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: Fakulti Pergigian

Plasma Proteins

Ts. RD.AIDIFITRINA BINTI KHIROTDIN


B.Biomedical Hons. (Clinical Biochemistry) (UKM) & M.Community Health Sc.(Hos.Management & Health Economics) (UKM)
Professional Technologist MBOT (PT 18050764), LAEP & Technical Assessor, SAMM MITI

Principal College, Students Affairs


& Lecturer (Biochemistry & Multi Disciplinary Hospital Health Management)
of UITM Branch Office Selangor, Sg.Buloh, Selayang and Teluk Intan Campus
Pejabat Rektor, Aras 1, Bangunan Akademik,

Centre of PreClinical Science Studies,


Faculty of Dentistry, Universiti Teknologi MARA Sungai Buloh Campus,
Jalan Hospital, 47000 Sungai Buloh, Selangor, Malaysia
: Fakulti Pergigian

STUDENT LEARNING OUTCOME:


1. Explain the basic of classification of plasma protein

2. Name the major types of plasma protein

3. List the major role of plasma proteins

4. Name the example of disease that use plasma protein as


diagnostic tools

5. Explain the basic principles of Electrophoresis through


experiment
SLO 1. Classes of Plasma Proteins
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Albumins - 60%  Plasma contains >300 different proteins


Globulins (α, β, γ) - 35%  Many pathological conditions affect level of
plasma protein
Fibrinogen - 4%
 Mostly synthesized in the liver
Other proteins - 1%  Some are produced in other sites
 A normal adult contains ~70 g/L of pps

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Site of synthesis of Plasma Proteins : Fakulti Pergigian

▪ Liver - 90% plasma proteins


▪ Plasma cell (B lymphocytes) - γ globulin
▪ Peptide hormones – Endocrine glands

Liver dysfunction - plasma proteins


A:G ratio (normal): 0.8-2.0., Decreased in liver
dysfunction and chronic inflammatory conditions.

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: Fakulti Pergigian

Types of Plasma Proteins


• Prealbumin
• Albumin
• α1-Globulins:
• a1-Antitrypsin, α-fetoprotein
• α2-Globulins:
• Ceruloplasmin, haptoglobin
• β-Globulins:
• CRP, transferrin, β2-microglobulin
• γ- Globulins

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SLO 2. Major type of Plasma Proteins : Fakulti Pergigian
Functions of pps : Fakulti Pergigian

• Transport (Albumin, prealbumin, globulins)


• Maintain plasma oncotic pressure (Albumin)
• Defense (Immunoglobulins and complement)
• Clotting and fibrinolysis (Thrombin and plasmin)
Functions of plasma proteins
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1. Colloid osmotic pressure in blood Colloid osmotic


2. Viscosity of blood
pressure (oncotic
3. Buffer action
4. Clotting and fibrinolysis pressure)
5. Defense function body
6. Transport function 80% of plasma oncotic
7. Plasma proteolytic enzyme system pressure is maintained by
8. Plasma protease inhibitor system albumin.
9. Nutritional reserve
It is the opposing force to
hydrostatic pressure.

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: Fakulti Pergigian

Colloid osmotic pressure (oncotic pressure) and filtration of fluid

Edema
Due to disturbance in
hydrostatic and/or
oncotic pressure between
intra-capillary and
interstitial component.

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Organ specific : Fakulti Pergigian

• Brain: Cerebral edema


• Lung: Intra-alveolar=pulmonary edema,
intra-pleural=pleural effusion
Clinically
• Peritoneum=ascites ▪Pitting ▪Non pitting
• Severe generalized edema=anasarca
• Leg edema- deep venous thrombosis
Edema▪Pitting ▪Non pitting

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SLO 4. Name the example of disease that use plasma protein as
diagnostic tools : Fakulti Pergigian

Prealbumin (Transthyretin)
 A transport protein for:
 Thyroid hormones
 Retinol (vitamin A)
• Migrates faster than albumin in electrophoresis
 Separated by immunoelectrophoresis
Lower levels found in:
 liver disease, nephrotic syndrome, acute phase
inflammatory response, malnutrition
 Short half-life (2 days)
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Albumin Functions : Fakulti Pergigian

• Most abundant plasma protein • Maintains oncotic pressure:


(~40 g/L) in normal adult • The osmotic pressure exerted by
plasma proteins that pulls water
 Synthesized in the liver as into the circulatory system
preproalbumin and secreted as
• Maintains fluid distribution in and
albumin outside cells and plasma volume
 Half-life in plasma: 20 days
 Decreases rapidly in injury, • 80% of plasma oncotic pressure is
infection and surgery maintained by albumin

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Functions : Fakulti Pergigian

• A non-specific carrier of
• hormones, calcium, free fatty acids,
drugs, etc.

