Serum protein electrophoresis & their clinical importance

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  • 1. M.PRASAD NAIDU Msc Medical Biochemistry, Ph.D Research scholar.

2. Introduction 3. SERUM PROTEINS Composition Albumin: Conc. 60%, M.W. 69000, 585 AAs with 17 disulphide bonds. 1. Synthesized from liver. 2. Maintains colloidal osmotic pressure. 3. Decreasing causes edema. 4. Serves as asource of AA 5. Decresed Alb cirrhosis,nephrotic syndrome, malnutrition. 6. Increased alb - dehydration 4. Globulins 1. Its a glycoprotein with m.w. 90000 130000. 2. Types. 3. The & helps to transport proteins, hormones, vitamins, minerals and lipids. 4. globulins functions as immunoglobulins. Total proteins - 6-8 gm/dl (100%) Alb - 3.5 5 gm/dl (60%) Glob - 2.5 3 gm/dl (40%) 1 - 3% 2 - 11% - 11% - 15% 5. METHODS FOR SEPARATION OF SERUM PROTEINS 1. Precipitation by salts. 2. Cohns fractional precipitation method. 3. Sedimentation by ultracentrifugation. 4. Paper chromatography. 5. Electrophoresis. 6. ELECTROPHORESIS Definition: Electrophoresis is the migration of charged molecules in an electric field. The negative charged particles (anions) moves towards positive charged electrodes (anode). Positively charged particles (cations) moves towards cathod (negatively charged electrode). Types: 1. Depending upon the nature of supporting medium a. Agar gel electrophoresis (AGE). b. PAGE, SDS PAGE, QPNC PAGE (Quantitative preparative native continuous PAGE). c. Cellulose acetate electrophoresis. d. Capillary electrophoresis. 7. 2. Depending upon the mode of technique. a. Slide gel electrophoresis. b. Tube gel electrophoresis. c. Disc electrophoresis. d. Low and high voltage electrophoresis. e. Two dimensional gel electrophoresis Applications of Electrophoresis: 1. Separating serum proteins for diagnostic purpose. 2. Haemoglobin separation. 3. Lipoprotein separation and identification. 4. Isoenzyme separation and their analysis. 5. Nucleic acid studies. 6. Determination of molecular weight of the proteins. 8. FACTORS AFFECTING ELECTROPHORESIS I. The electric field: a. Voltage - V M b. Current - C M c. Resistance- R 1/ M II. The sample: a. Charge - C M b. Size - S 1/ M c. Shape - Molecules of similar size but different shape such as fibrus and globular proteins exhibit diffeent migration characteristics. Because of the differential effect of frictional and electrophoretic force. 9. III. The buffers: This determines and stabilizes the pH of the supporting medium and hence affects the migration rate of compound in a number of ways. a. Composition: The buffer should be such that it does not binds with the compounds to be separated as this may alters the rate of migration. Therefore barbitone buffer is always preferred for the separation of proteins or lipoproteins. b. Concentration: As the ionic strength of the buffThe er increases the proportion of current carried by the buffer will increase and the share of the current carried by the sample will decrease thus slowing down the rate of migration. c. pH: For organic compounds pH determines the extent of ionization and therefore degree and direction of migration are pH dependent. d. The supporting medium: The composition of supporting medium may cause adsorption, electro osmosis and molecular sieving. Which may influence the rate of migration of compounds. The commonly using supporting medium in the laboratory are agarose, polyacrylamide and cellulose acetate membrane. 10. Types of buffers used in electrophoresis 1. Tris buffer. 2. Glycine buffer. 3. Sodium barbituric acid. 4. TAE buffer (Tris acidic acid EDTA). 11. Types of StainsFor serum proteins Amido block - Coomassie brilliant blue For isoenzymes- Nitro tetra zolium blue For lipoprotein zones- Fat red 7B - Oil red O - Sudan block B For DNA fragments - Ethidium bromide For CSA proteins - Silver nitrate 12. What is needed? Agarose - a polysaccharide made from seaweed. Agarose is dissolved in buffer and heated, then cools to a gelatinous solid with a network of crosslinked molecules Some gels are made with acrylamide if sharper bands are required 13. Buffer - in this case TBE The buffer provides ions in solution to ensure electrical conductivity. Not only is the agarose dissolved in buffer, but the gel slab is submerged (submarine gel) in buffer after hardening 14. Also needed are a power supply and a gel chamber Gel chambers come in a variety of models, from commercial through home-made, and a variety of sizes 15. A gel being run Agarose block Positive electrode DNA loaded in wells in the agarose Black background To make loading wells easier Comb Buffer 16. The comb is removed, leaving little wells and buffer is poured over the gel to cover it completely The serum samples are mixed with a dense loading dye so they sink into their wells and can be seen 17. The serum samples are put in the wells with a micropipette. Micropipettes have disposable tips and can accurately measure 1/1,000,000 