Plasma Proteins and Enzymes as Diagnostic Tools 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 1 Learning Objectives 1. Know the proteins in plasma that can be used for diagnosis of differ...

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 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 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 Colloid osmotic pressure (COP), the osmotic pressure exerted by large molecules, serves to hold water within the vascular space. It is normally created by plasma proteins, namely albumin, that do not diffuse readily across the capillary membrane. 3 Anticoagulants Som Heparin Citrate Oxalate EDTA Ima Citrate/oxalate/ EDTA bind Ca++ and other divalent cations. 4 Ethylenediaminetetraacetic acid (EDTA) Oxalate binds Ca++ 4 Plasma Proteins Are Mostly Glycoproteins 5 Nearly all plasma proteins are part of the glycoprotein family, with oligosaccharide chains linked either by N- or O-atoms. However, albumin is an important exception as it does not contain any saccharide residues. 5 Serum Electrophoresis Anode + Albumin α1 α2 β1 β2 γ Cathode - 6 Stain the gel to see the protein bands then scan to read the intensity of the stain. Nearly all plasma proteins are part of the glycoprotein family, with oligosaccharide chains linked either by N- or O-atoms. However, albumin is an important exception as it does not contain any saccharide residues. 6 Electrophoresis Band Major Proteins 1-Globulins 1-Antitrypsin - Lipoprotein 2-Globulins Haptoglobin Ceruloplasmin 2-Macroglobulin 1-Globulins Transferrin -Lipoprotein 2-Globulins C3 (complement protein) Fibrinogen (in plasma) -Globulins Immunoglobulins 7 7 Albumin Functions • • • Oncotic Pressure Transport e.g., Fatty acids, bilirubin, etc. Drug transport e.g., salicylates, barbiturates, sulfonamides, warfarin, penicillin 8 8 Normal Pathogenesis of edema in hypoalbuminemia 9 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 Venipuncture or venepuncture is the process of obtaining intravenous access for the purpose of venous blood sampling or intravenous therapy. 10 1-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 11 2-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 • 2-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 Ferroxidase also known as Fe(II):oxygen oxidoreductase is an enzyme that catalyzes the oxidization of iron II to iron III: 4 Fe2+ + 4 H+ + O2 = 4 Fe3+ + 2H2O. 12 -Globulin • Transport proteins and Plasminogen • -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 Hereditary hemochromatosis (HH) is a disorder of iron metabolism caused by common mutations in the gene HFE. The HFE protein binds to transferrin receptor-1 (TfR1) in competition with transferrin and reduces cellular iron by reducing iron uptake via stimulation of Hepcidin production. 13 -Globulin • Immunoglobulins ( 2 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 14 - 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 15 Alb S2 synthesis of netops 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 Prealbumin, also know as thyroxine binding prealbumin and transthyretin, is a glycoprotein of approximately 54,000 daltons that is synthesized in the liver. 17 Acute Phase Proteins -Fetoprotein 1-Antiprotease 2-Macroglobulin 2-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 Acute-phase proteins are a class of proteins whose plasma concentrations increase or decrease in response to inflammation. This response is called the acute-phase reaction. The acute-phase reaction characteristically involves fever, acceleration of peripheral leukocytes, circulating neutrophils and their precursors. 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) • 2-microglobulin – used in detection of Nephrotic Syndrome • 2-macroglobulin – used in detection of Nephrotic Syndrome 19 Kayser–Fleischer rings are dark rings that appear to encircle the iris of the eye. They are due to copper deposition in part of the Descemet's membrane as a result of liver diseases. The normal range for a ceruloplasmin serum test is 20 to 35 milligrams per deciliter (mg/dL). If the patient has Wilson disease, their ceruloplasmin level will probably be below 10 mg/dL Descemet's membrane -which is the basement membrane for the corneal endothelium- is a dense, thick, relatively transparent and cell-free matrix that separates the posterior corneal stroma from the underlying endothelium. Alpha-fetoprotein (AFP) is used as a tumor marker to help detect and diagnose cancers of the liver, testicles, and ovaries. The role of hemopexin is to bind and transport free heme to the liver, where it is internalized and degraded, thus preventing heme-mediated oxidative stress and heme-bound iron loss A haptoglobin test can detect whether a patient has hemolytic anemia or another type of anemia. It may also help determine the exact cause of increased red blood cell destruction. 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 C-Reactive Protein binds to phosphocholine on micro-organisms. It is thought to assist in complement binding to foreign and damaged cells and enhances phagocytosis by macrophages (opsonin-mediated phagocytosis), which express a receptor for CRP. It plays a role in innate immunity as an early defense system against infections. 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 Increased red blood cell destruction may occur as a result of: • inherited conditions that cause abnormalities in the size or shape of red blood cells, such as hereditary spherocytosis • spleen disorders • cirrhosis, or severe scarring of the liver • myelofibrosis, or scarring of the bone marrow A physician may decide to run a haptoglobin test if a patient is experiencing symptoms of hemolytic anemia. Hemopexin (Hx) is another plasma glycoprotein. Like hemoglobin is able to bind heme with high affinity. Hemopexin sequesters heme in an inert, non-toxic form and transports it to the liver for catabolism and excretion. The role of hemopexin is to bind and transport free heme to the liver, where it is internalized and degraded, thus preventing heme-mediated oxidative stress and heme-bound iron loss A haptoglobin test can detect whether a patient has hemolytic anemia or another type of anemia. It may also help determine the exact cause of increased red blood cell destruction. 