Clinical Enzymology CC LEC - MED226 PDF

CLINICAL ENZYMOLOGY CC LEC – MED226 ENZYMES Systematic Name Specific biologic proteins that catalyze biochemical reactions without altering the Practical/Trivial name:...

CLINICAL ENZYMOLOGY CC LEC – MED226 ENZYMES Systematic Name Specific biologic proteins that catalyze biochemical reactions without altering the Practical/Trivial name: equilibrium point of the reaction or being According to the name of the substrate consumed or changed in composition with the addition of the suffix “ase” Frequently appear in the serum after Examples: cellular injury, degradation of cells or from Enzymes acting on lipids – lipase storage areas. Enzymes acting on proteins – protease Abnormal large amounts of enzymes in serum are used clinically as evidence of Practical/Trivial name: organ damage. According to the type of reaction they catalyzed. TERMS ASSOCIATED WITH ENZYMES: Examples: Holoenzyme -an active substance formed Transfer of amino group from substrate by combination of a coenzyme (cofactor) to another - transferase and apoenzyme. Transfer to phosphate group from a high Apoenzyme - the protein portion subject energy phosphate compound to its to denaturation, in which the enzyme substrate - kinase loses its activity. Catalytically inactive Effect of hydrolysis on phosphate esters protein when cofactor is removed. They – phosphatase are heat labile and dialyzable. Removal of hydrogen atoms from its Isoenzyme – different forms of the same substrate – dehydrogenase enzyme capable of same catalytic function in the body Systematic Name According to the numerical designation TERMS ASSOCIATED WITH ENZYMES: given by the Enzyme Commission (E.C.) Metalloenzyme - enzyme whose metal Examples: ions are intrinsically part the molecule E. C. 1. 1. 1. 7 for lactate such as catalases and cytochrome oxidase. dehydrogenase Proenzyme - inactive precursor of E. C. 3. 2. 1.1 for amylase enzymes, also referred to as zymogens E. C. 2. 6.1. 2 for alanine Substrates - substances acted upon by the aminotransferase enzymes which are specific for each of First number defines the class to which their particular enzyme. the enzyme belongs Cofactors - a non-protein Next two numbers indicate subclass and substance/compounds needed by an sub class to which the enzyme is assigned enzyme before enzymatic activity can be Last number is a specific serial number to manifested. Cofactors are thermostable each enzyme in its sub-class. and dialyzable. Enzyme Nomenclature Practical or Trivial Name December 4, 2024 1  CLINICAL ENZYMOLOGY CC LEC – MED226 According to Michaelis and Menten, this process involves reversible interactions or conversion to product. Michaelis-Menten Equation give the means to determine total enzyme concentration in serum and other body fluids. accurately describes virtually all single-substrate enzyme catalyzed many reactions and many bisubstrate reactions in which the concentration of one substrate is constant throughout the course of the reaction. Michaelis-Menten Constant Cofactor Coenzyme- organic molecule It hastens enzymatic reaction but Types of Specificity undergoes a change or is consumed to Absolute Specificity – enzymes combine another product. with only one substrate and catalyzes only Examples: NAD and NADP one corresponding reaction Activator- inorganic/metal ions Group Specificity – enzymes combining with all substrates containing a particular ENZYME KINETICS: chemical group A. Enzyme-Substrate Interaction: Bond Specificity – enzymes are specific to Enzymes bind to substrates to form an chemical bonds enzyme-substrate complex (ES).The Stereoisomeric Specificity – enzymes enzyme- substrate complex (ES) can that predominantly combine with only one either: dissociate back to enzyme (E) and optical isomer of a certain compound substrate (S). Breakdown into product (P) and free Enzyme Theory enzyme (E), provided the product has low Emil Fisher's LOCK and KEY THEORY: affinity for the enzyme. December 4, 2024 2  CLINICAL ENZYMOLOGY CC LEC – MED226 - based on the rigid enzyme molecule into First Order Reaction - the rate of which the substrate fits. The shape of the reaction is determined by the key (substrate) must conform into the lock concentration of substrate as well as of (enzyme). enzymes (the rate of reaction changes continuously with time as the substrate is consumed. Factors Affecting Enzyme Reactions: 1. Enzyme concentration - increase in the concentration of