Enzymes: Chapter 10 PDF

ENZYMES Chapter 10 Preamble ◦ PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY. ◦ PowerPoints DO NOT cover the details needed for the Unit exam ◦ Each student is responsible for READING the TEXTBOOK for details to answer the UNIT OBJ...

ENZYMES Chapter 10 Preamble ◦ PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY. ◦ PowerPoints DO NOT cover the details needed for the Unit exam ◦ Each student is responsible for READING the TEXTBOOK for details to answer the UNIT OBJECTIVES ◦ Unit Objectives are your study guide (not this PowerPoint) ◦ Test questions cover the details of UNIT OBJECTIVES found only in your Textbook! Introduction Enzymes are biological catalysts present in all the cells of the body; each enzyme catalyzes a specific reaction. Enzymes supply the energy and/or chemical changes necessary for vital activities. Enzymes are proteins. – All enzymes are synthesized by the body under the control of a specific gene in the same way other proteins are formed. Enzyme Biochemistry (1) Enzymes have three properties: 1. Enzymes are not altered or consumed during the reaction. 2. Only small amounts of the enzyme are required because the enzyme is used over and over again. 3. Enzymes accelerate the speed at which a chemical reaction reaches equilibrium but do not alter the equilibrium constant. Have a unique sequence of amino acids that provides the primary structure Are composed of a heat-labile protein portion called the apoenzyme, which requires a coenzyme for full catalytic activity Catalyze reactions in which one or more substrate (S) molecules are converted to a product (P) Substrate ¾Enzyme ¾¾¾ ® Product Enzyme Biochemistry (2) Prosthetic groups – Bound coenzymes Holoenzyme – Apoenzyme and cofactor or coenzyme that form the catalytically active unit Cofactors – Organic or inorganic compounds that are required for enzyme function Coenzymes – Organic cofactors that commonly have a structure related to vitamins Enzyme Biochemistry (3) Enzymes are classified by the degree of substrate specificity. – Absolute specificity ▪ Catalyze only one specific substrate and one reaction – Group specificity ▪ Indicates an enzyme that catalyzes substrates with similar structural groups – Bond specificity ▪ Describes the catalyzing of a reaction with a certain type of bond – Stereospecificity ▪ Stereoisomer specificity ▪ Catalyze reactions with only certain optical isomers Enzyme Kinetics (1) Energy of Activation (EA) – Amount of energy required to energize one mole of the substrate to form the activated complex. Enzyme binds with the substrate to form an enzyme-substrate (ES) complex, which provides the free energy required for the reaction. E+X ® ES ® P+E Enzyme Kinetics (2) Vmax – When the substrate concentration is high enough that all enzyme molecules are bound to the substrate and all active sites are engaged Michealis-Menten Constant (K m ) – The substrate concentration in moles per liter when the initial velocity is 1 V 2max Km is a constant and remains the same for a given enzyme-substrate pair under given conditions. Copyright © 2018, 2011 Pearson Education, Inc. All Rights Reserved Enzyme Kinetics (3) First-order kinetics – The velocity is directly proportional to the substrate concentration. Zero-order kinetics – The reaction rate is independent of substrate concentration. Factors Influencing Enzyme Action (1) Enzymes are measured by activity and not by mass. – Because of the small amounts of enzyme normally present in serum International Unit – Amount of enzyme that produces 1 μmol of product per minute under standardized conditions of temperature, pH, substrate, and activators Substrate concentration – Increases the rate of enzymatic reaction to a certain point Temperature – Increases the velocities of most chemical reactions Factors Influencing Enzyme Action (2) pH – Optimal range for enzymes is relatively small. Cofactors – Activators, or coenzymes increase the rate of an enzymatic reaction. Inhibitors – Various substances that can decrease the rate of reaction Factors Influencing Enzyme Action (3) Competitive inhibition – Substance is similar to the normal substrate and competes with the substrate for the binding or active site of the enzyme. Noncompetitive inhibition – Inhibitor is structurally different than the substrate and binds to an allosteric site on the enzyme molecule. Uncompetitive inhibition – When an inhibitor binds to the ES complex to form an enzyme-substrate inhibiting complex that does not yield product. Diagnostic Enzymology Common enzyme measurements are measurements of either product formation or substrate depletion over time. – Not the actual enzyme concentration Abnormal serum enzyme levels are found in various diseases and in inflammation when more cells than normal are being destroyed. Concentrations of certain isoenzymes also provide evidence of a particular disease. Enzyme Classification Commission on Enzymes (Enzyme Commission of the International Union of Biochemistry) has established six classes. 1. Oxidoreductases 2. Transferases 3. Hydrolases 4. Lyases 5. Isomerases 6. Ligases Isoenzymes Multiple forms of an enzyme that can catalyze the enzyme’s characteristic reaction Existence of multiple forms can be used to determine the origin of a disease by ascertaining the particular isoenzyme present. Isoenzymes can be differentiated by various physical properties. Cardiac Enzymes (1) Creatine Kinase (CK) – A cytoplasmic and mitochondrial enzyme that catalyzes the reversible phosphorylation of creatine by adenosine triphosphate (A T P) Creatine + ATP ¾Mg ¾¾+2 ® Creatine phosphate + ADP CK – Equilibrium of the C K reaction is dependent on p H. – Creatine kinase is very important in muscle tissue. ▪ Allows high-energy phosphate to be stored in a more stable form than A T P Cardiac Enzymes (2) Creatine Kinase (CK) – Levels are highest in three major tissues: ▪ Muscle, brain, and heart, in descending order. – Creatine kinase is a dimer consisting of two subunits: ▪ B for brain, and M for muscle ▪ Results in three possible combinations – MM, MB, and BB Cardiac Enzymes (3) Creatine Kinase (CK) – CK-1 (BB) ▪ Most anodal isoenzyme ▪ Travels the farthest on electrophoresis ▪ Found predominantly in the brain and central nervous system (CNS) – CK-2 (MB) ▪ Found predominantly in heart muscle – To a minor degree in skeletal muscle ▪ Clinically important in the diagnosis of myocardial infarctions – CK-3 (CK-MM) ▪ Found in skeletal and cardiac muscle ▪ Major isoenzyme in normal serum Cardiac Enzymes (4) CK Clinical Significance – Elevations of CK are found primarily in conditions affecting brain, muscle, or cardiac muscle. ▪ Increases in both MM and MB isoenzymes indicate damage that is cardiac in origin. – Skeletal muscle diseases or conditions associated with an elevated MM fraction include muscular dystrophy. ▪ Other conditions include convulsive seizures (epilepsy), muscle trauma, extreme physical exercise, and viral myositis. – Creatine kinase (BB) found in brain and central nervous system disorders is associated with: ▪ Brain injury, including cerebral ischemia ▪ Acute cerebrovascular accident (CVA, or stroke) ▪ Cerebral trauma  Creatine Kinase: Clinical Significance Major Sources of Elevation (5) Skeletal Muscle Diseases (C K-3) (M M) Myocardial Tissue (Cardiac) (M M and M B) Muscular dystrophy, especially Duchenne type Convulsive disorders, e.g., epilepsy, grand mal seizures Myocardial infarction Muscle trauma Myocarditis Crush injuries Surgery Cardiac