• Tissue cells can take up albumin


by pinocytosis where it is
hydrolyzed to amino acids

• Useful in treatment of liver


diseases, hemorrhage, shock and
burns

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Hypoalbuminemia
• Causes
• Decreased albumin synthesis (liver cirrhosis, malnutrition)
• Increased losses of albumin
• Increased catabolism in infections
• Excessive excretion by the kidneys (nephrotic syndrome)
• Excessive loss in bowel
• Severe burns (plasma loss in the absence of skin barrier)

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Hypoalbuminemia : Fakulti Pergigian

Effects
• Edema due to low oncotic pressure
• Albumin level drops in liver disease causing low
oncotic pressure
• Fluid moves into the interstitial spaces causing
edema
• Reduced transport of drugs and other substances in
plasma
• Reduced protein-bound calcium
• Total plasma calcium level drops
• Ionized calcium level may remain normal

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Hyperalbuminemia : Fakulti Pergigian

• No clinical conditions are known that cause


the liver to produce large amounts of albumin
• The only cause of hyperalbuminemia is
dehydration

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Hypo-proteinemia
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• Liver failure
• Nephrotic syndrome
• Malnutrition
• Malabsorption
• Severe burns
• Infection (↑catabolism)
• Genetic

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a1-Antitrypsin
 Synthesized by the liver and macrophages
 An acute-phase protein that inhibits proteases
 Proteases are produced endogenously and from
leukocytes and bacteria
 Digestive enzymes (trypsin, chymotrypsin)
 Other proteases (elastase, thrombin)
 Infection leads to protease release from bacteria and
from leukocytes

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Types of a1-Antitrypsin
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• Over 30 types are known


• The most common is M type
• Genetic deficiency of a1-Antitrypsin
• Synthesis of the defective a1-Antitrypsin occurs in the
liver but it cannot secrete the protein
• a1-Antitrypsin accumulates in hepatocytes and is
deficient in plasma

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Clinical Consequences of a1-Antitrypsin : Fakulti Pergigian

Deficiency
• Neonatal jaundice with evidence of cholestasis
• Childhood liver cirrhosis
• Pulmonary emphysema in young adults

Laboratory Diagnosis
• Lack of a1-globulin band in protein electrophoresis

• Quantitative measurement of a1-Antitrypsin by:


• Radial immunodiffusion, isoelectric focusing or nephelometry

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: Fakulti Pergigian

a-Fetoprotein (AFP)
• Synthesized in the developing embryo and fetus by
the parenchymal cells of the liver
• AFP levels decrease gradually during intra-uterine
life and reach adult levels at birth
• Function is unknown but it may protect fetus from
immunologic attack by the mother
• No known physiological function in adults

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: Fakulti Pergigian

a-Fetoprotein (AFP)
• Elevated maternal AFP levels are associated with:
• Neural tube defect, anencephaly
• Decreased maternal AFP levels are associated with:
• Increased risk of Down’s syndrome
• AFP is a tumor marker for:
Hepatoma and testicular cancer

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: Fakulti Pergigian

Ceruloplasmin
• Synthesized by the liver
• Contains >90% of serum copper
• An oxidoreductase that inactivates ROS causing
tissue damage in acute phase response
• Important for iron absorption from the intestine
• Wilson’s disease:
• Due to low plasma levels of ceruloplasmin
• Copper is accumulated in the liver and brain

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: Fakulti Pergigian

Haptoglobin
• Synthesized by the liver

 Binds to free hemoglobin to form complexes that


are metabolized in the RES

 Limits iron losses by preventing Hb loss from kidneys

 Plasma level decreases during hemolysis

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: Fakulti Pergigian

Transferrin
 A major iron-transport protein in plasma
 30% saturated with iron

 Plasma level drops in:


