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Plasma Proteins & Protein Electrophoresis. HMIM224. Objectives of the lecture. 1- Recalling in brief general physiological functions & metabolism of plasma proteins. 2- Understanding main lines of plasma protein measurement - PowerPoint PPT Presentation
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HMIM224HMIM224
Plasma ProteinsPlasma Proteins & &Protein ElectrophoresisProtein Electrophoresis
Objectives of the lectureObjectives of the lecture
1- Recalling in brief general physiological functions & metabolism 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.
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:General functions of plasma proteins include: TransportTransport
Maintaining plasma oncotic pressureMaintaining plasma oncotic pressure
Buffering pH changesBuffering pH changes
Humoral immunityHumoral immunity
Enzyme activityEnzyme activity
ClottingClotting
The acute inflammatory response.The acute inflammatory response.
Plasma ProteinsPlasma Proteins
Metabolism of Plasma ProteinsMetabolism of Plasma Proteins
The concentration of plasma proteins is determined by 3 main factors:
A) A) SynthesisSynthesis::Most plasma proteins are synthesized in the liver, although some of them are produced in other sites e.g. Immunoglobulins by lymphocytes.
B) B) DistributionDistribution::In health, the total concentration of proteins in plasma is around 70 g/L in normal adult malesWater 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) C) CatabolismCatabolism::PP are degraded throughout the body.Most proteins are degraded after being taken up by cells within the body.
A) A) Quantitative measurement of a specific protein:Quantitative measurement of a specific protein: by chemical or immunological methods
B) B) Semiquantitative measurement by electrophoresisSemiquantitative measurement by electrophoresis::
• Proteins are separated on the basis of their electrical charge. • ProcedureProcedure:
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 fractionsfive broad fractions:
AlbuminAlbumin, , 1-globulin1-globulin, , 2-globulin2-globulin, , -globulin-globulin & & -globulin -globulin Each of the globulin fractions consists of a mixture of several proteins
Measurement of plasma proteinsMeasurement of plasma proteins
Protein ElectrophoresisProtein Electrophoresis
Albumin
Electrophoresis pattern for normal serum proteinsElectrophoresis pattern for normal serum proteins
-+
Normal Pattern of Plasma ProteinNormal Pattern of Plasma Protein ElectrophoresisElectrophoresis byby DensitometryDensitometry
Electrophoretic BAND Proteins
Albumin Albumin
1-globulin1-AntitrypsinProthrombin
1-Fetoprotein (AFT)
2-globulin CeruoplasminHaptaglobin
2-macroglobulin
- globulinC-Reactive Protein ( CRP)
Transferrin2-microglobulin
-globulin Immunoglobulins(A, G, M, D & F)
• PrealbuminPrealbumin migrates faster than albumin in the classic electrophoresis.
• It is the transport protein for:- Thyroid hormones - Retinol (vitamin A)
• PrealbuminPrealbumin is decreased in:
Liver disease
Nephrotic syndrome
Acute phase inflammatory response
Malnutrition.
Prealbumin Prealbumin
Some important facts to know about albumin:Some important facts to know about albumin:
• Albumin is present in higher concentrations than other plasma proteins ( ~ 40 g/L in normal adults~ 40 g/L in normal adults).
• Albumin is synthesized in the liverliver & 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.
• AlbuminuriaAlbuminuria : In this case, albumin can be detected in urine by ordinary laboratory means
due to physiological or pathological conditions.
AlbuminAlbumin
Functions of albumin:Functions of albumin: 1- Oncotic pressure1- 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.Hypoalbuminemia leads to edema.
2- Buffering2- Buffering.
3- Transport3- 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 Albumin (cont.)
Causes of hypoalbuminemiaCauses of hypoalbuminemia
Artefactual : Artefactual : Diluted sample
Physiological : Physiological : Pregnancy - Recumbence
Decreased amino acids : Decreased amino acids : Reduced essential amino acids in diet & reduced synthesis of nonessential aa
Manlnutrition
Malabsorption.
