Download Positive Acute Phase Proteins

Plasma Proteins
& Protein Electrophoresis
HMIM224
Objectives of the lecture
1- Recalling in brief general physiological functions of plasma proteins.
2- Understanding main lines of plasma protein measurement
3- Discussing the main concepts of plasma protein electrophoresis as
a semiquantitative method for protein measurement in clinical labs.
4- Recognizing the main biochemical, pathological & clinical aspects of some of
the plasma proteins as prealbumin, albumin, gamma globulins & acute phase
proteins.
Plasma Proteins
Over 300 proteins have been detected in plasma .
The concentration of many of these are affected by pathological processes.
General functions of plasma proteins include:
 Transport
 Maintaining plasma oncotic pressure
 Buffering pH changes
 Humoral immunity
 Enzyme activity
 Clotting
 The acute inflammatory response.
Metabolism of Plasma Proteins:
The concentration of plasma proteins (PP) is determined by 3 main
factors:
Rate of synthesis
Rate of catabolism
The volume of fluid in which proteins are distributed.
A) Synthesis:
Most plasma proteins are synthesized in the liver, although some of
them are produced in other sites e.g. Immunoglobulins by
lymphocytes.
B) Distribution:
In health, the total concentration of proteins in plasma is around 70 g/L
in normal adult males
Water passes more freely through capillary walls than proteins and
therefore the concentration of proteins in the vascular space is affected
by fluid distribution.
Application of a tourniquet for extended periods leads to fluid loss from the
occluded veins; increasing apparently plasma protein concentrations.
C) Catabolism:
PP are degraded throughout the body.
Most proteins are degraded after being taken up by cells within the body.
Measurement of plasma proteins
A) Quantitative measurement of a specific protein:
by chemical or immunological methods
B) Semiquantitative measurement by electrophoresis:
• Proteins are separated on the basis of their electrical charge.
• Procedure:
1- Serum or plasma is applied to a support medium (cellulose acetate or a gel)
2- Electrical current applied.
3- The gel is stained with a dye that visualizes proteins.
• In normal cases, electrophoreses separates the proteins into five broad fractions:
Albumin, a1-globulin, a2-globulin, b-globulin & g-globulin
Each of the globulin fractions consists of a mixture of several proteins
Protein Electrophoresis
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+
Albumin
a1
a2
b
g
Electrophoresis pattern for normal serum proteins
Normal Pattern of Plasma Protein Electrophoresis
by Densitometry
Electrophoretic BAND
Proteins
Albumin
Albumin
a1-globulin
a1-Antitrypsin
Prothrombin
a1-Fetoprotein (AFT)
a2-globulin
b- globulin
g-globulin
Ceruoplasmin
Haptaglobin
a2-macroglobulin
C-Reactive Protein ( CRP)
Transferrin
b2-microglobulin
Immunoglobulins
(A, G, M, D & F)
Prealbumin
• Prealbumin migrates faster than albumin in the classic electrophoresis.
• It is the transport protein for:
- Thyroid hormones
- Retinol (vitamin A)
• Prealbumin is decreased in:
Liver disease
Nephrotic syndrome
Acute phase inflammatory response
Malnutrition.
Albumin
Some important facts to know about albumin:
• Albumin is present in higher concentrations than other plasma proteins
( ~ 40 g/L in normal adults).
• Albumin is synthesized in the liver & has a half-life of 20 days.
• Very small amounts of albumin cross the glomerular capillary wall.
Accordingly, no more than traces of albumin may normally appear in urine
that can not be detected by ordinary laboratory means.
• Albuminuria :
In this case, albumin can be detected in urine by ordinary laboratory means
due to physiological or pathological conditions.
Albumin (cont.)
Functions of albumin:
1- Oncotic pressure:
Albumin is responsible for ~ 80% of the plasma oncotic pressure.
It is a major determinant of the distribution of fluids between intravascular & extravascular
compartments.
Hypoalbuminemia leads to edema.
2- Buffering.
3- Transport:
Many substances are transported in the blood bound to albumin e.g.
Lipid-soluble substances
Hormones e.g. thyroid hormones & steroid hormones
Calcium
Drugs e.g. salicylates
Free fatty acids (FFA)
Billirubin
Albumin (cont.)