of a litre 18. Pulsed field gel electrophoresis Pulsed Field Gel Electrophoresis (commonly abbreviated as PFGE) is a method for separating large DNA molecules, which may be used for genotyping or genetic fingerprinting. Under normal electrophoresis, large nucleic acid particles (above 30-50 kb) migrate at similar rates, regardless of size. By changing the direction of the electric field frequently, much greater size resolution can be obtained 19. Pulsed field gel electrophoresis 20. SDS-PAGE SDS-PAGE, officially sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a technique used in biochemistry, genetics and molecular biology to separate proteins according to their electrophoretic mobility . Quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE) is a new method for separating native metalloproteins in complex biological matrices. 21. Gel Electrophoresis 22. CAPILLARY ELECTROPHORESIS 23. MODES OF CAPILLARY ELECTROPHORESIS 24. TABLE 1 Indications for Serum Protein Electrophoresis Suspected multiple myeloma, Waldenstrm's macroglobulinemia, primary amyloidosis, or related disorder Unexplained peripheral neuropathy (not attributed to longstanding diabetes mellitus, toxin exposure, chemotherapy, etc.) New-onset anemia associated with renal failure or insufficiency and bone pain Back pain in which multiple myeloma is suspected Hypercalcemia attributed to possible malignancy (e.g., associated weight loss, fatigue, bone pain, abnormal bleeding) Rouleaux formations noted on peripheral blood smear Renal insufficiency with associated serum protein elevation Unexplained pathologic fracture or lytic lesion identified on radiograph Bence Jones proteinuria 25. NORMAL PATTERN OF SERUM ELECTROPHORESIS 1 i -globulin origin 2-globulin 1-globulin 2-globulin 1-globulin albumin 26. ABNORMAL PATTERN OF SERUM ELECTROPHORESIS MULTIPLE MYELOMAMULTIPLE MYELOMA i Extra M-Band is seen Albumin 27. NEPHROTIC SYNDROME i -globulin and 2-gloublin Albumin 28. AGAMMAGLOBULINEMIA i Absence or decrease of -globulin and others normal 29. LIVER DISEASES i -globulin origin 2-globulin 1-globulin albumin 30. Characteristic Patterns of Acute-Reaction Proteins Found on Serum Protein Electrophoresis and Associated Conditions or Disorders Increased albumin Dehydration Decreased albumin Chronic cachectic or wasting diseases Chronic infections Hemorrhage, burns, or protein-losing enteropathies Impaired liver function resulting from decreased synthesis of albumin Malnutrition Nephrotic syndrome Pregnancy Increased alpha1 globulins Pregnancy Decreased alpha1 globulins Alpha1-antitrypsin deficiency Increased alpha2 globulins Adrenal insufficiency Adrenocorticosteroid therapy Advanced diabetes mellitus Nephrotic syndrome Decreased alpha2 globulins Malnutrition Megaloblastic anemia Protein-losing enteropathies Severe liver disease Wilson's disease 31. Increased beta1 or beta2 globulins Biliary cirrhosis Carcinoma (sometimes) Cushing's disease Diabetes mellitus (some cases) Hypothyroidism Iron deficiency anemia Malignant hypertension Nephrosis Polyarteritis nodosa Obstructive jaundice Third-trimester pregnancy Decreased beta1 or beta2 globulins Protein malnutrition Increased gamma globulins Amyloidosis Chronic infections (granulomatous diseases) Chronic lymphocytic leukemia Cirrhosis Hodgkin's disease Malignant lymphoma Multiple myeloma Rheumatoid and collagen diseases (connective tissue disorders) Waldenstrm's macroglobulinemia Decreased gamma globulins Agammaglobulinemia Hypogammaglobulinemia 32. Differential Diagnosis of Polyclonal Gammopathy Infections Viral infections, especially hepatitis, human immunodeficiency virus infection, mononucleosis, and varicella Focal or systemic bacterial infections, including endocarditis, osteomyelitis, and bacteremia Tuberculosis Connective tissue diseases Systemic lupus erythematosus Mixed connective tissue Temporal arteritis Rheumatoid arthritis Sarcoid Liver diseases Cirrhosis Ethanol abuse Autoimmune hepatitis Viral-induced hepatitis Primary biliary cirrhosis Primary sclerosing cholangitis Malignancies Solid tumors Ovarian tumors Lung cancer Hepatocellular cancer Renal tumors Gastric tumors Hematologic cancers (see below) Hematologic and lymphoproliferative disorders Lymphoma Leukemia Thalassemia Sickle cell anemia Other inflammatory conditions Gastrointestinal conditions, including ulcerative colitis and Crohn's disease Pulmonary disorders, including bronchiectasis, cystic fibrosis, chronic bronchitis, and pneumonitis Endocrine diseases, including Graves' disease and Hashimoto's thyroiditis 33. Characteristic Features of Monoclonal Gammopathies Disease Distinctive features Multiple myeloma M protein appears as a narrow spike in the gamma, beta, or alpha2 regions. M-protein level is usually greater than 3 g per dL. Skeletal lesions (e.g., lytic lesions, diffuse osteopenia, vertebral compression fractures) are present in 80 percent of patients. Diagnosis requires 10 to 15 percent p