >400
mg/dL
● With px suspected this beta lipoprotenemia.
COMPUTE FOR LDL using FRIEDEWALD Equation:

TC – 140 mg/dl
HDL – 40 mg/dl
TAG – 60 mg/dl
Reagent Preparation
● The RGT and STD are ready for use
Reagent Stability
● The regents are stable up to the given expiry date, even
after opening, when stored at 2..8°C. The opened
reagent is stable for 2 weeks at 15...25°C. Contamination
must be avoided.
Specimen
PACKAGE INSERT ● Serum, heparinised or EDTA-plasma
Note: Lipemic specimens usually generate turbidity of the
sample/reagent mixture which leads to falsely elevated results.
The CHOLESTEROL liquicolor test avoids these falsely elevated
3
results through its built-in Lipid Clearing Factor (LCF). The LCF
clears up totally a turbidity caused by lipemic specimens.
Example:
Absorbance of Sample: 0.1250
Absorbance of STD: 0.1370
● Use formula for with standard
Question: Calculate the Concentration in Conventional Unit.
C= 200 x ( 0.1250/0.1370)
C = 182.48 mg/dl
● Wave length: 500 nm, Hg 546 NM
● Temperature: 20-25°C or 37°C If SI, substitute 200 with 5.17
● Measurement: Against reagent blank. Only one reagent
blank per series is required.
○ Reagent blank - Used to 0 the instrument Performance Characteristics
before measuring test sample and other blanks ● Linearity
○ The test is linear up to a cholesterol
concentration of 750 mg/dl (19.3 mmol/L).
Dilute samples with a higher cholesterol
concentration 1 + 2 with physiological saline
(0.9%) and repeat the determination.
○ Multiply the result by 3
● Prepare:
○ Reagent blank - purely 1000μl
○ Sample - 10μl (sample) & 1000μl (reagent)
○ Standard - 10μl (STD) & 1000μl reagent
● Mix, incubate for 10 mins at 20-25°C or 5 mins at 37°C
○ No need to mix in reagent blank Quality Control
● Measure the absorbance of the sample and STD against ● All control sera with values determined by this method
the reagent blank may be employed. We recommend to use our animal
serum-based HUMATROL or our human serum-based
SERODOS quality control sera.
Automation
● Proposals apply to reagents on analyzers are available at
request.
● Each lab has to validate the application in its own
responsibility
Notes
1. The test is not influenced by hemoglobin values up to
200mg/dL or by bilirubin values up to 5mg/dl
- If more than 200, it will interfere with your result
- Interferes with hydrogen peroxide causing a
● 1st: reagent blank (1000μl) false decrease
● 2nd: sample or STD 2. The reagents contain sodium azide as a preservative
(0.05%). Do not swallow. Avoid contact with skin and
mucous membranes
● With factor - absorbance lang ng sample

● Commonly used is with standard
4
PACKAGE INSERT HDL-CHOLESTEROL Pipette into cuvettes macro STD Sample (HDL
supernatant)
Distilled water 100 ul
STD 100 ul
HDL supernatant 100 ul
Reagent 1000 ul 1000 ul 1000 ul
● Mix, incubate for 5 minutes at 37 C or 10 minutes at

20-25 C. measure the absorbance of the sample and the
STD respectively, against the reagent blank within 60
minutes.
Calculation of the HDL cholesterol with factor
Calculation of the HDL cholesterol Concentration with

standard
1. Macro method
2. Semi-micro method
● Precipitant: phosphotungstic acid and magnesium

chloride
● PREC = precipitant
● STD = standard
○ Cholesterol Calculation of the LDL Cholesterol Concentration
● The LDL cholesterol is calculated from the total
Difference of macro assay and semi-macro assay cholesterol concentration (TC), the HDL Cholesterol
● If macro assay, mas malaki ang volume na gagamitin concentration and the triglycerides concentration
● If Semi, volume is lower according to Friedewald el.al.
● Absorbance of STD: 0.3650

● Absorbance of Sample: 0.1350
● Using a semi-micro method, calculate the concentration in
the SI Unit. (Female Patient)
○ C= 4.52 x ( 0.1350/0.3650)
○ C = 1.67 mmol/L
Clinical interpretation
HDL Men Women

Assay
CHOLESTEROL mg.dL mmol/L mg/dL mmol/L
1. Precipitation
● Uses phosphotungstic acid and magnesium chloride
Prognistablly >55 >1.42 >65 >1.68
Pipette into centrifuge tubes macro semi-macro
favourable
sample 500 ul 200 ul
Standard disk 35-55 0.9-1.42 45-65 1.16-1.68
PRECa 1000 ul level
PRECb 500 ul Risk indicator <35 <0.9 <45 <1.16
● Mix well, incubate for 10 minutes at room temperature.
Centrifuge for atleast 2 minutes at 10000 g, alternatively
for 10 minutes at 4000 g.
● After centrifugation, separate the clear supernatant from
the precipitate within 1 hour and determine the
cholesterol concentration using CHOLESTEROL
liquicolor reagent.
2. Cholesterol determination
5
NOTES
1. If the supernatant is not clear (High triglycerides level),
dilute the sample before the precipitation 1:1 with 0.9%
saline (multiply result by 2)
2. High concentrations of ascorbic acid (>2.5 mg/dL) will
give lower values
3. Hemoglobin levels higher than 100 mg/dL and bilirubin
levels higher than 10 mg/dL interfere with the test.
PACKAGE INSERT TRIGLYCERIDES
6
7
LECTURE 4: AMINO ACIDS AND PROTEINS:

LABORATORY METHODS AND PROCESSING
Prof. Bea Angeli D. Laude
Octobr 25, 2021
TOTAL PROTEIN METHODS 1. Digestion