Causes of hypoalbuminemia
Artfuctual :
Diluted sample
Physiological : Pregnancy - Recumbence
Decreased amino acids : Reduced essential amino acids in diet & reduced synthesis of nonessential aa
Manlnutrition
Malabsorption.
Increased catabolism :
Surgery
Trauma
Infections
Defective synthesis in liver: Chronic liver diseases
Increased loss : From the kidney:
Nephrotic syndrome
From GIT:
Protein loosing entropathies
Hypergammaglobulinemia
Increases immunoglobulins levels may result from stimulation of many clones of B
cells (polyclonal hypergammaglobulinemia) or monoclonal proliferation
(paraproteinemia).
Polyclonal hypergammaglobulinemia:
Stimulation of many clones of B cells produce a wide range of antibodies that appear
as diffuse increase in g-globulin on electrophoresis.
e.g. acute and chronic infections & autoimmune diseases
Monoclonal hypergammaglobulinemia
Proliferation of a single B-cell clone produces a single immunoglobulin which
appears as a discrete densely stained band (Paraprotein or M band) on
electrophoresis.
Paraproteins are characteristic of malignant B-cell proliferation.
Multiple myeloma is the commonest cause of paraproteinemia
Positive Acute Phase Proteins
• Stresses increases the levels of some of plasma proteins as occur in
infection, inflammation , malignancy, trauma or major surgery.
• These proteins are termed acute phase reactants and their synthesis is
a part of body’s response to injury.
a1-Antitypsin
Haptoglobin
Ceruloplasmin
Fibrinogen
C-reactive protein
Positive Acute Phase Proteins (cont.)
• Positive acute phase proteins are increased within 24 hours of injury in
response to humoral mediators (Cytokines – IL-1, IL-6, tumor necrosis
factors a and b, the interferons and platelet activating factors) which are
produced by tissue macrophages, monocytes & endothelial cells in
inflammation ,etc..
• Functions:
1. Binding to polysaccharides in bacterial walls
2. Activating complement
3. Stimulating phagocytosis
4. Protease inhibitors probably inactivate enzymes released from
lysosomes & minimize damage that may occur.
C-Reactive Protein (CRP)
It is an acute-phase protein synthesized by liver
It precipitates the polysaccharide (fraction C) of pneumococcal cell walls
It is important for phagocytosis .
Very large increase in plasma CRP occurs in many inflammatory conditions e.g.,
rheumatoid arthritis.
• CRP measurement with a sensitive assay (Ultra-sensitive CRP) is used for risk
assessment of patients with ischemic heart disease.
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Ceruloplasmin
• Synthesized by the liver
• Contains over 90% of serum copper
• It is important in acute phase response as it is able to inactivate
reactive oxygen species (ROS) that produce tissue damage
• It is important for iron absorption from the intestine.
• Plasma levels are usually low in Wilson’s disease in which copper
is accumulated in the liver leading to cirrhosis , and in the basal
ganglia of the brain.
Haptoglobin
• It is synthesized by the liver.
• It binds free hemoglobin to form complexes that are metabolized in the
RES.
• It limits iron losses which may occur as hemoglobin is small enough to be
filtered by the glomerului.
• Its plasma level decreases during hemolysis and increases in acute
inflammatory conditions (acute phase reactant).
a1-Antitrypsin
• Synthesized by the liver & macrophages
• It is acute-phase protein in order to inhibit proteases.
• Proteases arise from:
Endogenous production : by digestive enzymes such as trypsin & chymotrypsin ,etc
Infection: protease release from bacteria and from leucocytes (inflam. response)
Clinical consequences of a1-Antitrypsin deficiency:
• Neonatal jaundice evidence of cholestasis
• Childhood liver cirrhosis
• Pulmonary emphysema in young adults
Fibrinogen
Synthesized in the liver.
Its function is to form a fibrin clot (when activated by thrombin)
Fibrinogen is removed in the clotting process and is not seen in serum.
It is one of the acute phase proteins.
Its level rises with pregnancy and the use of oral contraceptives.
Decreased values generally reflects extensive coagulation during which
the fibrinogen is consumed.
Negative Acute Phase Proteins
Humoral effects of IL-1 and IL-6 include increased production of ACTH
and hence cortisol and inhibition of hepatic synthesis of proteins such as
albumin, prealbumin & transferrin (negative acute phase proteins)