Serum Proteins PDF

Plasma proteins Lorenzo Montanaro  Blood Components Blood is both a tissue and a fluid. It is a tissue because it is a collection of similar specialized cells that serve particular functions. These cells are suspended in a liquid matrix (plasma), which makes the blood a fluid....

Plasma proteins Lorenzo Montanaro  Blood Components Blood is both a tissue and a fluid. It is a tissue because it is a collection of similar specialized cells that serve particular functions. These cells are suspended in a liquid matrix (plasma), which makes the blood a fluid. Serum is the portion of plasma remaining after coagulation of blood, during this process the plasma protein fibrinogen is converted to fibrin and remains behind in the clot  Blood Centrifugation Blood is usually placed in tubes from 1.2mL to 15mL and centrifuged at 2500 rpm for 10 minutes The role of a centrifuge in testing blood is to separate whole blood into its various components. Blood is separated into it’s various components: – Red Blood Cells (RBC), erythrocytes. – White blood cells (WBC) – Plasma is a yellowish component in which the blood is normally suspended. Plasma contains proteins, glucose, clotting factors, hormones, and carbon dioxide.  Blood centrifugation allows separation of three phases Plasma Buffy coat Red blood cells  Plasma Components Physiologically normal components (glucose, albumin, clotting factors, lipoproteins, hormones) Metabolites (urea, bilirubin, uric acid, creatinine Factors released by tissues following damage or proliferation (creatin kinase, aminotransferases, alkaline phosphatases, PSA) Drugs and toxics (antibiotics, alcohol, toxic drugs)  Plasma proteins Plasma proteins are the most easily available proteins of the human body for study. Despite plasma proteins represent a small proportion of all human proteins they are important since their quantitative an qualitative variations are relevant for diagnostic purposes. We recognize more than 100 different major plasma proteins that are mainly synthesized in the liver and for a minor extent by plasmacells, macrophages and intestinal cells. In normal conditions the concentration of plasma proteins varies between 6 e 8 g/dl  Serum proteins Serum total protein, also known as total protein, is a biochemical test for measuring the total amount of protein in serum. Protein in the serum is made up of albumin and globulins. The globulins in turn are made up of α1, α2, β, and γ globulins. These fractions can be quantitated using protein electrophoresis, but the total protein test is a faster and cheaper test that estimates the total of all fractions together. I The traditional method for measuring total protein uses staining. The most common method of in-gel protein detection is staining with Comassie blue dye. In clinical diagnostics, the measurement is usually performed on automated analyzers along with other laboratory tests. ↓ Protein electrophoresis Electrophoresis is a technique in which the blood serum is applied to an acetate membrane soaked in a liquid buffer or to a buffered agarose gel matrix, or into liquid in a capillary tube, and exposed to an electric current to separate the serum protein components into five major fractions by size and electrical charge: serum albumin, alpha-1 globulins, alpha-2 globulins, beta and globulins, and gamma globulins. Acetate or gel electrophoresis: Proteins are separated by both electrical forces and electroendoosmostic forces. The net charge on a protein is based on the sum charge of its amino acids, and the pH of the buffer. Proteins are applied to a solid matrix such as an agarose gel, or a cellulose acetate membrane in a liquid buffer, and electric current is applied. Proteins with a negative charge will migrate towards the positively charged anode. Albumin has the most negative charge, and will migrate furthest towards the anode. Endoosmotic flow is the movement of liquid towards the cathode, which causes proteins with a weaker charge to move backwards from the application site. Gamma proteins are primarily separated by endoosmotic forces. Capillary electrophoresis: In capillary electrophoresis, there is no solid matrix. Proteins are separated primarily by strong electroendoosmotic forces. The sample is injected into a capillary with a negative surface charge. A high current is applied, and negatively charged proteins such as albumin try to move towards the anode. Liquid buffer flows towards the cathode, and drags proteins with a weaker charge. ACETATE ELECTROPHORESIS CAPILLARY ELECTROPHORESIS  Clinical pathology of serum proteins 1. ELECTROPHORETIC PATTERN OF SERUM PROTEINS 2. ACUTE PHASE PROTEINS 3. ERYTHROCYTE SEDIMENTATION RATE (VES in Italian)  Protein Fractions Electrophoresis separation yields 5 major protein fractions from the anode to the cathode, corresponding to: albumin (52-68%) 3.2-5.6 g/dL 1 globulin (2,4 – 5,3%) 0.1-0.4 g/dL 2 globulin (6,6 – 13,5%) 0.4-1.2 g/dL  globulin (8,5 – 14,5%) 0.6-1.3 g/dL  globulin (10,7 – 21%) 0.5-1.6 g/dL The albumin/globulin ratio generally varies from 1.2 to 1.7  IDENTIFICATION OF SPECIFIC SERUM GLOBULINS BY IMMUNOELECTROPHORESIS Human blood serum contains about 7 % protein, 2/3 of which is in the albumin fraction; the other 1/3 is in the globulin fraction. Electrophoresis of serum reveals a large albumin peak and three smaller globulin peaks, the alpha-, beta-, and gamma-globulins. The amounts of alpha-, beta-, and gamma-globulin in normal human serum are approximately 1.5, 1.9, and 1.1 %, respectively. Each globulin fraction is a mixture of many different proteins, as demonstrated by immunoelectrophoresis. In this method, serum from an animal injected with human serum is allowed to diffuse into the four protein bands obtained from the electrophoresis of human serum. Because the animal has previously been injected with human serum, its blood contains antibodies against each of the human serum proteins; each antibody combines with the serum protein antigen that caused its formation in the animal. The result is the formation of about 20 regions of insoluble antigen-antibody precipitate, which appear as white arcs in the transparent gel of the electrophoresis medium. Each region corresponds to a different human serum protein. Protein Levels in Human Plasma PLASMA PROTEIN NORMAL RANGE (mg/dL) Albumin 3,500 - 4,500 Prealbumin (Transthyretin) 10 - 40 α1-Globulins 300 - 600 α1 Acid Glycoprotein (orosomucoid) 55 - 140 α-Fetoprotein approx. 0.001 Retinol Binding Protein 3-6 Thyroxine Binding Globulin 1-2 Transcortin 3 - 3.5 α2 Globulins (excluding 400 - 900 lipoproteins)________ Ceruloplasmin 15 - 60 Haptoglobin Type 1-1 100 - 220 Haptoglobin Type 2-1 160 - 300 Haptoglobin Type 2-2 120 - 260 α2 Macroglobulin 100 - 280 β-Globulins (excluding lipoproteins) 600 - 1,100 C-Reactive Protein