● Will occur if you mix the sample with sulfuric acid and
Total Protein Specimen: heat between 340°C - 360°C
● Serum (sample of choice) ● To speed the reaction, a catalyst will be added. Cupric
● Need not be fasting sulfate is utilized. In some cases, another reagent is
○ Sample can be taken at any time of the day added that is potassium sulfate in order to increase the
whether or not the patient had a meal boiling point to improve the efficiency of the digestion
● Lipemia may interfere ● Sulfuric acid will oxidize the carbon, hydrogen, and sulfur
○ Must be contraindicated because it could in the protein to become CO2, CO, H2O, SO2
interferences in the procedures ● Nitrogen in the protein will be converted to become your
● Hemolysis ammonium bisulfite which is then utilized for
○ Elevate Total Protein because off the release of neutralization and distillation
RBC proteins in the serum
2. Neutralization and Distillation
Total Protein Methods ● In neutralization, your ammonium bisulfite was highly
● Kjeldahl Method exposed to an acid which is sulfuric acid which needs to
● Biuret Method be neutralized before proceeding to distillation
● Dye Binding ● The neutralizing agent is by the addition of an alkaline,
● Refractometry sodium hydroxide
● Once it is neutralized, proceed with distillation into a
Kjeldahl Method standard boric acid solution
● Classic method (determine nitrogen) ● From ammonium bisulfite, because of the presence of
● Not used in Clinical Laboratory your boric acid, it now becomes your ammonium borate
○ Time-consuming
○ The procedure is way too tedious for it to be a 3. Titration
part of routine activity ● Ammonium borate will be titrated with standard HCl so
○ Requires many reagents we can be able to determine the number of proteins that
● Average of 16% nitrogen as in proteins is present on the patient sample
○ In order to calculate the protein concentration
○ The actual nitrogen content of protein may Biuret Method
actually vary ranging from 15.1% - 16% ● Most widely used method
○ An error can be introduced if the protein ● Recommended by IFCC (International Federation of
standard which was calibrated by the Kjeldahl Clinical Chemistry) for determination of Total Protein
method that it may actually differ in protein ● Cupric ions complex with the groups involved in peptide
composition compared to the sample bond
● No proteins of significant concentration in the unknown ○ There will be a violet-colored soln if there are at
specimen are lost in the precipitation step which is the least two peptide bonds detected in the sample
first step of the method ● Biuret Reagent
○ Potassium hydroxide
■ Provides an alkali medium so the rxn
can take place
○ Sodium potassium tartrate
■ Complex cupric ions to prevent their
precipitation in the alkaline soln
○ Potassium iodide
■ Acts as antioxidant
● Absorbance is measured at 540 nm
● Also determines the size of the protein particles that is
present on the samples
● When small peptides react, the color of the chelate
produced has a different shade than that seen with
larger peptide
● Alkaline medium and at least 2 peptide bonds, a
violet-colored chelate formed
● Start with the precipitation of proteins
● Color varies from pink to a reddish violet
● Proteins will be precipitated with the presence of tungstic
● Color formed is proportional to the number of peptide
acid
bonds and reflects the Total Protein level
● Pag naa na si tungstic acid, the non-protein nitrogens
○ If pink = lower TP level
will also be removed with the supernatant
○ If reddish violet = higher TP level
● Proceed with digestion
● In (abnormally small proteins) multiple myeloma,
c-protein concentration is underestimated due to lighter
shade of color produced
1
○ Smaller peptides could produce lighter shade of the dye
compared to samples with larger protein Refractometry is a rapid, simple method, but is not commonly
● Lipemia in the sample is interferent used for total protein analysis.
○ High lipid content must be rejected as it will
interfere to the procedure
Fractionation, Identification, and Quantitation of Specific
Proteins
Dye-Binding Method ● Albumin
● Ability of most proteins in serum to bind dyes ● Globulin
● Most common dye utilized: Coomassie brilliant blue 250 ● These 2 proteins can give us useful diagnostic
relies on the binding to protein information to determine the presence of kidney or liver
○ When it forms complexes with the protein in the disease, we refer to it as A/G ratio
sample, it will be causing a shift of the ● Get total protein, and determine the levels of either
absorbance of the dye from 465-595 nm albumin or globulin (most often its albumin)
● A shift absorbance maximum of the dye from 465-595 ● Then, subtract to the total protein and we can get the
nm levels of globulin
● Absorbance at 595 nm is used to determine the protein ● If there’s a reversal or significant change in the ratio of
concentration albumin and total globulin, it will aid us in determining if
● Aside from coomassie brilliant blue, other dyes may be the patient has problems in the kidney or liver
used such as:
○ Bromothymol blue Methods for Fractionation, Identification and Quantification of
○ Ponceau S Specific Proteins
○ Amido black 10b ● Salt fractionation
○ Lissamine green ● Dye-binding
● Although it can be a simple and fast method, caution ● Serum protein electrophoresis
must still be applied in utilizing this method because
individual proteins tend to have different affinity to the Salt Fractionation
dyes to be utilized
● Fractionation of proteins is commonly done using the
precipitation technique:
Principle of Dye-binding Method in quantifying TP
● Globulins are separated from albumin by salting out,
using sodium salt to precipitate globulins
○ Globulin - precipitate
○ Albumin - supernatant fluid (measured)
■ Measured thru routine total protein
technique.
● Biuret test
● Dye binding method
○ Salting is no longer used bcoz we have
available methods that can directly react with ur
albumin in the given sample.
Dye-binding
● Dye-binding procedures
● Coomassie Brilliant Blue - dye utilized ○ most widely used methods for determining
● With the presence of the protein in the sample, it is going Albumin.
to form a dye-protein complex ● pH of the solution is adjusted so that albumin is positively
● With this formation, we have a change in the maximum charged.
absorbance of the dye which started at 465 - 490 nm to ○ If it is in complex with the albumin, maximum
shift to its maximum absorbance of 595nm absorbance is going to change
● Increased in absorbance at 595 nm, is utilized to ● Albumin is attracted to and binds to an ionic dye
determine the protein concentration of the sample ● amount of albumin
○ (calculated by) absorbance of the albumin-dye
Refractometry complex
■ Methyl orange
● A quick alternative to chemical analysis of serum total
■ 2,4’-hydroxyazobenzene-benzoic acid
protein when a rapid estimate is required
(HABA)
● Measurement of RI due to solutes in serum
■ Bromocresol Green (BCG)
○ Refractive Index (RI) can be accurate in
■ Bromocresol Purple (BCP)
measuring the serum protein concentrations,
but not in urine protein measurements because
Methyl Orange
of the excess amounts of solutes in relation to
● nonspecific for albumin
the protein
● Beta-lipoproteins, alpha-1 and alpha-2 globulins also
bind to dye
SUMMARY: TOTAL PROTEIN METHODS
METHOD PRINCIPLE COMMENT 2,4’-hydroxyazobenzene-benzoic acid (HABA)
Reference method; ● low sensitivity but more specific for albumin salicylates,
Digestion of protein;
assume average penicillin, conjugated bilirubin, and sulfonamides
Kjeldahl measurement of nitrogen
nitrogen content of ○ interfere albumin to HABA
content
16%
Formulation of Routine method; Bromocresol Green (Bcg)
violet-colored chelate requires at least two ● Not affected by any interfering substances that can be
Biuret present in the sample such as bilirubin and salicylates.
between Cu2+ ions and peptide bonds and an
peptide bonds alkaline medium ● hemoglobin binds to BCG
Protein binds to dye and ○ for every 100 mg/dL of Hgb, albumin increased
Dye binding causes a spectral shift in Research use by 0.1 g/dL
the absorbance maximum ● has been reported to overestimate low albumin values
○ nephrotic syndrome or end-stage renal disease
2
○ This pattern is going to be the basis as to what
Bromocresol Purple (Bcp) type of protein has caused the abnormality that
● alternate dye, binding specifically to albumin led to the px condition
● not subject to interferences, precise, exhibits excellent ● The scanning densitometer will compute the area under
correlation with immunodiffusion reference methods the absorbance curve for each band and the percentage
● Disadvantage: In renal insufficiency, BCP method of total dye that appears in each fraction.
underestimates serum albumin ○ This percentage will be multiplied to the total
● Bilirubin interferes with BCP binding to Albumin protein which is measured separately
● Concentration is calculated as a percentage of the total
Total Globins protein
● If in case that the methods for albumin determination is ● Computation also be made by cutting out the small
not available, we can still get the albumin levels by bands from the membrane and eluting the dye in 0.1
calculation mol/L NaOH
● Albumin can be calculated by subtraction of the globulin ● Absorbances are added to obtain total absorbance, and
from Total Protein. the percentage of the total absorbance found in each
● Total Globulin level- direct colorimetric method using fraction is calculated
glyoxylic acid.
● Glyoxylic acid, in the presence of Cu2+ and in an acid Reference values
medium (acetic acid and H2SO4), condenses with Fraction Percentage Concentration
tryptophan found in globulins to produce purple color.
Albumin 53% - 65% 3.5 - 5.0 g/dL
● Read spectrophotometrically, kukunin yung absorbance
and will be utilized to compute the total globulin levels. Alpha-1-globulin 2.5% - 5% 0.1 - 0.3 g/dL
● Determination of TG based on the presence of Alpha-2-globulin 7% - 13% 0.6 - 1.0 g/dL
tryptophan is not commonly used because mas easier Beta-globulin 8% - 14% 0.7 - 1.1 g/dL
gamitin ang dye binding method for albumin
Gamma-globulin 12% - 22% 0.8 - 1.6 g/dL
determination.
● Reference values for each fraction (Bishop, 7th ed.)
Electrophoresis ● Rank according to % in the total protein
● After determining the total albumin and globulin levels, if ○ 1st = Albumin
abnormalities are noticed then we can proceed to ○ 2nd = Gamma
electrophoresis. ○ 3rd = Beta
● separates proteins on the basis of their electric charge ○ 4th = Alpha
densities. ○ 5th = Alpha - 1
● protein will move according to their density determined
by pH of a surrounding buffer. SELECTED DENSITOMETRIC PATTERNS OF PROTEIN
● direction of movement depends on charge ELECTROPHORESIS
○ Cations → cathode terminal (-)
○ Anions → anode terminal (+) Reference Pattern
● the speed of migration can be estimated from the
difference between the pIof the protein and the pH of the
buffer.
● velocity depends on electric field strength, size, and
shape of molecule, temperature and characteristics of
buffer
● Cellulose acetate or agarose gel- support media
Serum Protein Electrophoresis