 Malnutrition, liver disease, inflammation,
malignancy

 Iron deficiency results in increased hepatic


synthesis

 A negative acute phase protein


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: Fakulti Pergigian

2–Microglobulin
• A component of human leukocyte antigen (HLA)
 Present on the surface of lymphocytes and most
nucleated cells
 Filtered by the renal glomeruli due to its small size
but most (>99%) is reabsorbed
 Elevated serum levels are found in
 Impaired kidney function
 Overproduction in disease
 May be a tumor marker for:
 Leukemia, lymphomas, multiple myeloma

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: Fakulti Pergigian

C-Reactive Protein (CRP)


• An acute-phase protein synthesized by the liver

• Important for phagocytosis

• High plasma levels are found in many inflammatory


conditions such as rheumatoid arthritis

• A marker for ischemic heart disease

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: Fakulti Pergigian

Hypergammaglobulinemia
• May result from stimulation of
• B cells (Polyclonal hypergammaglobulinemia)
• Monoclonal proliferation (Paraproteinemia)

Polyclonal hypergammaglobulinemia:
• Stimulation of many clones of B cells produce a wide
range of antibodies
• -globulin band appears large in electophoresis
• Clinical conditions: acute and chronic infections,
autoimmune diseases, chronic liver diseases
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: Fakulti Pergigian

Monoclonal Hypergammaglobulinemia

 Proliferation of a single B-cell clone produces a single type of Ig


 Appears as a separate dense band (paraprotein or M band) in
electrophoresis
 Paraproteins are characteristic of malignant B-cell proliferation
 Clinical condition: multiple myeloma

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: Fakulti Pergigian

Measurement of Plasma Proteins


A) Quantitativemeasurement of a
specific protein:
Chemical or immunological reactions

B) Semiquantitative measurement by
electrophoresis:
 Protes are separated by their electrical
charge in electrophoresis
 Five separate bands of proteins are
observed
 These bands change in disease

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Methods of plasma protein separation : Fakulti Pergigian

Common methods of protein separation into:


albumin, globulins (alpha, beta & gamma) and fibrinogen by –

▪ Electrophoresis
▪ Salting out
▪ Ultracentrifugation
▪ Affinity chromatography
▪ Fractional precipitation method
▪ Immune electrophoresis

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SLO 5. Explain the basic principles of Electrophoresis through
experiment : Fakulti Pergigian

Normal Pattern of Plasma Protein Electrophoresis

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Positive Acute Phase Proteins


• Plasma protein levels increase in:
• Infection, inflammation , malignancy, trauma, surgery

• These proteins are called acute phase reactants


• Synthesized due to body’s response to injury
• Examples: a1-Antitypsin, haptoglobin,
ceruloplasmin, fibrinogen, c-reactive protein

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: Fakulti Pergigian

Positive Acute Phase Proteins


Mediators cause these proteins to increase after
injury

Mediators: Cytokines (IL-1, IL-6), tumor necrosis


factors a and  , interferons, platelet activating
factor

Functions:
1. Bind to polysaccharides in bacterial walls
2. Activate complement system
3. Stimulate phagocytosis

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: Fakulti Pergigian

Negative Acute Phase Proteins

• These proteins decrease in inflammation


• Albumin, prealbumin, transferrin
• Mediated by inflammatory response via cytokines
and hormones
• Synthesis of these proteins decrease to save amino
acids for positive acute phase proteins

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Agarose Gel Electrophoresis : Fakulti Pergigian

Gel electrophoresis is a widely used technique for the analysis of


nucleic acids and proteins. Agarose gel electrophoresis is
routinely used for the preparation and analysis of DNA.

Gel electrophoresis is a procedure that separates molecules


on the basis of their rate of movement through a gel under
the influence of an electrical field.

We will be using agarose gel electrophoresis to determine the


presence and size of PCR products. PCR products indicate the
presence of Wolbachia.
• DNA is negatively charged.
• When placed in an electrical field, DNA will migrate toward the positive : Fakulti Pergigian

pole (anode).
• An agarose gel is used to slow the movement of DNA and separate by size.

