Enzyme Diagnosis PDF

Enzyme Diagnosis Dr Sathivel Arumugam Associate Professor in Biochemistry Faculty of Medicine  CLINICAL SIGNIFICANCE ENZYMES IN CLINICAL DIAGNOSIS Plasma enzymes can be classified into two major groups. First, a relatively small group of enzymes are actively secreted into the blood...

Enzyme Diagnosis Dr Sathivel Arumugam Associate Professor in Biochemistry Faculty of Medicine  CLINICAL SIGNIFICANCE ENZYMES IN CLINICAL DIAGNOSIS Plasma enzymes can be classified into two major groups. First, a relatively small group of enzymes are actively secreted into the blood by certain cell types. For example, the liver secretes zymogens (inactive precursors) of the Loading… enzymes involved in blood coagulation. Second, a large number of enzyme species are released from cells during normal cell turnover. These enzymes almost always function intracellularly and have no physiologic use in the plasma. In healthy individuals, the levels of these enzymes are fairly constant, and represent a steady state in which the rate of release from damaged cells into the plasma is balanced by an equal rate of removal of the enzyme protein from the plasma. The presence of elevated enzyme activity in the plasma may indicate tissue damage that is accompanied by increased release of intracellular enzymes (Figure 5.20). [Note: Plasma is the fluid, non-cellular part of blood. Laboratory assays of enzyme activity most often use serum, which is obtained by centrifugation of whole blood after it has been allowed to coagulate. Plasma is a physiologic fluid, whereas serum is prepared in the laboratory.] Loading…  Plasma Enzymes Plasma enzymes are divided into 2 groups: 1.Functional plasma enzymes (Secretory enzymes) Enzymes are actively secreted into the blood by certain cell types. eg: the liver secretes zymogens (inactive precursors) of the enzymes involved in blood coagulation eg plasminogen PLASMA FUNCTIONAL ENZYME: Enzymes that are present in plasma and have specific function are called plasma functional enzyme. Activities of these enzymes are higher in plasma than tissues. The are mostly synthesized in liver and enter the circulation. e.g. lipoprotein lipase, plasmin, thrombin, choline, esterase, ceruloplasmin etc. 2. Non Functional Plasma Enzymes Present in plasma in very low concentration in comparison to tissue. Have no known physiological function in blood. Their substrate is absent in plasma and appear incidentally in blood. Increased levels of non functional plasma enzymes in plasma indicates tissue damage. These enzymes can be used for diagnosis.  2. Non functional plasma enzymes (Intracellular enzymes) Large number of enzyme species are released from cells during normal cell turnover. These enzymes almost always function intracellularly, and have no physiologic use in the plasma. In healthy individuals, the levels of these enzymes are fairly constant Increased plasma levels of these enzymes may indicate tissue damage Source of non functional Plasma Enzymes Cell Damage: Loading… e.g. Myocardial infarction and viral hepatitis. Obstruction of Normal pathways: B e.g. Obstruction of bile duct increases alkaline phosphatase. Release of enzymes from normal and diseased cells Medical importance of non functional enzymes Measurement of non functional enzymes is important for: Diagnosis of diseases; As disease of different organs cause elevation of different plasma enzymes. Prognosis of the disease; To follow up of the treatment by measuring plasma enzymes before and after.  Examples Creatine kinase Lactate dehydrogenase Transaminase Acid phosphataseE Amylase Alkaline phosphatase Lactate Dehydrogenase (LDH) Enzyme that catalyze the conversion of sugar into energy. Found in liver, heart, pancreas, kidney. It is released during tissue damage. It is a marker of common injuries and diseases. Diagnostic Application Used for the diagnosis of; Myocardial infraction Infective hepatitis Leukemia Muscular dystrophy LDH have five isoenzyme LDH 1: Heart and RBC LDH 2: WBCs LDH 3:Lungs LDH 4:Kidneys LDH 5: Liver and skeletal muscles. High LDH Level: High level of LDH indicate some form of tissue damage (For instance a person with high level of LDH will indicate muscle damage). Low LDH Level: It is very rare for a person to have low LDH level  Intracellular Distribution of Diagnostic Enzymes Liver Heart Pancreas Salivary Bone Muscle Biliary Prostate Glands Tract LD5 LD1 AMS AMS ALP CK ALP ACP AST AST LPS GGT ALT CK LD – Lactate Dehydrogenase ACP – ACid Phosphatase ALT – ALanine Transaminase ALP – ALkaline Phosphatase AST – ASpartate Transaminase GGT – Gamma glutamyl peptidase CK – Creatinine Kinase AMS – Alpha amylase Enzymes