● Serum are applied close to the cathode end of a support
medium (ph 8.6)
● All major serum proteins carry a net negative charge at
ph 8.6 and migrate toward the anode.
● 5 bands:
○ Albumin- travels farthest to the anode
○ alpha-1-globulins
○ alpha-2-globulins
○ beta-globulins
○ Gamma-globulins
● Width of the band = number of proteins present in that
fraction.
● After separation, protein fractions are fixed by immersing ● All protein fractions are in normal level
the support medium in an acid solution (acetic acid)
● Next step, proteins are stained.
○ Dyes
■ Ponceau S
■ Amido black
■ Coomassie blue
● Cleared transparent medium is placed in a scanning
densitometer for reading
● The pattern on the membrane moves past a slit through
which light is transmitted to a phototube to record the
absorbance of the dye that is bound to each protein
fraction
○ The absorbance is going to determine the
quantity of the protein that is present
● Absorbance is recorded on a strip-chart recorder to
obtain a pattern of the fraction
3
Monoclonal Increase Inflammation
● Sharp peak in the gamma globulin area

● Increased production of gamma globulins ● Decrease in albumin but increase in alpha-1, alpha-2,
and beta bcs of increased production of acute phase
Alpha-1-Antitrypsin Deficiency reactants
● Alpha-1 = acid glycoprotein, alpha-1-antitrypsin
● Alpha-2 = ceruloplasmin & haptoglobin
● Beta = C reactive protein
● Commonly seen in trauma, burns, infarction, malignancy,
and liver disease
● Why acute phase reactants?
○ Bcs they are increased mainly in the serum
within a few days following trauma or exposure
to inflammatory reagents
○ Fibrinogen, haptoglobin, ceruloplasmin, serum
amyloid a = will increase several folds
○ C reactive protein & alpha-2 macroglobulin will
increase several hundred folds
○ Good indicators of the presence of
inflammation
● If gamma is increased = chronic infection
Cirrhosis
● Deficiency in alpha-1
Nephrotic Syndrome
● Distinct bcs of the presence of beta gamma bridge

○ Caused by the presence of fast moving gamma
globulins in the px sample that prevents distinct
bands from appearing between the gamma and
beta globulin fractions therefore, forming a
bridge between two
● Px tend to lose some of their serum albumin and low ○ Continuous pattern
molecular weight proteins including some globulin ■ Caused by fast moving
gamma
● Increased alpha-2 macroglobulin, beta lipoprotein, High Resolution Protein Electrophoresis
complement components and haptoglobin ● Modifying electrophoretic parameters (12 bands)
● Uses higher voltage coupled with a cooling system and
4
more conc. buffer.
● Agarose gel
○ support medium
● semiquantitative estimates of protein
○ Densitometer
● useful in detecting small monoclonal bands and
differentiating unusual bands
*
PROTEINS IN OTHER BODY FLUIDS
Urinary Protein
● Can originate from the kidneys and the urinary tract, lso
vagina and the prostate
● Proteinuria
○ resulting from glomerular or tubular dysfunction
○ Useful indicator for problems of glomerular or
tubular portions of the kidney
● Qualitative test: Reagent test strip
○ Dip the strip in the urine
○ Observe for color
○ Protein error of indicators
● Quantitative test: 12-24 hour urine specimen
○ Results are generally reported in terms of
weight of protein for 24hrs by calculating the
amount of protein that is present in the total
volume of the urine collected during that time
● Precipitation methods: Sulfosalicylic Acid (SSA),
Trichloroacetic acid (TCA), Benzethonium chloride
● Chemical Methods: Biuret Method, Folin-Lowry Method
○ Biuret agent is mainly with the principle of the
complex formed by the cupric ions of the biuret
reagent with the peptide bonds which gives off
a pink to reddish-violet color
○ Folin-Lowry Method, it is going to use
Folin-Ciocalteu′s reagent which is a
phosphotungstomolybdic acid solution or
frequently called as phenol reagent because it
is able to oxidize phenolic compounds
■ The reagent will change in color from
Prealbumin - fastest to migrate yellow to blue during the reaction with
the following amino acids mainly
Other Methods: tyrosine, tryptophan, and histidine that
● Capillary Electrophoresis is present in the urine sample
○ separation of molecules takes place in silica ● Dye-binding methods: Coomasie blue, Ponceau S
capillaries
○ capillaries are typically 30-50 cm long Urine Protein Methods
○ Amount of sample is nm
● Isoelectric Focusing
Method Principle Comment
○ zone electrophoresis that separates proteins on
the basis of pI
○ When a protein is electrophoresed in the gel, it Turbidimetric Proteins are Rapid, easy to
migrates to a place in the gel where the pH is methods precipitated as fine use; (problem)
the same as its isoelectric point (sulfosalicylic acid, particles, turbidity is unequal
● Immunochemical Methods trichloroacetic acid, measured sensitivity for
○ specific proteins may be identified by or benzethonium spectrophotometrically individual person
immunochemical assays in which the reaction chloride)
of protein (antigen) and antibody is measured
○ radial immunodiffusion, immunoelectrophoresis, Biuret Proteins are Accurate
immunofixation electrophoresis, electro concentrated by
immunodiffusion, immunoturbidimetry, precipitation,
immunonephelometry redissolved in alkali,
○ Protein serves as the antigen (same with then reacted with Cu2+,
racket) Cu2+ forms colored
complex with peptide
bonds
Folin-Lowry Initial biuret reaction; Very sensitive