enzyme produces an increase in the rate of reaction, provided that the other conditions remain the same and that a constant but excess amount of substrate is present. Meaning, if the amount of enzyme is doubled, the reaction proceeds twice as fast. 2. Substrate Concentration - An increase in the Kochland’s Induced-Fit Theory concentration of substrate produces also an - substrate binding to the enzyme’s active site increase in the rate of reaction, provided all causes confirmational changes. other conditions are kept constant. - more acceptable because proteins are flexible, However, the rate of the reaction reaches a allowing shape changes and explaining the maximal value at a particular concentration influence of hormones on enzymatic activities. of substrate, and higher concentrations of substrate do not result in increased rate of reaction (Saturation kinetics). 3. Temperature – rate of any chemical reaction is usually increased 2-3 times for every 10 degrees Celcius rise in temperature. (25, 30, 37 degree celcius) 4. Hydrogen Ion Concentration or pH – Enzymatic reactions proceed at their fastest rate at an optimum pH and are considerably slowed or even stopped at higher or lower pH values. Most physiologic reactions occur in pH 7-8.. 5. Storage: Types of Reaction Order: -20 degree Celcius – for longer period of Zero Order Reaction - the rate of time reaction linear with time, independent of 2-8 degree Celcius – for substrate and substrate concentration and directly coenzymes proportional to enzyme concentration. December 4, 2024 3  CLINICAL ENZYMOLOGY CC LEC – MED226 Room temperature – ideal for storage of Measurement is done at the end of the LDH (LD4 and LD5) reaction 6. Hemolysis: mostly increase enzyme Disadvantage: underestimation of the concentration “true” enzyme activity and linearity of 7. Lactescense or milky specimen – decreases reaction cannot be observed enzyme concentration Multi-point and Kinetic Assay ENZYME INHIBITION: Change in concentration of the indicator Competitive Inhibitor – competes with substance at several intervals substrate for the active site. The Continuous measurement of change in inhibition is reversible. concentration as function of time Non- competitive Inhibitor – substances Use of Coupled Reactions that do not resemble the substrate and Enzymatic activity is measured by bind to the enzyme in areas other than coupling the activity with colorimetric the active site (called allosteric site) reaction Uncompetitive Inhibition – binds to the enzyme-substrate complex, so that an Units for Expressing Enzymatic Activity increasing the substrate concentration International Unit (I.U. or U) Equivalent to the amount of enzyme that catalyzes the conversion of 1 micromole of substrate per minute under controlled conditions. Katal Unit (K.U.) Equivalent to the amount of enzyme that catalyzes the conversion of 1 mole of substrate per second under controlled conditions. MEANS OF MEASURING ENZYME ACTIVITY Change in Coenzyme Concentration Increase in Product Concentration ENZYME INDUCTION: Decrease in Substrate Concentration This phenomenon states that a certain enzyme has the ability to adapt to their biochemical SPECIFIC ENZYMES systems. I. Phosphatases Characterized by its ability to hydrolyze a Types of Enzyme Assays large variety of organic phosphate esters Endpoint Analysis with the formation of an alcohol and a Reaction is initiated by addition of phosphate ion substrate Hydrolases Reaction is allowed to proceed for a Class 3 (Classification of Enzyme) period of time A. Alkaline Phosphatase (EC 3.1.3.1) or ALP December 4, 2024 4  CLINICAL ENZYMOLOGY CC LEC – MED226 Alkaline Orthophosphoric Monoester Osteoblastic tumors Phosphohydrolase Bone Cancer involved in the cleavage of phosphate Osteosarcoma containing compounds in alkaline pH. Sprue It facilitates movement of substances Hyperparathyroidism across cell membranes. Diabetes, renal failure Liver and bone diseases- most common Germ cell tumors (such as seminoma, causes of elevated ALP dysgerminoma) causes elevated placenta Reference Value: 30-90 U/L – like isoenzymes; Ovarian serous carcinoma, nonseminomatous germ cell tumors, endometrial carcinoma, and leukemia. Clinical Significance: Carcinoplacental ALP Regan ALP – found in lung, breast, ovarian and gynecological cancers; bone ALP co-migrator; - most heat stable, inhibited by phenylalanine reagent Nagao ALP – adenocarcinoma of the pancreas and bile duct, pleural