Increased catabolism : Increased catabolism : Surgery Trauma Infections
Defective synthesis in liver: Defective synthesis in liver: Chronic liver diseases
Increased loss : Increased loss : From the kidneyFrom the kidney: Nephrotic syndrome From GITFrom GIT: Protein losing enteropathies
Albumin Albumin (cont.)
Increases immunoglobulins levels may result from stimulation of many clones of B cells (polyclonal hypergammaglobulinemia) or monoclonal proliferation (paraproteinemia).
Polyclonal hypergammaglobulinemia:Polyclonal hypergammaglobulinemia:Stimulation of many clones of B cells produce a wide range of antibodies that
appear as diffuse increase in diffuse increase in --globulin on electrophoresisglobulin on electrophoresis.e.g. acute and chronic infections & autoimmune diseases
Monoclonal Monoclonal hypergammaglobulinemiahypergammaglobulinemiaProliferation of a single B-cell clone produces a single immunoglobulin which
appears as a discrete densely stained band (Paraprotein or M band) on a discrete densely stained band (Paraprotein or M band) on electrophoresiselectrophoresis.
Paraproteins are characteristic of malignant B-cell proliferation.malignant B-cell proliferation.
Multiple myeloma Multiple myeloma is the commonest cause of paraproteinemia
HypergammaglobulinemiaHypergammaglobulinemia
• 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 acute phase reactants and their synthesis is a part of body’s response to injury.
11-Antitypsin-Antitypsin
HaptoglobinHaptoglobinCeruloplasminCeruloplasminFibrinogenFibrinogenC-reactive proteinC-reactive protein
Positive Acute Phase ProteinsPositive Acute Phase Proteins
• Positive acute phase proteins are increased within 24 hours of injury in response to humoral mediators (Cytokines – IL-1, IL-6, tumor necrosis factors and , the interferons and platelet activating factors) which are produced by tissue macrophages, monocytes & endothelial cells in inflammation ,etc..
• FunctionsFunctions:
1. Binding to polysaccharides in bacterial wallsBinding to polysaccharides in bacterial walls2. Activating complementcomplement3. Stimulating phagocytosisphagocytosis4. Protease inhibitors Protease inhibitors probably inactivate enzymes released from
lysosomes & minimize damage that may occur.
Positive Acute Phase Proteins Positive Acute Phase Proteins (cont.)
• It is an acute-phase protein acute-phase protein synthesized by liver• It precipitates the polysaccharide (fraction C) of
pneumococcal cell walls• It is important for phagocytosisphagocytosis .• Very large increase in plasma CRP 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..
C-Reactive Protein (CRP)C-Reactive Protein (CRP)
• Synthesized by the liver• Contains over 90% of serum copper
• It is important in acute phase responseacute phase response as it is able to inactivate reactive oxygen species (ROS) that produce tissue damage
• It is important for iron absorption iron absorption from the intestine.
• Plasma levels are usually low in Wilson’s disease Wilson’s disease in which copper is accumulated in the liver leading to cirrhosiscirrhosis , and in the basal ganglia of the brain.
CeruloplasminCeruloplasmin
• 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 glomeruli.• Its plasma level decreasesdecreases during hemolysis and
increasesincreases in acute inflammatory conditions (acute phase reactant).
HaptoglobinHaptoglobin
• Synthesized by the liver & macrophages• It is acute-phase protein acute-phase protein in order to inhibit proteases.• Proteases arise from: Endogenous production Endogenous production : by digestive enzymes such as trypsin &
chymotrypsin ,etc
Infection:Infection: protease release from bacteria and from leucocytes (inflam. response)
Clinical consequences of Clinical consequences of 11-Antitrypsin deficiency:-Antitrypsin deficiency:• Neonatal jaundicejaundice evidence of cholestasis• Childhood liver cirrhosis liver cirrhosis • Pulmonary emphysema Pulmonary emphysema in young adults
11-Antitrypsin-Antitrypsin
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 serumnot seen in serum.
It is one of the acute phase proteinsacute phase proteins.
Its level risesrises with pregnancy and the use of oral contraceptives.
DecreasedDecreased values generally reflects extensive coagulation during which
the fibrinogen is consumed.
FibrinogenFibrinogen
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)
Negative Acute Phase ProteinsNegative Acute Phase Proteins