oxidation of tyrosine,
5
● CSF is one of the most carefully handled
tryptophan, and samples in the lab because of the difficulty of its
histidine residues by extraction
Folin phenol reagent ● Means of collecting: lumbar of spinal tap
(mixture of ● The doctor will do the collection by inserting a
phosphotungstic and syringe between lumbar 4 and 5 vertebra
phosphomolybdic 1. Lie on table in fetal position
acids); measurement of 2. Doctor injects anesthetic to numb lower back
resultant blue color 3. Needle inserted between lower back bones
4. Small amount of cerebrospinal fluid drawn
Dye-binding Protein binds to dye, Limited linearity;
(Coomassie blue, causes shift in unequal
Ponceau S) absorption maximum sensitivity for
individual
proteins
CSF Protein
● The presence of proteins in the CSF is indicative of
infection, inflammation, or any other types of nervous
system disorder
○ Also, it can indicate a problem in the
capillary-endothelial barrier through which
ultrafiltration occurs
● Reference interval (10-40 years old)
○ 15-45 mg/dL
● Abnormal increased of total CSF Proteins
○ Conditions will include bacterial ,viral and
fungal meningitis, traumatic tap during CSF
collection, multiple sclerosis, obstruction in the ● The CSF must be separated into three tubes
neoplasm, and cerebral infarction ○ Tube 1: chemistry / serology
● Degree of permeability can be evaluated by measuring ○ Tube 2: microbiology
the CSF albumin comparing it with the serum albumin ○ Tue 3: hematology
○ Albumin is a very good indicator of nervous ○ The excess CSF must be kept in another tube
system problems because it is not produced in just in case there are additional tests to be
the CSF , but in the liver performed in the sample
○ In the event that albumin is higher in CSF than ● The accepted sample must appear clear
serum albumin, that is indicating increased ● If there is presence of blood, hemolysis, xanthochromia
permeability of capillaries or a problem in the because of the presence of bilirubin - it must be rejected
blood-brain barrier ● But if after centrifugation, you have a CSF with clear
supernatant and formation of red cell button in the bottom
- can still be accepted for processing
What to expect during a Spinal Tap
6
LECTURE 1: RENAL FUNCTION: LABORATORY

APPLICATIONS
Prof. Josephine Cuajotor, RMT
November 8, 2021
Introduction ○ Gold standard; reference method

● Discuss the Non-Protein Hydrogen Compounds (NPNs) ○ Isotopic dilution is a method that determines the
that are considered as waste products formed in the quality of the chemical substance
body as a result of the degradative metabolism of nucleic ○ Mass Spectrometry is a medical method or
acids as well as amino acids and proteins technique that ionizes the chemical species and
● The excretion of these compounds is an important sorts the ions based on mass ratio
function of the kidneys
● Three important principal compounds of the NPNs 1. Conventional Method
○ Urea ● Hydrolysis of urea by urease prepared by the
○ Creatinine jack beans
○ Uric Acid ● Considered as the indirect method in
calculating the concentration of urea
BLOOD UREA NITROGEN (BUN) ● After the urease reaction, the ammonium ions
● One of the most important and abundant NPN produced is treated with
● Why measure? ● Quantification of NH4+
○ To evaluate renal function ○ Nessler’s reaction
○ To assess hydration status ○ Berthelot’s reaction with Nitrop (?)
■ BUN is a good indicator of the ● The ammonia and carbon dioxide produced
nitrogen state and state of hydration are measured by the various methods to
○ To determine nitrogen balance calculator the concentration of urea in the
○ To aid in the diagnosis of renal disease original sample
○ To verify the adequacy of dialysis ● The measurement of ammonium is often used
■ Since it can be easily removed by
dialysis 2. Kinetic Method (Coupled-Enzyme)
● Urease and GLDH
Specimen Requirements ○ Urea is hydrolyzed enzymatically by the
● A fasting sample is not required enzyme urease to produce ammonia and CO2
○ Although the protein content of the diet ● Measures disappearance of NADH @ 340 nm
influences urea concentration, the effect of a ○ Rate of change of the absorbance at 340 nm
single protein-containing meal is minimal due to disappearance of NADH is directly
● Ammonium ions and high concentrations of sodium proportional to the urea concentration
fluoride and sodium citrate should be avoided because ● Used on common automated machines
they will inhibit urease
○ To avoid false results
● Thiosemicarbazide and ferric ions are added to enhance
color development in the methods to be used in the
determination of urea concentration
● Urinary urea measurements- calculation of nitrogen
balance
● In terms of using the urine as a specimen, especially the
24-hour urine specimen, urine should be refrigerated
Conversion Factors:
● Urea is hydrolyzed by urease to ammonia and carbon
● Urea Nitrogen x 2.14 = Urea (mg/dL)
dioxide
● Urea Nitrogen (mg/dL) x 0.36 = Urea (mmol/L)
● From the ammonium ions reacts with GLDH to form
products glutamate and water
● GLDH - Glutamate dehydrogenase (EC 1.4.1.3)
Take note: Calculating the concentration of Urea is only expressed
by the nitrogen content of the area and not the pure urea 3. Chemical Methods
substance hence, it is called the Blood Urea Nitrogen and Urine
a. Diacetyl monoxime (direct method)
Urea Nitrogen is more appropriate
● Urea reacts directly with diacetyl monoxime in
strong acidic condition to form a yellow diazine
Analytical/Laboratory Methods derivatives at 540 nm
● 3 methods: ○ Direct method in the determination of
○ Conventional Method urea concentration in the sample
■ Or enzymatic method is commonly ● Ferric iron and thiosemicarbazide
used in the laboratory ○ Stabilizes color; intensifies reaction
○ Kinetic method b. ɑ-Phthalaldehyde method (direct method)
○ Chemical method ● ɑ-Phthalaldehyde
● Isotopic dilution mass spectrometry ● Naphthylethylenediamine
1
c. Urine
● Should be refrigerated if cannot be analyzed 2. Kinetic Jaffe Reaction
within an hour ● In order to increase the specificity of the
○ Urea is susceptible to bacterial creatinine level; to give more accurate results,
decomposition Kinetic Jaffe reaction is being established.
○ May cause false positive or false ● performed directly on sample
negative ● serum is mixed with alkaline picrate and the
rate of change in absorbance is measured
● Interferences:
○ α-ketoacids and cephalosporins
3. Coupled-Enzymatic Method
In order to further enhance the specificity of the reaction to the
creatine examination
● Creatininase
○ Creatininase-CK Method
■ Creatininase
■ CK, PK, LDH
● NADH to NAD+
Reference Intervals: UREA NITROGEN (340 nm)
○ Creatininase-Hydrogen Peroxide
Adult Method
■ Creatininase
Plasma or serum 6-20 mg/dL 2.1-7.1 mmol/L
● Creatinase
Urine, 24h 12-20 g/d 0.43-0.71 mol urea/d ● Sarcosine oxidase
● Peroxidase
CREATININE
4. Reference standard
Analytical/ Lab Methods: Glomerular Filtration Tests (GFTs) ● Isotope dilution-mass spectrometry (IDMS)
1. Direct Jaffe Reaction (1886)

