W1 Plasma Proteins (Damuni).pdf

Plasma Proteins Enzymes As Diagnostic Tools Presented by Dr. Zahi Damuni Professor of Biochemistry Reading: Simmons, Gerhard Meisenberg, W. Principles of Medical Biochemistry (3rd Edition), Chapter 15 1 Learning Objectives 1. Know the proteins in plasma that can be used for diagnosis of different di...

Plasma Proteins Enzymes As Diagnostic Tools Presented by Dr. Zahi Damuni Professor of Biochemistry Reading: Simmons, Gerhard Meisenberg, W. Principles of Medical Biochemistry (3rd Edition), Chapter 15 1 Learning Objectives 1. Know the proteins in plasma that can be used for diagnosis of different diseases 2. Know which proteins are used for diagnosis of what diseases 3. Be able to connect certain clinical symptoms with laboratory test and arrive at a close diagnosis 4. Know the enzymes used in the diagnosis of different organ diseases 5. Know what enzymes should be analyzed to arrive at a diagnosis 6. Be able to match certain symptoms with enzyme results and arrive at a close diagnosis 2 Introduction to Plasma Proteins Colloid Osmotic Pressure (the osmotic pressure normally created by plasma proteins that do not diffuse readily across the capillary membrane). Transport Acute Phase Proteins (proteins whose plasma concentrations increase or decrease in response to inflammation) Enzymes Note: With the exception of immunoglobulins, most proteins are synthesized in the liver. 3 Anticoagulants Heparin Citrate Oxalate EDTA Citrate/oxalate/ EDTA bind Ca++ and other divalent cations. 4 Plasma Proteins Are Mostly Glycoproteins 5 Serum Electrophoresis Anode + Albumin α1 α2 β1 β2 γ Cathode - 6 Electrophoresis Band Major Proteins α1-Globulins α1-Antitrypsin α- Lipoprotein α2-Globulins Haptoglobin Ceruloplasmin α2-Macroglobulin β1-Globulins Transferrin β-Lipoprotein β2-Globulins C3 (complement protein) Fibrinogen (in plasma) γ-Globulins Immunoglobulins 7 Albumin Functions Oncotic Pressure Transport e.g., Fatty acids, bilirubin, etc. Drug transport e.g., salicylates, barbiturates, sulfonamides, warfarin, penicillin 8 Normal Pathogenesis of edema in hypoalbuminemia 9 Abnormalities of Albumin Metabolism Hypoalbuminemia Reduced synthesis - IL6/stress response Altered distribution - Increased capillary permeability, decreased lymph clearance Increased catabolism - Chronic infections/trauma Abnormal losses - Burns, renal disease, GI loss, hemorrhage Hyperalbuminemia Dehydration: Loss of water and concentration of substances in the vascular system Excessive stasis during venepuncture 10 α1-Globulin Mostly transport proteins and protease inhibitors (limit inflammation and vascular damage) α1-antitrypsin – inhibits trypsin and elastase (neutrophils) and other proteases Serum amyloid A – apolipoprotein of HDL binding cholesterol, immune cell recruitment in acute inflammatory phase High density lipoprotein – apolipoproteins of HDL, Apo A1 activates LCAT 11 α2-Globulin Protease inhibitors and transport proteins Haptoglobin – binds free hemoglobin, haptoglobin-hemoglobin complex targets hemoglobin for removal by spleen α2-macroglobulin (transcuprein) – protease inhibitor of plasmin, thrombin and kallikrein, carrier of cytokines and growth factors, zinc and copper transporter Ceruloplasmin – ferroxidase activity and copper transporter (> 95% of copper transport) Thyroxine-binding globulin – highest affinity T3 and T4 transporter α2-antiplasmin – inhibits plasmin and neutrophil elastase Protein C (autoprothrombin IIa, factor XIV) – Activated Protein C (APC) inhibits Factor Va and VIIIa Angiotensinogen – zymogen of Angiotensin, regulates blood pressure 12 β-Globulin Transport proteins and Plasminogen β-2 microglobulin – binds MHC I related proteins MHC I, Qa and CD1 – involved in recognition of self vs. non self Hemochromatosis protein (HFE protein) – regulates transferrin binding to its receptor. Plasminogen – zymogen of plasmin and angiostatin; Plasmin activates clotting and fibrinolysis Sex hormone-binding globulin – androgen and estrogen binding protein Transferrin – high affinity Iron binding protein 13 β-Globulin Immunoglobulins (β2 peak and tail) IgA – Rare in plasma Mono or multimeric Regulates Fc Receptor mediated inflammatory responses; includes antibody-dependent cell-mediated cytotoxicity (ADCC) and degranulation of granulocytes IgM – Pentameric, 10 antigen binding sites