H O2
 

DNA

- +

Power • Polymerized agarose is porous,


allowing for the movement of DNA
Scanning Electron Micrograph of
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Agarose Gel (1×1 µm)  44
How fast will the DNA migrate?
: Fakulti Pergigian

strength of the electrical field, buffer, density of agarose gel…


Size of the DNA!
*Small DNA move faster than large DNA
…gel electrophoresis separates DNA according to size

DNA

small
large

- +

Power
Within an agarose gel, linear DNA migrate inversely
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to the log10 of their molecular weight. 45
Agarose
: Fakulti Pergigian

D-galactose 3,6-anhydro
L-galactose

•Sweetened agarose gels have been


eaten in the Far East since the 17th
century.

•Agarose was first used in biology


when Robert Koch* used it as a
culture medium for Tuberculosis
bacteria in 1882

*Lina Hesse, technician and illustrator for a colleague of Koch was


the first to suggest agar for use in culturing bacteria

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Agarose is a linear polymer extracted from seaweed.
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Buffer
Making an Agarose Gel
An agarose gel is prepared by combining
agarose powder and a buffer solution.

Flask for boiling

Agarose
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Electrophoresis Equipment : Fakulti Pergigian

Power supply

Gel tank Cover

Electrical leads

Casting tray Gel combs

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Gel casting tray & combs

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Preparing the Casting Tray
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Seal the edges of the casting tray and put in the combs. Place the casting tray on
a level surface. None of the gel combs should be touching the surface of the
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casting tray.
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Agarose Buffer Solution

Combine the agarose powder and buffer solution. Use a flask that is
several times larger than the volume of buffer.

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Melting the Agarose
: Fakulti Pergigian

Agarose is insoluble at room temperature (left).


The agarose solution is boiled until clear (right).

Gently swirl the solution periodically when heating to allow all the grains of agarose to
dissolve.
***Be careful when boiling - the agarose solution may become superheated and may boil
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violently if it has been heated too long in a microwave oven.
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Pouring the gel
: Fakulti Pergigian

Allow the agarose solution to cool slightly (~60ºC) and then


carefully pour the melted agarose solution into the casting tray.
Avoid air bubbles.

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Each of the gel combs should be submerged in the melted agarose solution.
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When cooled, the agarose polymerizes, forming a flexible gel. It should appear
lighter in color when completely cooled (30-45 minutes). Carefully remove the
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Place the gel in the electrophoresis chamber.

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DNA

buffer     
wells

Anode
Cathode (positive)
(negative)

Add enough electrophoresis buffer to cover the gel to a depth of at


least 1 mm. Make sure each well is filled with buffer.

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Sample Preparation
: Fakulti Pergigian

Mix the samples of DNA with the 6X


sample loading buffer (w/ tracking
dye). This allows the samples to be
seen when loading onto the gel, and
increases the density of the samples,
causing them to sink into the gel
wells.

6X Loading Buffer: 
 Bromophenol Blue (for color)
 Glycerol (for weight)

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Loading the Gel
: Fakulti Pergigian

Carefully place the pipette tip over a well and gently expel the sample. The
sample should sink into the well. Be careful not to puncture the gel with the
pipette tip.
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Running the Gel
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Place the cover on the electrophoresis chamber, connecting the electrical leads.
Connect the electrical leads to the power supply. Be sure the leads are attached
correctly - DNA migrates toward the anode (red). When the power is turned on,
bubbles should form on the electrodes in the electrophoresis chamber.
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Cathode : Fakulti Pergigian

(-)

 wells
DNA  Bromophenol Blue
(-)

Gel

Anode
(+)

After the current is applied, make sure the Gel is running in the correct
direction. Bromophenol blue will run in the same direction as the DNA.
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DNA Ladder Standard
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-
 12,000 bp

 5,000

DNA
migration  2,000
 1,650
Note: bromophenol
blue migrates at  1,000
approximately the same  850
rate as a 300 bp DNA  650
molecule  500
 400
bromophenol blue  300
 200
+  100
Inclusion of a DNA ladder (DNAs of know sizes) on the gel makes it easy to determine the
sizes of unknown DNAs.
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As an alternative to purchasing costly DNA ladders, one can be created using
meal worm (Tenebrio molitor) DNA and a restriction enzyme. : Fakulti Pergigian

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http://people.uis.edu/rmosh1/DNAexerciseVIIa02.pdf

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