● Creatinine reacts with picric acid (trinitrophenol)
in alkaline solution to form a red-orange
chromogen
● Folin-Wu Method (1919)
● Fuller’s earth
● Method of Hare (addition to Lloyds reagent)
Folin-Wu Method (1919)

● Nonspecific
● Interferences:
○ acetoacetate, acetone, ascorbate, glucose, and
pyruvate
○ May falsely decrease and increase creatinine
levels of the blood.
Fuller’s Earth
● PFF + aluminum magnesium silicate
● Enhances the specificity of the Jaffe reaction
● Creatinine levels which is used in the protein free filtrate
(PFF) is absorbed into fuller’s earth reagent and that is ● coupled-Enzyme method
aluminum magnesium silicate ○ Called as such because of the different enzyme
involved in the reaction.
Method of Hare (addition to Lloyd’s Reagent)
● PFF + sodium aluminum silicate.
● Creatinine in the PFF is absorbed unto the sodium
aluminum silicate and reacted with alkaline picrate
○ Eluted and reacted with alkaline picrate
Fullers Earth and Method of hare is not routinely done in the lab
setting because they are time consuming and they are not readily
automated.
Specimen requirement
● Plasma, serum
○ Hemolyzed and icteric samples should be
Pic: general principle that is involved in the Jaffe reaction avoided
2
○ Lipemic samples produce erroneous results with the same lot number as well.
○ Fasting is not required ● Pwede ba dili same lot number?
■ Bcs creatinine is formed by the body ○ Yes, but entails a lot of work and u have to run
with a relatively (?) constant weight another quality control for that reagent
depending on the muscle’s mass
*hemolysis and bilirubin can cause false decrease
● Urine
○ Urine specimens should be refrigerated or
frozen if longer than 4 days specimen
requirement
● Creatinine in serum or urine is stable for at least 7 days
at 4ºC
Interfering Substances
False increase; reacts with False decrease ● Reagent 1 and Reagent 2 (in an amber bottle)
picrate solution
● Ascorbic Acid ● Hemoglobin and

○ interfere peroxidase bilirubin
● Glucose ● Uric acid
● Protein, urea ○ Decreased in
● Alpha-keto acids/ kinetic methods
ketone
● Cephalosporins,
Dopamine, Lidocaine
URIC ACID
● Readily oxidized to alantoine which is a more water
Creatinine soluble end product and therefore can function as
reducing agents
Population Plasma, Jaffe Method Enzymatic
Serum, or Method Analytical Methods: Other Renal Function Tests
Urine
1. Caraway Method
Adult Male Plasma or 0.9-1.3 mg/dL 0.6-1.1 mg/dL ● Based on the oxidation of UA in PFF, with
serum (80-115 (55-96 subsequent reduction of phosphotungstic
μmol/L) μmol/L) acid to tungsten blue
○ Sodium carbonate
Adult Female Plasma or 0.6-1.1 mg/dL 0.5-0.8 mg/dL ■ Provides alkaline pH for
serum (56-97 (40-66μmol/L color development
μmol/L) ) ● Lacks specificity
Child Plasma or 0.3-0.7 mg/dL 0.0-0.6mg/dL

serum (27-625 (0-52 μmol/L)
μmol/L)
Adult Male Urine 24-H 800-2,000

mg/day
(7.1-17.7 2. Uricase method
mmol/day) ● Uricase catalyzes the oxidation of UA to
allantoin
Adult Female Urine 24-H 600-1,800 ○ Measures the differential absorption
mg/day of uric acid and allantoin at 293 nm
(5.3-15.9 ● More specific
mmol/day) ● hemoglobin and xanthine can cause negative
interference
3. Couple Enzymatic Methods

● Measures the hydrogen peroxide produced as
UA is converted to allantoin
○ Peroxidase or catalase
■ Catalyze a chemical
indicator reaction
● Interference:
○ Bilirubin and ascorbic acid (destroy
● Always read the storage, lot number and expiration date peroxide)
● When replacing the reagents from the auto analyzer,
kailangan yung gi replace mo na lot number na reagent
must have the same lot number
○ Ex. if lot number is F3945, it must be replaced
3
Enzymatic Colorimetric
● Uric acid is oxidized to allantoin by uricase

● The generated hydrogen peroxide reacts with
4-aminophenazone/ESPT to quinoneimine
● Aminophenazone will react with the
3,5-dichloro-2-hydroxybenzenesulfonic acid with
hydrogen peroxide (using the hydrogen peroxidase) =
quinoneimine and water
Other renal function tests uric acid
Specimen requirement
● Heparinized Plasma, serum, or urine
● Serum
○ Should be removed from cells ASAP to prevent
dilution by intracellular contents
○ Fasting is not required
■ Diet may affect the uric acid
concentration overall but a recent
meal has no significant effect and
fasting specimens are unnecessary
for this type of test
○ Avoid lipemic and hemolyzed specimens
○ May be refrigerated for 3-5 days
Interfering substances
● High bilirubin Concentration
● Ascorbic acid
○ Falsely decrease results to Peroxidase
methods
○ Addition of Potassium ferricyanide and
ascorbate oxidase (decrease)
● Drugs
○ Salicylates
○ Thiazides
○ Falsely increase results
Uric Acid (URICASE METHOD)
Adult Male Plasma or 3.5-7.2 mg/dL (0.210.42 mmol/L)

serum
Adult Plasma or 2.6-6.0 mg/dL (0.16-0.36 mmol/L)

Female serum
Child Plasma or 2.0-5.5 mg/dL (0.12-0.33 mmol/L)

serum
Adult Urine, 24 h 250-750 (1.5-4.4 mmol/day)

mg/day
4
LECTURE 2: LIVER FUNCTION: LABORATORY

METHODS AND PROCESSING
Prof. FRITDEY JAD DOCTOLERO, RMT
NOVEMBER 26, 2021
INTRODUCTION gumaana yung Vitamin K