trauma Extreme physical exercise Open-heart surgery Viral myositis/polymyositis Intramuscular injections (variable) Cardiac catheterization Rhabdomyolysis Hyperthermia  Creatine Kinase: Clinical Significance Major Sources of Elevation (6) Brain (C K-1) (B B) Miscellaneous elevations of C K-1 (B B) Cerebral ischemia Acute C V A (stroke) Normal childbirth (muscle Head injuries, cerebral trauma contraction) Brain tumor Small cell carcinoma of the Cerebral thrombosis lung Pulmonary infarction Hypothermia Reye’s syndrome Prostate, colon, ovary, and Hypothyroidism breast tumors Cardiac Enzymes (7) CK Methodologies – Serum is the specimen of choice. – CK activity is unstable and rapidly lost during storage. C K Reference Range Range is Affected by: Males Age Physical activity 52-236 U/L Race Females Bed rest (even overnight can 38-176 U/L decrease C K) Cardiac Enzymes (8) CK Isoenzyme Methodologies – CK isoenzymes are measured by immunoinhibition, mass assay, and electrophoresis. ▪ Immunoinhibition – Requires a specific antibody against the M sub-unit that reacts with the M on the MB isoenzyme and completely inhibits the MM isoenzyme – Has been replaced in many laboratories by mass assay ▪ Mass assay – Sandwich technique is used with two antibodies One against the M subunit (anti-M) One against the B subunit (anti-B) Cardiac Enzymes (9) CK Isoenzyme Methodologies – CK isoenzymes are measured by immunoinhibition, mass assay, and electrophoresis. ▪ CK electrophoresis – Semiquantitative – Can be performed on agarose or cellulose acetate. – CK isoenzymes are separated by electromotive force at a pH of 8.6. CK-BB (CK-1) migrates most rapidly toward the anode or positive pole. CK-MB migrates midway. CK-MM remains near the point of origin. Cardiac Enzymes (10) C K Isoenzyme Reference Range and Relative Index – In a normal gel electrophoresis, C K-B B is absent or trace, C K-M B is ≤6% of the total, and C K-M M is 94-100%. – Relative Index ▪ Calculated by dividing the C K-2 mass (μg/L) by the total C K activity (U/L) CK-2(mg/L or ng/ML) ´ 100 Relative Index (RI) = Total CK (U/L) CK Isoenzyme Reference Range and Relative Index – Relative Index ▪ Reference range for the relative index is less than 3%. ▪ Ratios greater than 5 are indicative of a cardiac source. ▪ Ratios between 3 and 5 are a gray zone. – Requires serial determination to diagnose or rule out myocardial infarction Lactate Dehydrogenase (LD) (1) An oxidoreductase A hydrogen transfer enzyme that catalyzes the oxidation of L-lactate to pyruvate with NAD+ as a hydrogen acceptor Lactase dehydrogenase L-Lactate + NAD ¬¾¾¾¾¾¾¾ + ® Pyruvate + NADH + H+ pH 8.8 to 9.8 pH 7.4 to 7.8 Found in the cytoplasm of most cells in the body – Elevations of LD without any other information is nonspecific for any disease or disorder. Concentration in tissues is approximately 500 times higher than serum levels. LD is a tetramer consisting of four polypeptide chains. In healthy individuals, the concentration of LD isoenzymes are: – LD-2 > LD-1 > LD-3 > LD-4 > L D-5. Lactate Dehydrogenase (LD) (2) Two subunits H (heart) and M (muscle) form five isoenzymes. – LD-1 (HHHH) ▪ Heart, RBCs, and kidney – LD-2 (HHHM) ▪ Heart, RBCs, and kidney – LD-3 (HHMM) ▪ Spleen, lungs, and many tissues – LD-4 (HMMM) ▪ Liver and skeletal muscle – LD-5 (MMMM) ▪ Liver and skeletal muscle Lactate Dehydrogenase: Clinical Significance (1) Cardiac or Heart Liver Disease Cirrhosis Myocardial infarction Obstructive jaundice Myocarditis Viral hepatitis Shock Toxic hepatitis Infectious mononucleosis Congestive heart failure Hepatic cancer or metastasis Hemolytic Disease Muscle Muscular dystrophy Hemolytic anemias Muscle trauma Pernicious anemia: highest levels Physical exercise Acute leukemias Crush injuries Lactate