Regulates opsonization 14 γ- Globulin Predominantely Immunoglobulins IgA – Rare in plasma Mono or multimeric Regulates Fc Receptor mediated inflammatory responses; includes Antibody dependent cell-mediated cytotoxicity (ADCC) and degranulation of granulocytes IgG – 75% of free immunoglobulins in sera 2 antigen binding site Functions include roles in agglutination, opsonization, pathogen recognition, type II and III hypersensitivity, etc. IgM – Pentameric, 10 antigen binding sites Regulates opsonization 15 Plasma Proteins in Disease Arrows indicate PEAK shape changes and Relative Ratios of proteins compared to Albumin Actual concentration of a specifc protein may be unchanged Normal Liver Cirrhosis Acute Phase Response Nephrotic Syndrome 16 The Binding Proteins Prealbumin/ Transthyretin Retinol and Thyroid Hormones Albumin Osmotic Pressure and Binding Protein for Multiple Substances Retinol Binding Protein Retinol Transport Thyroxine Binding Globulin Thyroid Hormones (T3 And T4) Cortisol-Binding Globulin Or Transcortin Binds Glucocorticoids 17 Acute Phase Proteins α-Fetoprotein α1-Antiprotease α2-Macroglobulin β2-Microglobulin Ceruloplasmin Haptoglobin Hemopexin Transferrin Fetal liver Protease inhibitor Protease inhibitor Subunit of MHC class I molecules Contains copper Binds hemoglobin Binds heme Binds iron 18 Alpha-fetoprotein used in detection of Neural Tube Defects, Down’s Syndrome; Hepatocellular, Germ Cell and Other Cancers (in elderly). Pregnancy-Low levels (Down’s); high levels Neural Tube Defects Ceruloplasmin lower levels used in detection of Wilson’s disease (Copper deposition, Kayser-Fleisher rings, Hepatic damage, arthritic changes) β2-microglobulin – used in detection of Nephrotic Syndrome α2-macroglobulin – used in detection of Nephrotic Syndrome 19 Acute Phase Proteins - Immune Proteins Fibrinogen Clot formation C-Reactive Protein Acute phase reactant binds extracts of pneumococcal cell walls: The first protein to rise in an acute infection or inflammation, or in response to injury Immunoglobulins Heterogeneous 20 The Role of Haptoglobin and Hemopexin Haptoglobin is a protein used to clear free hemoglobin from the circulation. Haptoglobin is an "acute-phase" protein that is elevated in many inflammatory diseases, such as ulcerative colitis, acute rheumatic disease, heart attack, and severe infection. 21 α1-Antitrypsin Measured by the trypsin inhibitory capacity Main function is antiprotease activity in lung Pulmonary emphysema Synthesized by hepatocytes 22 Acute-Reaction Proteins Associated Conditions or Disorders Found on Serum Protein Electrophoresis Increased Albumin Dehydration Decreased Albumin Malnutrition, chronic infections, burns, hemorrhage, impaired liver function, nephrotic syndrome, pregnancy Increased α-Globulins Pregnancy Decreased α-Globulins α1-Antitrypsin deficiency Increased α2-Globulins Nephrotic syndrome Decreased α2-Globulins Severe liver disease, Wilson’s disease, malnutrition Increased β-Globulins Obstructive jaundice, biliary cirrhosis, iron deficiency anemia 23 Other Enzymes As Diagnostic Tools 24 Constitutive Plasma Enzymes While most enzymes are present in cells, others are secreted into plasma by specific organs (mainly liver) Low concentrations of such enzymes could signal liver disease e.g., lecithin:cholesterol acyltransferase (LCAT) 25 Tissue-Derived Enzymes Certain enzymes are present in higher concentrations in specific tissues. An increase in levels of these enzymes in plasma may reflect damage to corresponding tissues. The level of specific enzyme activity in plasma usually correlates with the extent of tissue damage. 26 Normal Cell Turnover Blood Different Cell Types 27 Cell Damage vs Proliferation 28 Why Are Enzymes Measured? Release of enzymes from cells can be due to: Necrosis or severe damage to cells Increased concentration of enzymes in cells Duct obstruction Reduced urinary excretion 29 Clinically Important Enzymes Plasma Cholinesterase (BuChE, butyrylcholinesterase) Aspartate and Alanine transaminases (AST/ALT) Alkaline phosphatase (ALP) Acid phosphatase (ACP) Lactate dehydrogenase (LDH) Creatine kinase (CK) Gamma-glutamyltransferase (GGT) Amylase and lipase 30 Selecting Enzyme Test Has tissue damage occurred, and if so, what is the extent? Which tissues have been damaged? 