21 Hemopexin-Heme complex binds to a receptor, such as LRP1, on hepatocytes principally or macrophages within the spleen, liver and bone marrow. 21 1-Antitrypsin • Measured by the trypsin inhibitory capacity • Main function is antiprotease activity in lung • Pulmonary emphysema • Synthesized by hepatocytes 22 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 1-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 Wilson disease is a rare genetic disorder characterized by excess copper stored in various body tissues, particularly the liver, brain, and corneas of the eyes. The disease is progressive and, if left untreated, it may cause liver (hepatic) disease, central nervous system dysfunction, and death. 23 Other Enzymes As Diagnostic Tools 24 24 Constitutive Plasma Enzymes • While most enzymes are present in most 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 Lecithin–cholesterol acyltransferase is an enzyme that converts free cholesterol into cholesteryl ester (a more hydrophobic form of cholesterol), which is then sequestered into the core of a lipoprotein particle, eventually making the newly synthesized HDL spherical and forcing the reaction to become unidirectional since the particles are removed from the surface. The enzyme is bound to high-density lipoproteins (HDLs) and low-density lipoproteins in the blood plasma. LCAT deficiency can cause impaired vision due to cholesterol corneal opacities, anemia, and kidney damage. 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 26 Normal Cell Turnover Blood Different Cell Types 27 27 Cell Damage vs Proliferation 28 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 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 30 Selecting Enzyme Test • Has tissue damage occurred, and if so, what is the extent? • Which tissues have been damaged? 31 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 32 Acetylcholinester ase (ACHE) found principally in nervous tissue and erythrocytes Na+ Muscle Action Potential 33 33 Decreased Levels of Plasma Cholinesterase (BuChe) • Hepatic disease: synthesis • Inherited abnormal variants of plasma cholinesterase with low biological activity 34 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 35 Increased Levels of Plasma Cholinesterase (BuChe) • Nephrotic syndrome • Rapidly growing cells: liver recovery 36 36 The Transaminases A group of enzymes that catalyze the transfer of an amino group from amino acids to -ketoacids 37 37 Alanine Transaminase (ALT) Present in high concentrations in cells of • Liver • Skeletal and cardiac muscle (lesser extent) • Kidney (lesser extent) 38 38 Marked Increases Moderate increases Circulatory failure Liver cirrhosis * Acute viral hepatitis * Cholestatic jaundice * Post cardiac surgery Skeletal muscle disease 39 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 40 Causes of High Levels of AST • Artifactual (hemolysis) • Physiological in neonatal Very High Levels Observed In: • Viral hepatitis • Myocardial infarction • Circulatory failure 41 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 Alcoholic liver disease is a result of overconsuming alcohol that damages the liver, leading to a buildup of fats, inflammation, and scarring. It can be fatal. The condition is a primary cause of chronic liver disease in Western nations. Nonalcoholic fatty liver disease (NAFLD) is an umbrella term for a range of liver conditions affecting people who drink little to no alcohol. As the name implies, the main characteristic of NAFLD is too much fat stored in liver cells. 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 43 Alkaline Phosphatase (ALP) Cathode Anode 44 44 Physiological Changes In ALP Levels • • • • • Normal pregnancy Infancy and childhood High fat meals Diseases that affect the bile duct * Bone diseases * 45 45 Acid Phosphatase (AP) Found in cells of: • Prostate • Liver • Erythrocytes • Platelets • Bone Note: Prostate Specific Antigen (PSA) (tumor marker) 46 PSA is kallikrein-3, is a glycoprotein enzyme encoded in humans by the KLK3 gene. PSA is a member of the kallikrein-related peptidase family and is secreted by the epithelial cells of the prostate gland. 46 Isoenzymes of Lactate Dehydrogenase LDH1:LDH2 used to detect MI. LDH5 used to detect acute hepatitis. 47 47 Muscle Creatine Kinase (CK) Creatine Phosphate ADP Creatine ATP 48 48 Brain (Tumor) Heart Muscle (MI) Skeletal Muscle (MD) 49 49 Gamma-Glutamyltransferase (GGT) Found mainly in cell membranes of • Liver and Bile duct * • Kidney • Pancreas • Several other organs 50 50 Causes of Raised GGT • Enzyme induction – Drugs and alcohol • Anticonvulsants: e.g., Phenobarbital, Phenytoin, Carbamazepine • Cholestatic liver disease • Hepatocellular damage 51 Cholestasis is a liver disease. It occurs when the flow of bile from your liver is reduced or blocked. Bile is fluid produced by your liver that aids in the digestion of food, especially fats. When bile flow is altered, it can lead to a buildup of bilirubin. 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 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 Acute pancreatitis is a condition where the pancreas becomes inflamed (swollen) over a short period of time. The most common cause of acute pancreatitis is having gallstones. Gallstones cause inflammation of your pancreas as stones pass through and get stuck in a bile or pancreatic duct. This condition is called gallstone pancreatitis. 53 54 54 55 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 10-24 hours but released as specific as Troponin 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 CKMB is the heart specific isoenzyme and has been the gold standard method for the diagnosis of AMI in many laboratories. It exists in large quantity in heart muscle but is not totally cardiac specific and exists also in skeletal muscles and other tissues. 56