Dehydrogenase: Clinical Significance (2) Miscellaneous Pulmonary infarct or embolism Hemolyzed specimen Renal disease: glomerulonephritis, pyelonephritis Renal and bladder malignancies Malignancies Hodgkins Abdominal cancer Lung cancer Lactate Dehydrogenase (LD) (3) LD Clinical Significance – Increases in LD may be: ▪ Absolute (total LD) ▪ Relative (increase in one of the isoenzymes) Lactate Dehydrogenase (LD) (4) L D Methodologies – Most current methods measure the interconversion of NAD+ to NADH at 340 nm Wacker procedure ▪ Lactate to pyruvate reaction ▪ Most often used – Wacker procedure ▪ Lactate to pyruvate reaction L-Lactate + NAD+ ¾Lactase ¾¾¾¾¾¾ dehydrogenase ® Pyruvate + NADH + H+ ▪ Most often used – Wroblewski and LaDue (P→L) ▪ Reverse reaction Pyruvate + NADH + H+ ¾Lactase ¾¾¾¾¾¾ dehydrogenase ® L-Lactate + NAD+ ▪ Less expensive Lactate Dehydrogenase (LD) (5) LD Reference Range L→P 100-224 U/L @ 37°C P→L 80-280 U/L @ 37°C Hemolysis will falsely elevate L D because of R B C L D-1 and L D-2. Enzymes in Cardiac Disease Two major cardiac enzymes are CK and CK-MB. – CK ▪ Increases to above the upper reference limit 4 to 6 hours after onset of a myocardial infarction ▪ Peaks at 24 hours ▪ Returns to normal within 2 to 3 days – CK-MB rises and returns to baseline more rapidly than CK. Enzymes in Liver Disease (1) Marked elevations of AST and ALT are associated with hepatocellular disease or damage to hepatocytes. Marked elevation of ALP is associated with hepatobiliary disease or obstructive liver disease. Enzymes in Liver Disease (2) Aspartate Aminotransferase (AST) – Catalyzes the interconversion of amino acids and α-oxoacids by the transfer of amino groups – Transamination is important in the synthesis and degradation of amino acids that occurs during intermediary metabolism. – Found predominantly in the cytoplasm of the cells of the heart, liver, skeletal muscle, and kidney – AST Clinical Significance ▪ Elevations of AST occur mostly in myocardial (cardiac), liver, and muscle diseases or conditions. – Now generally accepted AST is of little value due to lack of tissue specificity. Aspartate Aminotransferase: Clinical Significance (2) Liver Cardiac and Heart Viral hepatitis Alcoholic hepatitis Myocardial infarction Liver cancer or tumor Congestive heart Nonalcoholic steatohepatitis failure Toxic hepatitis (drugs or chemicals) Cirrhosis Pericarditis Bile duct obstruction Myocarditis Hepatic carcinoma Reye’s syndrome Infectious mononucleosis Aspartate Aminotransferase: Clinical Significance (3) Muscle Miscellaneous Muscular dystrophy Pulmonary infarct Dermatomyositis Hemochromatosis Skeletal muscle Pulmonary embolism injury (trauma) Acute pancreatitis Gangrene Enzymes in Liver Disease (4) Aspartate Aminotransferase (AST) – AST Methodologies ▪ AST is measured using a modification of the Karmen method with the addition of coenzyme P-5-P to ensure full catalytic activity. L-Aspartate + a -ketoglutarate ¬¾¾¾¾¾¾¾ Aspartate aminotransferase P-5-P ¾® Oxaloacetate + L-Glutamate ▪ Malate dehydrogenase is the indicator reaction measuring the decrease in absorbance at 340 nm as NADH is oxidized to NAD+ Oxaloacetate + NADH + H+ ¾Malate ¾¾¾¾¾¾ dehydrogenase ® Malate + NAD+ Enzymes in Liver Disease (5) Aspartate Aminotransferase (A S T) A S T Reference Range At 37°C is 5-30 U/L No clinically significant gender differences Hemolysis should be avoided. A S T is 10 to 15 times higher in R B Cs than in serum. Enzymes in Liver Disease (6) Alanine Aminotransferase (ALT) – An aminotransferase that catalyzes the deamination of alanine – Found in greatest concentration within the cytoplasm of the liver ▪ Predominantly a liver-specific transaminase ▪ Present in smaller amounts in the kidney, the heart, and skeletal muscle