& Protein Electrophoresis 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 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

Plasma Proteins General functions of plasma proteins include: 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 is determined by 3 main factors: 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 - + Albumin a1 a2 b g Electrophoresis pattern for normal serum proteins

Normal Pattern of Plasma Protein Electrophoresis by Densitometry

C-Reactive Protein ( CRP) Electrophoretic BAND Proteins Albumin a1-globulin a1-Antitrypsin Prothrombin a1-Fetoprotein (AFT) a2-globulin Ceruoplasmin Haptaglobin a2-macroglobulin b- globulin C-Reactive Protein ( CRP) Transferrin b2-microglobulin g-globulin 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.) 1- Oncotic pressure: 2- Buffering. 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 Malnutrition 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 -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. 1-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  and , 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.

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).

1-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 1-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)