31 Cholinesterases Plasma cholinesterase (BuChE, butyrylcholinesterase) - found in plasma and synthesized in liver Catalyzes hydrolytic cleavage of various esters of choline Acetylcholinesterase (ACHE) only degrades acetylcholine Plasma cholinesterase (BuChE) degrades drugs such as scoline and cocaine 32 Acetylcholinester ase (ACHE) found principally in nervous tissue and erythrocytes Na+ Muscle Action Potential 33 Decreased Levels of Plasma Cholinesterase (BuChe) Hepatic disease: synthesis Inherited abnormal variants of plasma cholinesterase with low biological activity 34 Ingestion of organophosphates (OP’s) or absorption via skin interferes with the activity of both BuChe and ACHE. OPs are irreversible inhibitors of acetylcholinesterase. Clinical symptoms arise from build up of acetylcholine. Atropine sulfate (competitive antagonist of acetylcholine receptors). Atropine Sulfate Injection is an antimuscarinic agent used to treat bradycardia (low heart rate), reduce salivation and bronchial secretions before surgery, as an antidote for overdose of cholinergic drugs or mushroom poisoning. 35 Increased Levels of Plasma Cholinesterase (BuChe) Nephrotic syndrome Rapidly growing cells: liver recovery 36 The Transaminases A group of enzymes that catalyze the transfer of an amino group from amino acids to α-ketoacids 37 Alanine Transaminase (ALT) Present in high concentrations in cells of Liver Skeletal and cardiac muscle (lesser extent) Kidney (lesser extent) 38 Marked Increases Moderate increases Circulatory failure Liver cirrhosis * Acute viral hepatitis * Cholestatic jaundice * Post cardiac surgery Skeletal muscle disease 39 Aspartate Transaminase (AST) L-aspartate + 2-oxoglutarate  oxaloacetate + L-glutamate Present in high concentrations in cells of Cardiac and skeletal muscle Liver Kidney Erythrocytes 40 Causes of High Levels of AST Artifactual (hemolysis) Physiological in neonatal Very High Levels Observed In: Viral hepatitis Myocardial infarction Circulatory failure 41 AST/ALT ratio The normal AST/ALT ratio is approximately ~ 0.8 An AST/ALT ratio of 2.0 or higher or ALT level exceeding 300 U/L may be indicative of alcoholic liver disease However, the AST/ALT ratio is usually 1.0 or less in nonalcoholic fatty liver disease 42 Alkaline & Acid Phosphatases A group of enzymes that display maximum activity at pH 5 or 10. Attached to cell membranes, suggesting an association between their activity and membrane transport. 43 Alkaline Phosphatase (ALP) Cathode Anode 44 Physiological Changes In ALP Levels Normal pregnancy Infancy and childhood High fat meals Diseases that affect the bile duct * Bone diseases * 45 Acid Phosphatase (AP) Found in cells of: Prostate Liver Erythrocytes Platelets Bone Note: Prostate Specific Antigen (PSA) (tumor marker) 46 Isoenzymes of Lactate Dehydrogenase LDH1:LDH2 used to detect MI. LDH5 used to detect acute hepatitis. 47 Muscle Creatine Kinase (CK) Creatine Phosphate ADP Creatine ATP 48 Brain (Tumor) Heart Muscle (MI) Skeletal Muscle (MD) 49 Gamma-Glutamyltransferase (GGT) Found mainly in cell membranes of Liver and Bile duct * Kidney Pancreas Several other organs 50 Causes of Raised GGT Enzyme induction – Drugs and alcohol Anticonvulsants: e.g., Phenobarbital, Phenytoin, Carbamazepine Cholestatic liver disease Hepatocellular damage 51 Note: GGT is not associated with bone and is used to differentiate tissues of origin of ALP For example: In diseases of the bile duct, both GGT and ALP are raised. In diseases of the bone, GGT is normal, and ALP is raised 52 Amylase & Lipase Amylase and lipase (intestinal) are produced by the pancreas for digestion of starch and fat Amylase is also found in saliva Amylase and lipase are increased in the plasma in acute pancreatitis and are necessary to differentiate between different causes of abdominal pain Both enzymes are excreted in the urine and are elevated in plasma in renal failure 53 54 55 Test Sensitivity Peak time Origin Troponin I Most sensitive and specific 12 hours but released 2-4 hours after MI Released from cytosolic pool of the myocytes Creatine Kinase CK-MB Relatively specific but not as specific as Troponin 10-24 hours but released 3 hours after MI Resides in cytosol Lactate Dehydrogenase Not as specific as troponin 72 hours High LDH1:LDH2 suggest MI Aspartate transaminase (AST) Not specific for heart damage 48 hours Also one of liver function test 56