L-Aspartate + a -ketoglutarate ¾Aspartate ¾¾¾¾¾¾¾ aminotransferase P-5-P ® Pyruvate + L-Glutamate – ALT Clinical Significance ▪ Diagnostic significance is confined mainly to hepatocellular disorders. – More specific than AST ▪ Highest elevation – Acute viral hepatitis and toxic hepatitis. ▪ Moderate elevations – Obstructive liver disease, hepatic cancer, and cirrhosis. Enzymes in Liver Disease (7) Alanine Aminotransferase (A L T) A L T Clinical Significance Diagnostic significance is confined mainly to hepatocellular disorders. More specific than A S T Highest elevation Acute viral hepatitis and toxic hepatitis. Moderate elevations Obstructive liver disease, hepatic cancer, and cirrhosis. De Ritis Ratio (A S T/A L T) Alcoholic liver disease and cirrhosis A S T >A L T. Ratio of A S T/A L T is >1.0. Viral hepatitis, acute inflammatory disease and obstructive liver disease are associated with A L T >A S T. Ratio of A S T/A L T is A L T. Ratio of A S T/A L T is >2.0. Enzymes in Liver Disease (8) Alanine Aminotransferase (ALT) – ALT Methodologies ▪ Wroblewski and LaDue – Uses lactate dehydrogenase (LD) as the indicator reaction. (A ST → LD) Alanine + a -ketoglutarate ¾Alanine ¾¾¾¾¾¾¾ aminotransferase P-5-P ® Pyruvate + L-Glutamate Pyruvate + NADH ¾Lactase ¾¾¾¾¾¾ dehydrogenase ® L-Lactate + NAD+ Enzymes in Liver Disease (9) Alanine Aminotransferase (A L T) A L T Reference Range At 37°C is 6-37 U/L Hemolysis should be avoided. A L T levels in R B Cs are 5 to 8 times higher than serum levels. Enzymes in Liver Disease (10) Alkaline Phosphatase (ALP) – Generic name for a group of enzymes with maximum activity in pH range of 9.0 to 10.0. – ALP frees inorganic phosphate from an organic phosphate monoester, resulting in the production of an alcohol at an optimal pH of 10. - Phosphomonoester + H2O ¾Alkaline ¾¾¾¾¾¾ phosphatase +2 Zn , Mg +2 ® Alcohol + HPO4 – Present is practically all tissues of the body ▪ Especially at or near the cell membranes – Richest sources are the cell membranes of hepatocytes lining the sinusoidal border of the parenchymal cells and the bile canaliculi, and osteoblasts in the bone. Enzymes in Liver Disease (11) Alkaline Phosphatase (ALP) – ALP Clinical Significance ▪ Highest levels of ALP are found in hepatobiliary disease involving biliary tract disorders and hepatobiliary obstruction. ▪ Elevations because of osteoblastic activity are associated with Paget’s disease (osteitis deformans). Alkaline Phosphatase: Clinical Significance – Elevated ALP Carcinoplacental Hepatic Bone (Osteoblastic) (Regan) Physiological increase Hepatobiliary Paget’s disease Healing bone disease (e.g., (osteitis deformans) fractures hepatitis, cirrhosis) Osteomalacia (osteoblastic Biliary tract disorders Rickets activity) Hepatobiliary Osteogenic Pregnancy obstruction sarcoma (placental A L P) Liver cancer Hyperparathyroidism Infants and children Infectious and viral (growth spurts) hepatitis Alcoholic cirrhosis Enzymes in Liver Disease (13) Alkaline Phosphatase (ALP) – ALP Methodologies ▪ Bowers and McComb is the most common procedure. – 4-nitrophenol phosphate (4-NPP) [formerly Ρ-nitrophenylphosphate (Ρ-NPP)] is the substrate, and the yellow product, 4-nitrophenoxide, is measured at 405 nm. 4-Nitrophenylphosphate(4-NPP) ¾¾¾¾¾¾¾ ® Alkaline phosphatase Mg+2 pH 10.3 4-Nitrophenoxide + Phosphate Enzymes in Liver Disease (14) Alkaline Phosphatase (A L P) A L P Reference Range 44-147 U/L Dependent on the specific procedure or modification used and the instrument Pediatric and adolescent A L P levels are 2 to 3 times higher than adult levels. Pregnancy is also associated with an elevated A L P as a result of placental A L P. Biliary Tract Enzymes (1) Gamma Glutamyl Transferase (GGT) – Transfers the γ-glutamyl group from