in Liver disorder Enzymes of Clinical Significance Enzyme Source of blood elevation ALT hepatopathy AST MI, hepatopathy GMT hepatopathy (alcohol, drugs) ALP biliary tract diseases, bone diseases ACP prostatic cancer CK MI (CK-MB), muscle diseases AMS pancreatitis LPS pancreatitis CHS hepatopathy (alcohol, drugs) – decreased 0  Alteration of Plasma enzyme levels in Disease states Many diseases that cause tissue damage result in an increased release of intracellular enzymes into the plasma. These enzymes are routinely determined for diagnostic purposes in diseases of the heart, liver, skeletal muscle, and other tissues. The level of specific enzyme in the plasma correlates with the extent of tissue damage. Thus, determining the degree of elevation of a particular enzyme activity in the plasma is useful in evaluating the prognosis for the patient  Plasma enzymes as diagnostic tools The presence of increased levels of these enzymes in plasma reflects damage to the corresponding tissue. For example, The enzyme alanine aminotransferase (ALT) is abundant in the liver. Elevated levels of ALT in plasma signals possible damage to hepatic tissue.  Isoenzymes (Isozymes) and Diseases Definition Isoenzymes are groups of enzymes having the same enzymatic activity but differ in physical, chemical and immunological properties because of genetically determined differences in amino acid sequences. They can be separated by electrophoresis  Isoenzymes of CK Creatine kinase – 3 isoforms CK consists of two protein subunits, M (for muscle) and B (for brain), which combine to form three isoenzymes. BB (CK-1), MB (CK-2) and MM (CK-3). All CK in the brain is the BB isoform, whereas in skeletal muscle it is MM. In cardiac muscle, about one-third is MB with the rest as MM CK-MB accounts for about 35 per cent of the total CK activity in cardiac muscle and less than five per cent in skeletal muscle: its plasma activity is always high after myocardial infarction Subunit structure, electrophoretic mobility and enzyme activity of Creatine kinase (CK) isoenzymes 1. Cardiac Disorders: e.g. Acute Myocardial Infarction (AMI). C) 1) The myocardium becomes ischemic and undergoes necrosis. (death of tissue) 2) Cellular contents are released into the circulation. Blood levels of the following enzymes increase: AST LD1 CK  2. Hepatic Disorders Hepatocellular Disorders: (1) Viral hepatitis: Hepatitis B & Hepatitis C. (2) Toxic hepatitis: caused by chemicals & Toxins (e.g. aflatoxin, Asp. flavus) Increased levels of the following enzymes: ALT AST LD5 Biliary tract disorders: The plasma levels of the following enzymes increase: ALP GGT  3. Skeletal Muscle Disorders Muscle dystrophy. Muscle trauma. Muscle hypoxia. Frequent Intra-Muscular Injections. Loading… The plasma levels of the following enzymes increase: CK AST 4. Bone Disorders: 1) Paget’s Bone Disease: caused by increased osteoclastic activity. 2) Rickets 3) Osteomalacia: The plasma levels of the following enzyme increase: ALP 5. Acute Pancreatitis The plasma levels of the following enzymes increase: Lipase AMS 6. Salivary Gland Inflammation: In Mumps: The levels of -Amylase (AMS) is significantly increased  7. Malignancies a) Plasma (Acid phosphatase) ACP levels increase in: Prostatic carcinoma. Bone metastatic carcinoma  b) Plasma levels of Alkaline phosphatase (ALP) increase in: Pancreatic carcinoma. Bile duct carcinoma. Liver metastasis.  c) Plasma levels of Total Lactate dehydrogenase (LDH) increase in: Leukemia Lymphomas. Liver metastasis.  Isoenzymes and Heart Diseases Isoenzymes (or isozymes) are a group of enzymes that catalyze the same reaction. However, these enzymes do not have the same physical properties (as they differ in amino acid sequence). Thus, they differ in electrophoretic mobility. The plasma level of certain isozymes of the enzyme Creatine kinase (CK) level is determined in the diagnosis of myocardial infarction.  Many isoenzymes contain different subunits in various combinations. CK occurs in 3 isoenzymes, each is a dimer composed of 2 subunits (B & M): CK1 = BB, CK2 = MB CK3 = MM each CK isozyme shows a characteristic electrophoretic mobility.  Myocardial muscle is the only tissue that contains high level of CK2 (MB) isoenzyme. Appearance of CK2 (MB) in plasma is specific for heart infarction. Following an acute myocardial infarction, CK2 appears in plasma 4-8 hours following onset of chest pain (peak is reached after 24 hours).  