cancer, Clinical Significance: variant of Regan Decreased serum ALP: – inhibited by L-leucine and Hypophosphatasia- characterized by phenylalanine insufficient bone calcification Kasahara ALP – After blood transfusions or hepatoma/hepatocellular carcinoma cardiopulmonary bypass (transiently decrease) Clinical Significance: Malnutrition Increased ALP is increased in the ff. conditions: Hypophosphatemia (prolonged, severely Liver Disease low levels) Biliary Obstruction Zinc deficiency (prolonged, severely low Hepatitis and cirrhosis levels. Liver tumors Zinc is a necessary cofactor in ALP Cholestasis activity. Bone Disease ALP Level are normally higher in: Osteitis deformans/Paget’s disease Children than adults Osteomalacia Women during pregnancy Rickets December 4, 2024 5  CLINICAL ENZYMOLOGY CC LEC – MED226 B. Acid Phosphatase (E.C 3.1.2.3 or ACP) Active osteoclast-mediated bone Acid Orthophosphoric Monoester resorption Phosphohydrolase Gaucher's cells Catalyzes same reaction made by ALP Hairy cell leukemia Active at pH 5.0 Diagnostic significance: detection of ACP Elevations prostatic carcinoma Moderate elevation of Total ACP Tissue sources: prostate (major source), Female Breast CA RBC, platelets and bone Paget’s disease Hyperparathyrodism Reference values: Non-prostatic ACP elevations 2.5-11.7 U/L (total ACP male) Niemann-Pick disease 0-3.5 ng/mL (prostatic ACP) Gaucher’s disease Myelocytic leukemia ACP Isoenzymes Band 1, the major source is prostate III. Aminotransferases gland. Prostatic ACP activity is inhibited Catalyzes the transfer of an amino group by tartrate. of one amino acid to a hydrocarbon to Band 2 and 4 isoenzymes are from form a different amino acid granulocytes. Aminotransferases are of two types: Band 3 is the major form present in Aspartate Aminotransferase (EC plasma. This isoenzyme (band 3) is 2.6.1.1) or AST derived from platelets, erythrocytes, and L-aspartate + 2-oxoglutarate monocytes. oxaloacetate + glutamate Band 5 is found mainly in osteoclasts. Alanine Aminotransferase (EC This isoenzyme, (band 5) is resistant to 2.6.1.2) or ALT tartrate inhibition. L-alanine + alpha ketoglutarate pyruvate + glutamate Tartrate-inhibited ACP (prostatic isoenzyme) Cofactor - pyridoxal-5'-phosphate or P- Prostatic Cancer 5’-P Benign prostatic hyperplasia Prostatic infarction AST (Aspartate Aminotransferase) Urinary tract obstruction, carcinoid serum glutamic-oxaloacetic tumors of the rectum, and prostatic transaminase massage Involved in the transfer of an amino Medico-legal cases. ACP also has group between aspartate and α- implications in suspected rape. Positive ketoacids with the formation of ACP is evident in vaginal swab if semen is oxaloacetate and glutamate present for the first 12 hours up to four 2 isoenzyme fractions: cytoplasm and days from the incident. mitochondrial AST Major tissue source: cardiac tissue, liver Tartrate-resistant ACP (bone isoenzyme) and skeletal muscles December 4, 2024 6  CLINICAL ENZYMOLOGY CC LEC – MED226 Other sources: kidney, pancreas and RBC Reference values: 5-37 U/L Aminotransferase levels are altered in: Hepatocyte injury (increase in AST, and Clinical Significance ALT but to a lesser degree) Increased AST: Muscle injury (increase in both enzymes) In the evaluation of myocardial Kidney infarcts (increase in both enzymes) infarction, hepatocellular disorders and Renal failure (falsely lowered) skeletal muscle involvement MI -> AST level is usually 4-10 times the III. Creatine Kinase (EC 2.7.3.2) or CK upper limit of normal Involved in the reversible phosphorylation Diagnosis of chronic alcohol abuse and of creatine by ATP drug hepatotoxicity CK is an enzyme is involved in energy Pulmonary infarction, pericarditis, acute storage of tissues. hepatitis On the course of active muscle Decreased AST: contraction. ATP is used up and creatine seen during pregnancy phosphate is converted by CK to creatine and ATP. ALT (Alanine aminotransferase) This process allows continued contraction. serum glutamic pyruvic transaminase During periods of rest, ATP is converted to enzymatic activity similar to AST creatine phosphate by CK to serve as Highest concentration in the liver energy reservoir. Other sources: kidney, pancreas, RBC, Creatine kinase requires magnesium as a heart, skeletal muscles, cofactor. lungs Reference Values: 6-37 U/L CK Isoenzymes CK1 or CK-BB- found predominantly in the Diagnostic significance brain and smooth muscles and most Significant