● There are many liver tests but they can be grouped administration
according to what their purpose is ● Since Hindi siya gumana,
● Hepatic Synthesis Ability Vitamin K supply is not the
○ Determines the synthetic ability of the liver problem
● Conjugation/Excretion Function ● The main reason for the
● Detoxification Function deficiency of clotting factors
is because there is a
HEPATIC SYNTHESIS ABILITY problem in the synthetic
● The liver is the one to synthesize the majority of the ability of the liver
proteins ■ Prothrombin Time is also called
● Most of the tests are familiar because they are also tests Vitamin K Response Test
for proteins ■ Abnormal result, problonged ang
● Total Protein Determination tawag
○ Kjeldahl Method ● The result is expressed in
■ Standard reference method for seconds and the result is
protein determination more that the reference
○ Biuret Method range
○ Folin-Ciocalteu (Lowry) Method ○ Albumin
○ UV Absorption Method ■ A/G ratio
■ The absorbance the proteins at 210 ● Applicable for performing
nm is because of the absorbance of the procedures for the
the peptide bonds present in these determination of total protein
proteins and albumin
○ Electrophoresis Conjugation/Excretion Function
○ Refractometry ● Bilirubin Assay
○ Turbidimetric/Nephelometric Methods ○ Malloy-Evelyn Method
○ Salt Fractionation ○ Jendrassik-Grof Method
● Prothrombin Time ● Bromosulfophthalein Test (BSP) Dye
○ Hematologic Test ○ Function and potency of the bile ducts
○ Test that allows us to determine intrahepatic ○ Rarely used
disorders from extrahepatic obstructive liver ○ Dye Extraction Test
diseases ■ Rosenthal White Method
■ When the patient is experiencing ● Double collection method
intrahepatic disorders, prolong ang ● Inject ng dye (BSP) to the
pro-time body of the px.
■ But if it is an extrahepatic obstructive ● 2mg per kilogram of body
liver disease, expect that th results weight
would be normal because hindi na ● Collect after 5 minutes and
mismo yung liver ang may problema after 30 minutes
so the liver’s capability to synthesize ● Normal values after 5
clotting factors is not affected minutes: 50% dye retention
○ Sa intrahepatic disorders ang problem is sa ● Normal values after 30
mismong liver. minutes: 0% dye retention
■ The liver cannot synthesize ■ MacDonald Method
substances like your clotting factors ● Single collection method
● Plays a role on the ● 5mg per kilogram of body
coagulation in the blood weight
● Seen in the plasma ● Collect after 45 minutes
● The liver needs Vitamin k to ● Normal value after
produce those clotting 45minutes: +/- 5% dye
factors retention
■ Ang isa na iniisip na treatment with ● Urobilinogen
abnormal results is the administration ○ Ehrlich Method
of Vitamin K ■ Utilizes pDAB
● After administration, the ■ Para-dimethylaminobenzaldehyde
patient is tested again for ● Detoxification Function
prothrombin time, abnormal ○ Enzyme Test
or prolonged parin yung ■ ALP
result and matagal pa parin ■ ALT
ang coagulation process ■ AST
and that means hindi ■ 5’N
1
■ GGT Jendrassik-Grof Method (1938)
■ LAP ● Serum bilirubin
■ LDG ● Still uses the Diazo reagent
■ Assess the extent of liver damage ● Caffeine-benzoate-acetate (Accelerator)
and differentiate the hepatocellular ○ Ascorbic acid – terminates reaction
and disruptive diseases ■ To terminate the reaction of the Diazo
■ Why? When the liver is injured, there reagent with the bilirubin
will be cytolysis and necrosis occurs ■ Destroys the excess Diazo reagent
the enzyme inside the cells will be ○ Alkaline tartrate solution
liberated and be present in the blood ■ To shift the absorbance of the
● Ammonia - toxic form (converted to urea through the azobilirubin to a more intense blue
urea cycle in the liver) color.
○ Nesslerization Reaction ■ If the blue color is more intense, there
is less interference.
■ Measures the azobilirubin at 600 nm
● Can get the results for the three fractions of your
bilirubin:
■ Allow ammonia to react with ○ Conjugated/Direct bilirubin
potassium mercuric iodide (Nessler’s ○ Total bilirubin
reagent) ○ Unconjugated/Indirect bilirubin
○ Berthelot Reaction ● However, only the conjugated and total can be
measured. Why?
Detoxification Function ○ For unconjugated bilirubin, it is calculated.
● Ammonia ○ Subtract total bilirubin from conjugated/direct
○ Nesslerization Reaction bilirubin
○ Allow ammonia to react with potassium
Formula for unconjugated bilirubin
mercuric iodide and make sure the reaction is
B1= TB-B2
with the presence of gum ghatti = ↑ ammonia =
● B1 = Unconjugated/Indirect bilirubin
Brown precipitate
● B2 = Conjugated/Direct bilirubin
■ potassium mercuric iodide- Nessler's
● TB = Total Bilirubin
reagent
*In total bilirubin, aside from B1 and B2, if the px is sick, your total
bilirubin (TB) would also include Delta Bilirubin
● Delta Bilirubin
○ Berthelot Reaction ○ conjugated bilirubin binded to albumin
■ Allow ammonia to react with phenol ○ has a longer half-life compared to the other
and hypochlorite make sure reaction forms of bilirubin
happens in the presence of sodium ○ seen only in hepatic obstruction
nitroprusside= ↑ammonia = Blue ■ In a normal individual, the total
bilirubin is only made up of B1 and
ANALYSIS OF BILIRUBIN B2. But in patients with hepatic
obstruction, the delta bilirubin will take
Classic Diazo reaction (Ehrlich, 1883) part in the total bilirubin.
● Urine bilirubin reacts to diazotized sulfanilic acid to
produce red azo dye (540 nm). Among those methods for analysis of bilirubin, what is the
● Azo/ diazotized - a pair of nitrogen atoms preferred reference method?
● Classic - siya yung first na nakadevelop ● Candidate: Modified Jendrassik-Grof Method
● Modified Jendrassik-Grof Method vs. Original
Van den Bergh, 1913 Jendrassik-Grof Method is the ACCELERATOR.
● Using serum ○ Original: Caffeine-benzoate-acetate
● Used alcohol accelerator for the coupling of bilirubin to ○ Modified: Caffeine-benzoate
diazotized sulfanilic acid ■ No acetate
○ Accelerator = Solubilizer
■ Allows your B1 unconjugated bilirubin Advantages of Jendrassik-Grof over Malloy-Evelyn:
to react with the color reagent ● Not affected by pH changes
■ If wala ang accelerator, ang ma ○ Insensitive to a 50-fold variation in protein
memeasure lang ay conjugated concentration of the sample
bilirubin ○ Maintains optical sensitivity even at low bilirubin
concentrations
○ Has minimal turbidity and a relatively constant
Malloy-Evelyn Method (1937) serum blank
● Developed a method to quantify bilirubin in serum ○ Is not affected by hemoglobin up to 750 mg/dL
samples but still utilizing the classing diazo reaction
● Serum bilirubin Bilirubinometry
● 50% methanol (Accelerator) ● Used in neonatal population
● Performed at the pH of 1.2 ○ Because in adults, carotenoids are present,
● Has a diazo reagent which can interfere with the measurement of
○ contains sulfanilic acid in HCl acid and sodium the bilirubin.
nitrite ○ Effect: Positive interference
● Diazotized Sulfanilic Acid (DSA) will react to the central ■ Increased level of bilirubin
methylene carbon bilirubin and after that reaction, mag ● Measurement of reflected light from skin using two
s-split yung bilirubin molecule into two molecule of azo wavelengths
bilirubin = RED PURPLE → will be measured at 560 nm ○ provides numerical index based on spectral
wavelength. reflectance
2
Icterus Index
● Yellowishness of the serum or plasma
● 0.01% potassium dichromate
○ Involves diluting serum with saline until it
visually matches the color of your 0.01%
potassium dichromate.
○ Hence, comparison is done
● The number of times that the serum will be diluted is
called the ICTERUS INDEX.
SPECIMEN REQUIREMENTS
● Serum or Plasma
○ Serum is preferred for MalloyEvelyn
■ Addition of alcohol may precipitate
protein – interference!
○ Fasting sample is preferred
■ Presence of lipemia may interfere
■ If there are many fats in the
serum/plasma, the lipemia will
increase the measured bilirubin
concentrations
○ In cases of hemolysis/hemolyzed sample
■ Falsely decrease bcs it will interfere
with the reaction of bilirubin with the
diazo reagent
○ Protect from light
■ Bcs bilirubin is an unstable compound
■ Bilirubin is easily or rapidly photo
oxidized to biliverdin when exposed to
light
■ Falsely decreased by 30% - 50% per
hour when the sample is exposed to
light.
REFERENCE VALUES
3
PACKAGE INSERT
4
● reagents are stable kahit na open na
● Specimen = serum or plasma
○ No hemolytic samples
○ Stable up to 3 days protected from light,
refrigerated at 2-8 C
● Measure against a sample blank
● For the sample blank, u need the Total bilirubin reagent
(TBR)
○ 1000 ul
● Before u get 1000 ul from the bottle of TBR, u need to
thoroughly mix it first
● Aside from mixing the bottle, before placing the sample
itself, u need to mix it thoroughly again and incubate for 5
mins (at room temperature)
● 2 test tubes will be used and do not mix vigorously (just
swirl)
● Measure the absorbance of sample against the sample
blank
○ Measure first the sample blank at 546 nm and
then measure the sample tube itself
5
LECTURE 3: BLOOD GASSES: LABORATORY