Diagnosis of Myocardial infarction(MI) Following an acute MI, CK-MB (CK2) appears approximately 4–8 hours following onset of chest pain, reaches a peak of activity at approximately 24 hours, and returns to baseline after 48–72 hours Troponin T and Troponin I are regulatory proteins involved in myocardial contractility. They are released into the plasma in response to cardiac damage. Cardiac troponin I (cTnI) is highly sensitive and specific for damage to cardiac tissue. cTnI appears in plasma within 4–6 hours after an MI, peaks in 8–28 hours, and remains elevated for 3–10 days. Elevated serum troponins are more predictive of adverse outcomes in unstable angina or myocardial infarction than CK2 Appearance of CK and cardiac troponin in plasma after a myocardial infarction  Isoenzymes of Alkaline phosphatase The commonest causes of an increased total alkaline phosphatase activity Bone disease with increased osteoblastic activity liver disease with involvement of the biliary tracts  Examples of enzymes commonly assayed for diagnostic purposes Enzyme Location Cause of elevated plasma level Acid phosphatase - ACP Prostate Prostatic cancer Alkaline phosphatase Bone, liver Rickets, hypoparathyroidism, (ALP) osteomalacia, obstructive jaundice, cancer of bone/liver Alanine aminotransferase – Liver (muscle, Hepatitis, jaundice, circulatory (ALT) heart, kidney) faillure with liver congestion Aspartate aminotransferase – Heart, muscle, Myocardial infarction, muscle (AST) red cells, liver damage, anemia, hepatitis, circulatory faillure with liver congestion Amylase - AM Pancres Acute pancreatitis, peptic ulcer -Glutamyl transferase – GMT Liver, kidney, Hepatitis, alcoholic liver  Enzyme profiles in some important Clinical conditions 1) Myocardial infarction 3) Muscle diseases 2. Creatine kinase (CK- 1.CK-MM MB) 2.AST 3. AST 3.Aldolase 4. LDH 4) Bone diseases 2) Liver diseases 4.ALP 5. ALT 6. AST 5) Prostate cancer 7. ALP 5.Acid phosphatase (ACP) 8. GGT 6.Prostate specific antigen (PSA)  Enzymes use in Laboratory Assays Enzyme methods - Enzymes isolated from different sources - used for determination of various substances in the blood, plasma/serum and urine - Enzymatic methods are much more specific than chemical methods Commercial kits or diagnostic strips - determination of glucose - glucose oxidase, peroxidase cholesterol - cholesterol esterase, cholesterol oxidase urea – urease, Samples- blood, plasma, serum - Glucose oxidase …….. blood or urine (strips) -Markers in the immunochemical analysis - ELISA (enzyme-linked immunoadsorbent assay) – peroxidase, alkaline phosphatase  Enzymes in Therapy Thrombolytic Drugs (Fibrinolytics) Streptokinase, Urokinase, Altepase (r-tPA) These drugs are used to lyse thrombi /clot to recanalyse occluded blood vessels They are curative rather than prophylactic and act by activating the natural fibrinolytic system  L- Asparaginase (Oncolytic) Normal tissues synthesize L-asparagine in sufficient quantities for protein synthesis Most neoplastic tissues require its exogenous supply from circulation L-asparaginase breakdowns circulating asparagine to L- aspartate and ammonia, prevents protein synthesis of neoplasms It produces cell death  Pancreatic enzymes – Digestive aids Lipases– digestion of fats. Deficiency leads to malabsorption of fats and fat- soluble vitamins Amylases– break down starch molecules into smaller sugars. Secreted by salivary glands and the pancreas Proteases– (trypsin, chymotrypsin, carboxypeptidases) break down proteins to amino acids  Other uses of Pancreatic enzymes Anti-inflammatory agent in cases of trauma, inflammation, thrombophlebitis In autoimmune conditions such as Rheumatoid arthritis, systemic lupus erythmatosus, scleroderma and multiple sclerosis, ulcerative colitis, Crohn’s disease and AIDS These enzymes reduce the number of circulating immune complexes which are responsible for the disease  Proteolytic and glycolytic enzymes for treating damaged tissue Debrase Gel Dressing Used for the mechanical debridement of damaged tissue from burns VibrilaseTM (recombinant vibriolysin) Used for the treatment of denatured proteins such as those found in burned skin Enzymes for infectious diseases Lysozyme, RNAse A, Urinary RNAse U - Used in antiHIV Uricase - it is used in the treatment of hyperuricemia and gout Urate + H2O Allantoin + H2O2 + CO2  Ribonuclease - It is used as an antiviral agent to hydrolyse the viral RNA Hyaluronidase - It breaks down hyaluronic acid in connective tissue - Also enhances the diffusion of subcutaneously injected agents - Typically used as an adjunctive agent with local anaesthetics, here it increases the speed of onset of action In cataract surgery - to increase the hypotonic effect of local anesthetics  REFERENCES Lippincott Biochemistry 5th Edition. Harper’s Illustrated Biochemistry Basic Medical Biochemistry-A Clinical Approach Download from Internet