in the evaluation of hepatic rapidly moving enzyme disorders CK2 or CK-MB - hybrid, normal muscle Diagnosis of acute or chronic viral contains 14% to 20% of CK-MB in skeletal hepatitis -> ALT increases to a greater muscle, CK-MB comprises 0% to 1% of degree than AST total CK in type 1 fibers, and 2% to 6% of Monitors the course of hepatitis total CK in type 2 fibers. treatment and the effect of drug therapy CK3 or CK-MM - found in skeletal muscles and slowest and most common form Aminotransferase levels are altered in: Macro-CK - an oligomer present in Hepatocyte injury (increase in AST, and mitochondria and is seldom released into ALT but to a lesser degree) circulation. Muscle injury (increase in both enzymes) Kidney infarcts (increase in both Reference Values: enzymes) Male = 15-160 U/L Renal failure (falsely lowered) Female =15-130 U/L December 4, 2024 7  CLINICAL ENZYMOLOGY CC LEC – MED226 CK-MB = < 6% of total CK Diagnostic significance IV. Gamma- Glutamyl Transferase (EC 2.3.2.1) or Very sensitive indicator of acute myocardial GGT infarction (AMI) and Duchenne disorder GGT catalyzes the transfer of glutamyl Duchenne's Muscular dystrophy - highest moiety from peptides to amino acids, other elevation of total CK peptides, or water molecules. Following AMI, CK-MB levels begins to rise GGT are plasma membrane bound on cells within 4-8 hours, peak at 12-24 hours and that has high secretory or absorptive normalize within 48-72 hours. properties (such as liver, canaliculi cells CK-MB is not elevated in angina. proximal renal tubules, intestinal epithelium, and prostate gland). CK levels are increased in: Most sensitive indicator of alcoholism. − Progressive muscular dystrophy Half-life is 7 to 10 days. In alcoholic liver − Poliomvelitis disease, half-life increases to 28 days. − Acute psychosis Sensitive indicator of alcoholism (occult − Alcoholic myopathy alcoholism) - most sensitive marker of acute − Delirium tremens alcoholic hepatitis − Hypothyroidism − Malignant hyperthermia Reference Values: − Acute cerebrovascular disease Male = 6 - 45 U/L − Trichinosis and dermatomyositis Female = 5 - 30 U/L − Cardiac or skeletal muscle damage GGT elevations − Chronic myopathies Liver damage is the major source of GGT − Chronic renal failure release. − Acute respiratory excretion Smoking -> Moderate smoking raises GT levels by 10%, while heavy smoking by 20%. CK-BB is increased in: Medications increase GGT levels up to five Smooth muscle injury (intestinal ischemia) times than normal, these drugs include: Malignancies (prostate cancer, small cell ethanol, warfarin, phenytoin, barbiturates carcinoma of the lung, and intestinal carbamazepine, and valproic acid. malignancies). GGT decrease Macro-CK2 is present in Pregnancy -> First trimester of pregnancy Malignancies causes 25% decrease in GGT levels Myocardial infarction (when Macro-CK2 is Oral contraceptives reduce GGT by 20%. present, it is usually associated with poor prognosis) V. Lactate Dehydrogenase (EC 1.1.1.27) or LD MI (less than 5% of the causes) LD is a zinc-containing enzyme, and its activity is part of the glycolytic pathway. LD is a tetramer of two active subunits: December 4, 2024 8  CLINICAL ENZYMOLOGY CC LEC – MED226 H (heart) and M (muscle). Isoenzymes: S-type (ptyalin); P-type Combinations of the subunits produce five (amylopsin) isoenzymes Reference Values: LD1 (HHHH; H4) 60-180 SU/dL (Somogyi units) LD2 (HHHM; H3М) 95-290 U/L LD3 (HHMM; H2M2) Optimum pH: 6.9-7.0 LD4 (HMMM; HM3) L Diagnostic Significance D5 (MMMM; M4) Acute pancreatitis Rise at 2-12 hrs, peak at 24 hrs and Lactate Dehydrogenase normalize within 3-5 days Normal electrophoretic pattern AMS in urine remains elevated up to 7 All bands of isoenzymes are low days LD2 is always higher than LD1 Tissue Sources: heart, RBC, Kidney (LD1 VIlI. Lipase (EC 3.1.1.3) and LD2); lungs, pancreas, spleen (LD3); An enzyme that hydrolyzes the ester skeletal muscles, liver, intestine (LD4 and linkages of fats to produce alcohol and LD5) fatty acids RBC and Cardiac tissues contain high Most specific pancreatic marker: secreted levels of LD-1 exclusively in the pancreas, not affected Reference Values: by renal disorders 100-225 U/L (forward reaction) Reference Values: 0-1.0 U/mL 80-280 U/L (reverse reaction) Optimum pH: 7.8-8.0 Diagnostic Significance Diagnostic Significance Highest level are seen in pernicious In acute pancreatitis, lipase levels rise 6 anemia and hemolytic disorders