METHODS AND PROCESSING
November 22, 2021
LABORATORY APPLICATION ○ A change in voltage indicates the activity of

each analyte
○ Severinghaus electrode
Specimen Collection
● Heparinized plastic syringe
○ Usually seen in hospitals
○ Usually dapat 2 mL or less na syringe
○ Pre-treated with heparin
○ Flush entire syringe up to the tip with 0.5mL
heparin and the expel the excess
○ Disadvantages:
■ Excess heparin causes downward
shifting of blood pH
● That’s why we have to be
careful sa amount ng
heparin na ilagay
■ Leaking of gas through plastic
● It will lead to an erroneous
result of the ABG
■ Should have precautionary measures ● Basic diagram for PO2 determination
with these following disadvantages ○ An oxygen-permeable membrane covers the tip
○ Addvantages: of electrode to selectively allow O2 to diffuse
■ Ease of usage into the electrolyte solution
■ Availability and resources ○ A small constant polarizing potential is applied
● Glass syringe pretreated with heparin between anode (Ag/AgCl Reference Electrode)
○ expensive and cathode (platinum wire)
● Heparinized evacuated tubes ○ As a result, the electrons are drawn from the
○ With oxygen contamination = ↑ pO2 anode to the cathode
● The physicians usually collect the sample because they ○ Then, the oxygen diffuses from the sample
are trained to do so path towards the cathode
● Sample comes from the arteries ○ The current produced in the circuit is
● Medtechs are not trained to do arterial blood collection proportional to the oxygen reduced at the
● For CBCs and Clin Chem, we should not collect the cathode
sample from the arteries because the machines are ○ Amperometric - amount of current flow
sensitive
● Machines usually detect venous blood
● It should be tratined RTs or Physicians or a trained
medtech can collect the sample
Specimen Handling and Storage

● Blood samples should be chilled with the use of ice chips
○ Prevents O2 consumption by the RBCs and
release of acidic metabolites
● Sources of error:
○ Specimen exposed to air
■ Decrease pCO2, increase pH,
increase pO2
○ Specimen at RT more than 30 mins
■ Decrease pO2, decrease pH, increase
pCO2
Instrumentation: Arterial Blood Gas Analyzer ● pCO2 electrode system

● Uses 3 electrodes as sensing devices to measure pO2, ○ PCO2 is determined with a modified pH
pCO2, and pH electrode (severinghaus electrode)
● pO2 measurement is amperometric ○ An outer semipermeable membrane will allow
○ Amount of current flow is an indication of the CO2 diffuse to the layer of electrolyte
oxygen present ○ A bicarbonate buffer covers the covers the
○ Measurement of the electric current flowing glass of the pH electrode
under an applied and potential difference ○ The CO2 that diffuses across the membrane
between two electrodes in a solution will react with the buffer, forming the carbonic
○ Clark electrode acid
● pCO2, and pH measurements are potentiometric ○ Which then dissociate into bicarbonate and
1
hydrogen
○ The change in the activity of the hydrogen is
measured by the ph and electrode related to
PCO2
○ Talking about the change in activity of hydrogen
ion
STEP 1 Evaluate the pH

● pH = 7.35 – 7.45
○ less than 7.35 = acidosis
○ more than 7.45 = alkalosis
● If pH is not given in the problem, calculate the pH using
Henderson-Hasselbalch Equation:
● pH electrode system
○ A glass membrane sensitive to hydrogen is
placed around an internal Ag/AgCl to form
measuring electrode
○ The reference electrode (calomel or Ag/AgCl
electrode) provides a steady reference voltage
against which voltage changes from the ● wherein: pk’ is 6.1 (constant)
measuring electrode are compared Example: In healthy individual..
○ A comparison to the measuring electrodes ● HCO3 – 24mmol/L, pCO2 – 40 mm Hg
○ Detect changes in voltage, then detect pH ● pH = 6.1 + log ( 24 / 0.0307 x 40)
● pH = 7.35
Reference electrodes:
● Calomel electrode ● If dC02 is given, calculate the pH using this formula:
○ mercury/mercurous chloride
● Silver/silver chloride
○ overall better and faster
● Normal hydrogen electrode
Arterial blood gas analyzer

● wherein: pk’ is 6.1 (constant)
STEP 2 Evaluate the ventilation (lungs)
● pCO2 = 35 – 45 mm Hg
○ < 35 = respiratory alkalosis
○ > 45 = respiratory acidosis
STEP 2 Evaluate the metabolic process (kidneys)

[HCO3-] = 22 – 26 mmol/L (mEq/L)
● < 22 = metabolic acidosis
● > 26 = metabolic alkalosis
STEP 3 Determine which is the primary and compensating

V. ABG INTERPRETATION disorder
● To assess whether primary problem is respiratory or
Normal Values metabolic in origin, compare changes HCO3 and PCO2
from baseline
● If the change in HCO3 from baseline is larger, then the
problem is primarily metabolic and vice versa
Check pH Check △HCO3 & △ Primary disturbance