glutathione and other γ-glutamyl peptides to amino acids or small peptides to form the γ amino acids and cysteinyl-glycine. Glutathione + Amino acid ⎯⎯⎯⎯⎯⎯⎯⎯⎯ Gamma glutamyltransferase → Glutamyl-peptide + L-Cysteinylglycine – Present in serum and all cells except muscle – Mainly from the kidney, liver, pancreas, and intestine, where it is found primarily in the cell membrane – Most serum activity is from the liver. ▪ Therefore, GGT is used to evaluate liver function, especially hepatobiliary tract disorders. Biliary Tract Enzymes (2) Gamma Glutamyl Transferase (GGT) – GGT Clinical Significance ▪ Primary role of GGT is detection and differential diagnosis of hepatobiliary disease with cholestasis or biliary obstruction associated with the highest elevation. ▪ Only moderate increases are seen in hepatocellular disease. ▪ One useful role is to differentiate liver disease from bone disease. ▪ GGT is not elevated in bone disease. – Therefore, if a patient has an elevated ALP and a normal GGT, the ALP would most likely be of bone origin. Biliary Tract Enzymes (3) 5’-Nucleotidase (5’-NT, NTP) – Hydrolyzes the phosphate group from nucleoside-5’-phosphates – Microsomal and membrane-associated enzyme found in a variety of tissues ▪ Most specifically in liver tissue – 5’-NT Clinical Significance ▪ Useful with ALP and GGT results in determining whether ALP elevation is from bone or liver disease. ▪ Predominantly elevated in diseases of the biliary tract where presence of bile salts stimulates its release from hepatocytes Biliary Tract Enzymes (7 of 8) 5’-Nucleotidase (5’-NT, NTP) – 5’-NT Methology ▪ Two most common substrates are: – AMP – Inosine-5’-phosphate (INP) 5’-N T Reference Range 2-15 U/L No clinically significant gender differences Digestive and Pancreatic Enzymes (1) Pancreatic function is ascertained using two major screening enzymes. – Amylase – Lipase Historically, lipase methodologies were too time-consuming and their sensitivity left something to be desired. Digestive and Pancreatic Enzymes (2) Amylase (AMY) – A hydrolase that catalyzes the hydrolysis of complex carbohydrates including starch, amylopectin, glycogen, and their partially hydrolyzed products. – The main sources of AMY are: ▪ Salivary glands ▪ Acinar cells of the pancreas – Clinical significance ▪ AMY is increased in acute pancreatitis, obstructive liver disease, acute alcoholism, and other conditions that affect the pancreas. Amylase: Clinical Significance - Hyperamylasemia Pancreas Acute pancreatitis Salivary Gland Chronic pancreatitis Mumps Alcoholic liver disease Parotitis Obstructive liver disease, cholecystitis Salivary gland lesions Pancreatic cancer Maxillofacial surgery Pancreatic trauma Amylase: Clinical Significance - Hyperamylasemia Intraabdominal Conditions Miscellaneous Perforated peptic ulcer Intestinal obstruction Septic shock Acute appendicitis Cardiac surgery Ruptured ectopic pregnancy Tumors Cholecystitis Peritonitis Diabetic ketoacidosis Macroamylasemia Gastritis, duodenitis Digestive and Pancreatic Enzymes (5) Macroamylasemia – Artifactual increase in serum AMY found in 1% to 2% of the population – AMY binds with IgG or IgA ▪ Forms a complex too large to be filtered by the kidney ▪ Therefore, it remains in circulation. – Serum AMY is elevated up to 6 to 8 times the upper reference limit but is not associated with any disease and patients are asymptomatic. Digestive and Pancreatic Enzymes (6) Amylase (A M Y) – AMY Methodologies ▪ Saccharogenic methods – Measure the enzyme activity by quantitating the reducing substances formed (sugars, dextrins) by their reducing properties. ▪ Amyloclastic methods – Determine the decrease in substrate (starch) concentration by the addition of iodine, which turns blue when it binds to