hours after onset of attack, peak at 24 Hepatic carcinoma and toxic hepatitis - 10 hours, remains elevated for 7 days, and fold increase normalize in 8-14 days Viral hepatitis and cirrhosis - 2-3x In chronic acute pancreatitis, acinar cell increased degradation occurs resulting in loss of LD 1 and LD2 "flipped pattern" - MI, amylase and lipase production hemolytic anemia LPS is also elevated -> pancreatic duct LD5 - moderately increased in acute viral obstruction and tumors of the pancreas hepatitis, and cirrhosis; markedly increased in hepatic carcinoma and toxic Other Enzymes hepatitis Glucose-6-Phosphate Dehydrogenase Oxidoreductase VII. Amylase/Diastase (E.C. 3.2.1.1) Catalyzes the oxidation of glucose-6- Catalyzes the breakdown of starch phosphate -> 6-phosphogluconate Smallest enzyme Important as the 1st step in the pentose Earliest pancreatic marker phosphate shunt Reference Value: 7.9 - 16.3 U/g Hgb December 4, 2024 9  CLINICAL ENZYMOLOGY CC LEC – MED226 It is important in cleavage of Leucine Aminopeptidase (LAP) succinylcholine. (muscle relaxant Exhibits napthylamidase activity -> used during surgery) enzyme attacks the free amino end of the It is primarily produced in the liver, but is peptide chain also synthesized by myocardium and Substrate: Acyl B napthylamide pancreas. Rich in pancreas Increased LAP: Clinical Significance Hepatobiliary disease Enzyme measurement is done: Pancreatic Cancer To monitor those exposed to Last trimester of pregnancy cholinesterase inhibitors (pesticides such Obstructive biliary disease as organophosphate insecticides), Pseudocholinesterase activity falls before 5' Nucleotidase (5' N) cholinesterase in RB\s with poisoning. Used to differentiate obstructive from As a liver function test. hepatocellular jaundice and hepatobiliary Pseudocholinesterase production reflects from osseous disease synthetic function of the liver. Activity is Increased -> post-hepatic jaundice, low in malnutrition. intrahepatic cholestasis and infiltrative For diagnosis of genetic variants -> lesions of liver prolonged apnea after using Slightly increased -> hepatocellular succinylcholine during anesthesia. jaundice Normal -> bone disease Angiotensin-Converting Enzyme (EC: 3.4.15.1) or ACE Ornithine carbamoyl transferase (OCT) It is also known as kininase II or peptidyl Found most exclusively in the liver dipeptidase A. Marker for hepatobiliary diseases ACE is responsible for conversion of Excellent marker for liver disease but it is angiotensin I to angiotensin Il and rarely used inactivation of bradykinin, enkephalin, tachykinins. Cholinesterase (EC 3.1.1.7) Enzyme structure is described as a single and Pseudocholinesterase (EC 3.1.1.8) polypeptide chain with zinc at the active These enzymes cleave one of the body's center. (Activity lost with chelating major neurotransmitter known as agents). acetylcholine. Most activity is present in the lungs, but it True Cholinesterase is found in endothelial cells throughout It has high activity in CNS, red the body. blood cells, lung, and spleen. Most common reason for testing is to Pseudocholinesterase or acylcholine diagnose and monitor sarcoidosis acylhydrolase If progressed to fibrosis, ACE declines Elevation is noticeable in pulmonary involvement. December 4, 2024 10  CLINICAL ENZYMOLOGY CC LEC – MED226 Enzymes as Cardiac Markers Myeloperoxidase Enzymes for Myocardial Infarction Hydrogen peroxide oxidoreductase Stored in azurophilic granules of PMNs and monocytes Catalyzes the conversion of chloride anion and hydrogen peroxide to hypochlorite Clinical significance: MPO is released into the extracellular fluid and general circulation during inflammatory conditions Active mediator in the atherosclerotic CV disease Aldolase Enzyme involved in the conversion of fructose 1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde-3-phosphate Aldolase A - muscle, RBC and brain Aldolase B - liver, kidney, enterocytes Aldolase C - brain Elevated aldolase level can be seen in skeletal muscle damage, IM, muscular dystrophy Enzymes as Markers of Hepatic Disorders Hepatic Disorders Acute injury (Acute hepatitis) and Necrosis ALT, AST, ALP, Bilirubin (B1 and B2), LD4 and LD5 Normal Total Protein and Albumin Cirrhosis Bilirubin (B1 and B2), NH3 Slightly increased/Normal - ALT, AST, ALP and LD Decreased/Low total protein and albumin Increased Globulin Biliary Tract Obstruction Increased ALP, Bilirubin (B2), GGT, 5'- nucleotidase, LAP December 4, 2024 11