CO2
pH <7.4 △HCO3 > △CO2 Metabolic acidosis
△CO2 > △HCO3 Respiratory acidosis
pH >7.4 △HCO3 > △CO2 Metabolic alkalosis
△CO2 > △HCO3 Respiratory alkalosis
2
*kung sino yung the same sa step 1, yun ang primary disorder B. partial compensation
Ex. acidosis ang step 1, acidosis din ang primary ● 7.31 – 7.34 acidosis
● 7.46 – 7.49 alkalosis
STEP 4 Determine the degree of compensation: C. complete compensation
● Non-compensatory Partial = Implies that the pH is approaching normal
● Partial compensation Complete = Implies that the pH has returned to the normal range
○ 7.31 – 7.34 acidosis
○ 7.46 – 7.49 alkalosis ABG INTERPRETATION
● Complete compensation
STEP 5 Evaluate the degree of oxygenation
Partial = Implies that the pH is approaching normal ● pO2 = 80 – 110 mm Hg
Complete = Implies that the pH has returned to the normal range ● (adequate oxygenation
Non-compensatory = 7.30 below or 7.49 above ● Hypoxemia
○ Mild = 60 – 79 mm Hg
○ Moderate = 40 – 59 mm Hg
STEP 5 Evaluate the degree of oxygenation
○ Severe = 39 mm Hg or less
pO2 = 80 – 110 mm Hg (adequate oxygenation)
● Hypoxemia:
STEP 6 Final Interpretation
○ Mild = 60 – 79 mm Hg
○ Moderate = 40 – 59 mm Hg a. Degree of Compensation
○ Severe = 39 mm Hg or less b. Primary Disorder
c. Degree of Oxygenation
● Partially Compensated Metabolic Acidosis with
STEP 6 FINAL INTERPRETATION
Mild Hypoxemia
● Degree of Compensation ● In clinical settings, you still have to compute on
● Primary Disorder the FIO2 to know if we should put nasal prong,
● Degree of Oxygenation mask, or incubate the patient
ABG INTERPRETATION:SUMMARY
SAMPLE CASE
20/M with diarrhea of greater than 5x/day.
● Interpret the ABG result: Case
○ pH: 7.32
○ pC02 = 28 mmHg Step 1 Evaluate pH (and calculate if Acidosis
○ HCO3 = 14 meq/L applicable)
○ PO2 = 78%
Step 2 Evaluate the Ventilation Metabolic Acidosis
STEP 1: Evaluate the pH
pH = 7.35 – 7.45 Step 2 Evaluate the Metabolic Metabolic Acidosis
● less than 7.35 = acidosis Process
● more than 7.45 = alkalosis
Step 3 Determine the Respiratory Alkalosis
STEP 2: Evaluate the ventilation (lungs) Compensating Disturbance
pCO2 = 35 – 45 mm Hg
● < 35 = respiratory alkalosis Step 4 Degree of Compensation Partially Compensated
● > 45 = respiratory acidosis
Step 5 Check Oxygenation Mild Hypoxemia
STEP 2: Evaluate the metabolic process (kidneys)
[HCO3-] = 22 – 26 mmol/L (mEq/L) Step 6 Final Interpretation Partially Compensated
● < 22 = metabolic acidosis Metabolic Acidosis
● > 26 = metabolic alkalosis with Mild Hypoxemia
STEP 3: Determine which is the primary and compensating

disorder ● There are cases that the pH level is normal if fully
compensated and that’s the time where you should
compute for the FIO2
○ To determine primary and compensating
disorder
In this case, change in HCO3 is greater than pCo2, therefore the
acidosis is METABOLIC in origin
Basis:
● Change in HCO3 = (24-14)/ 24 = 0.42
● Note: (24 is the normal HCO3 level, 14 is the given
HCO3 value in the case)
● Change in pCoz = (40-28)/ 40 = 0.30
Note: (40 is the normal pCO2 level, 28 is the given value in the
case)
● Therefore 0.42 is greater than 0.30, change in HCO3 is

greater than pCoz.
Primary Disturbance: Metabolic Acidosis
Compensating Disturbance: Respiratory Alkalosis
STEP 4 Determine the degree of compensation:

A. non-compensatory
3

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CLINICAL CHEMISTRY LEC

LECTURE 1: LABORATORY SAFETY AND

● Example on how we plan and ensure safety to the

Examples of Engineering Controls Administrative Controls

HAZARDS CLASSIFICATION: TYPES OF HAZARDS

Safety Hazards General Classification

POSTURE AND POSITION

LECTURE 2: EXPRESSING CONCENTRATIONS

Which among the 4 is very concentrated and the least 𝑥 (𝑚𝐿)

𝑣𝑜𝑙𝑢𝑚𝑒 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 𝑥 (𝑔)

𝑤𝑒𝑖𝑔ℎ𝑡 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒

Volume of solution= volume of solute + volume of solvent

Addressing the first question

𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒

● The final answer should be in a sentence form

𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒

Calculate the barium chloride concentration in mEq/L of a

How many grams of NaCl would be in a 100g of a 1 molal

𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒 𝑚𝑤 137.33 + 35.45 (2)

What is the normality of a solution that contains 150 grams

𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒

How do you make 500 mL of a 2N NaCl?

𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒

Example: 250 𝑚𝑔 10 𝑑𝐿 𝑔 𝑚𝑜𝑙𝑒 1𝑒𝑞 1000 𝑚𝑒𝑞

% = g/100mL 𝑚𝑔 𝑑𝐿 𝑔 𝑚𝑜𝑙𝑒 𝑒𝑞 𝑚𝑒𝑞

10 𝑚𝑔 10 𝑑𝐿 𝑔 𝑚𝑜𝑙𝑒 1000 𝑚𝑚𝑜𝑙

LECTURE 4: OPTICAL TECHNIQUES

● Can be described as discrete packets of energy

Important things to know:

● It means that it would take ½ second for a cycle to occur

● Wavelengths ranging from 1mm to 1m

● Different textbooks will give different wavelengths

Visible Light (Red)

Visible Light (Orange)

Visible Light (Yellow)

Visible Light (Green)

● What your eyes perceive, the thing absorbs

waves are in-phase

waves are out-of-phase

Pic left: true interference pattern of white light.

● If the concentration of the solution is low, and when u

● Glucose with Dinitrosalicylic acid (DNS)

○ Photodetector at the end

● Spectrum of visible light

Entrance and Exit Slits

Bandpass or Bandpass width

● Prism is the simplest monochromator

II. Interference Filters

● The polychromatic light is coming from this direction, but

● For blue wavelengths, once it is reflected back, it is

● analytical cell or sample holder

● Converts transmitted light energy into an equivalent

Barrier-Layer Cell (Photocell)

● Depletion Or intrinsic region

● How do we know that spectrophotometer is really giving

● used for Group 1 metals (1+ charge)

● Na+ – emits yellow light (589 nm)

ATOMIC ABSORPTION SPECTOPHOTOMETRY

LUMINESCENCE both electrons get combined, like a magnet = triplet state