starch. ▪ Chromogenic assay – Use of a dye-labeled AMY substrate (amylose or amylopectin). ▪ Enzymatic – Based on the amylolytic hydrolysis of small oligosaccharides, which results in better controlled and more consistent reactions. – Maltotetraose – Maltopentose Digestive and Pancreatic Enzymes (7) Amylase (A M Y) – AMY Methodologies ▪ Maltotetraose reaction – Used in many instruments – AMY → Maltose phosphorylase → β-Phosphoglucose mutase → Glucose-6- phosphate dehydrogenase Maltotetraose + H2O ¾a¾¾¾¾ - Amylase Ca+2 , Cl- ® 2 Maltose Maltose + Pi ¾Maltose ¾¾¾¾¾¾ phosphorylase ® Glucose + b -Glucose-1-P b -Glucose-1-P ¾b¾¾¾¾¾¾¾ -Phosphoglucose mutase ® Glucose-6-P Glucose-6-P + NAD+ ¾Glucose-6-phosphate ¾¾¾¾¾¾¾¾¾¾ dehydrogenase ® Glucose-6-P + NADH + H+ Digestive and Pancreatic Enzymes (8) Amylase (A M Y) Reference Range Very dependent on methodology and instrument Approximately 40-140 U/L for serum 24-400 U/L for urine Digestive and Pancreatic Enzymes (9) Amylase Creatinine Clearance Ratio (ACCR) – Compares the renal clearance of AMY to the clearance of CR on the same urine and serum. – ACCR is elevated in acute pancreatitis (>8%), because the renal clearance of AM Y is greater than that of CR, but returns to normal levels after the AMY is cleared from the serum. Urine AMY (U/L)  Serum creatinine (mg/dL) ACCR(%) = Serum AMY (U/L)  Urine creatinine (mg/dL) – In macroamylasemia, the ACCR is < 2% because the large complex cannot be filtered by the glomeruli and the decreased ACCR differentiates macroamylasemia from other causes of hyperamylasemia. Digestive and Pancreatic Enzymes (10) Lipase (L P S) – Hydrolyzes glycerol esters of long-chain fatty acids (triglycerides) to produce alcohol and fatty acids – Only the ester bonds at carbons 1 and 3 (a and a1 positions) are attacked , not β – Acts only at the interface between water and the substrate when the substrate is present in emulsified form – Pancreatic LPS is the only LPS of clinical significance. Digestive and Pancreatic Enzymes (13 of 21) Lipase – L P S Clinical Significance ▪ L P S is produced by the acinar cells of the pancreas. ▪ L P S is less affected by intraabdominal conditions described under A M Y, making it more specific for acute pancreatitis but less sensitive. ▪ LPS Can Be Associated with Other Conditions – Chronic pancreatitis – Duodenal ulcer – Peptic ulcer – Intestinal obstruction – Acute cholecystitis – Acute alcohol poisoning – Trauma to the abdomen (surgery or an accident) Digestive and Pancreatic Enzymes (15 of 21) Lipase – LPS Methodologies ▪ Enzymatic LPS reactions have largely replaced titrimetric and turbidimetric methodologies. ▪ (LPS → Glycerol kinase → L-α-glycerophosphate kinase → Peroxidase) L P S Reference Range Depends on the procedure used Upper reference limit for the T O O S methodology is 45 U/L at 37°C. Digestive and Pancreatic Enzymes (17 of 21) Pancreatic Enzymes – Serum amylase and lipase and urine amylase provide valuable information. ▪ Useful in differentiating acute pancreatitis from other intraabdominal conditions ▪ LPS is not as affected by intraabdominal conditions as AMY. ▪ Urine AMY remains elevated longer than either serum AMY or LP S. Digestive and Pancreatic Enzymes (18 of 21) Trypsin (T R Y) – Proteinase that hydrolyzes the peptide bonds formed by the carboxyl groups of lysine or arginine with other amino acids – TRY Clinical Significance ▪ Important in screening for cystic fibrosis and chronic pancreatitis – In healthy individuals, TRY-1 is major form found in serum. – In acute pancreatitis, increase in the levels of TRY-1 parallels amylase values. – TRY Methodologies ▪ Enzymatic assays have been developed to measure TRY-1. ▪ Free TRY-1 is not normally present in serum. – Always complexed to another protein Digestive and Pancreatic Enzymes (20 of 21) Chymotrypsin (CHY) – Serine proteinase that hydrolyzes peptide bonds connecting the hydroxyl group of tryptophan, leucine, tyrosine, or phenylalanine – Prefers the carboxyl group of the aromatic amino acid residues as opposed to trypsin – CHY Clinical Significance ▪ Investigate chronic pancreatic insufficiency ▪ CHY levels can determine whether oral pancreatic enzyme supplements are sufficient and whether the dosage needs to be adjusted. ▪ More resistant to catabolism in the intestine than TRY – Enzyme of choice for detecting pancreatic enzymes in the feces Miscellaneous Clinically Significant Enzymes (1 of 9) Acid Phosphatase (ACP) – Group of hydrolases similar to alkaline phosphatases ▪ Major difference is the pH of the reaction. – Tissue richest in ACP is the prostate. ▪ Levels are 1000 times greater than in other tissues. – ACP Clinical Significance ▪ Conditions involving the prostate associated with elevated A C P are benign prostate hypertrophy (B P H) and prostate surgery. ▪ Was also used in forensics in the investigation of rape – Seminal fluid in vaginal washings will result in A C P activity for 4 days following rape (intercourse). ▪ Bone disease is a third category of elevated A C P. Miscellaneous Clinically Significant Enzymes (3 of 9) Acid Phosphatase (ACP) – ACP Methodology ▪ Group-specific enzyme that will catalyze reactions with most phosphomonoesters ▪ Reaction products are colorless at an acid pH, but with the addition of alkali to end the reaction, they are changed to chromagens that can be measured spectrophotometrically. ▪ Total ACP − ACP after tartrate inhibition = Prostatic ACP – ACP is unstable at room temperature and requires immediate freezing or buffering. Miscellaneous Clinically Significant Enzymes (4 of 9) Aldolase (ALD) – Catalyzes the cleavage of D-fructose-1,6-diphosphate to D-glyceraldehyde-3- phosphate (GLAP) and dihydroxyacetone phosphate (DAP) – An important enzyme in the glycolytic breakdown of glucose to lactate – ALD Clinical Significance ▪ Diagnosing and monitoring skeletal muscle diseases ▪ Three subclasses of aldolase: – Aldolase A is found in muscle, erythrocytes, and the brain. – Aldolase B is expressed in the liver, kidneys, and enterocytes – Aldolase C is found in the brain. ▪ Aldolase A is clinically significant for determining the primary pathology between muscular versus neurological myopathy. Miscellaneous Clinically Significant Enzymes (6 of 9) Cholinesterase (CHE) – Hydrolyase enzyme – Catalyzes the hydrolysis of choline esters to form choline and the corresponding fatty acid. – Divided into two groups: ▪ Acetylcholinesterase (true cholinesterase) ▪ Butyrylcholinesterase (acylcholine acylhydrolase) – CHE Clinical Significance ▪ Detection of: – Pesticide poisoning – Liver function test – Abnormal genetic variants ▪ Chronic exposure to organophosphates by inhalation or through the skin results in a decrease of both acetylcholinesterase and pseudocholinesterase. Miscellaneous Clinically Significant Enzymes (8 of 9) Cholinesterase (CHE) – CHE Methodology ▪ Various substrates including butyrylthiocholine ▪ Other substrates include the iodide salts of acetylthiocholine, proprionylthiocholine, and succinylthiocholine. Miscellaneous Clinically Significant Enzymes (9 of 9) Cholinesterase (C H E) C H E Reference Range Depends on the methodology Using succinylthiocholine, the range for women is 33-76 U/L and 40-78 U/L for men in patients with the normal C H E genotype. Postamble ◦ READ the TEXTBOOK for the details to answer the UNIT OBJECTIVES. ◦ USE THE UNIT OBJECTIVES AS A STUDY GUIDE ◦ All test questions come from detailed material found in the TEXTBOOK (Not this PowerPoint) and relate back to the Unit Objectives