Enzymes PDF - Preliminary Examination 2024

ENZYMES PBMLS 019 LEC PRELIMS| 2024| PROF. REGINE L. PACE, RMT GENERAL PROPERTIES & DEFINITION ENZYME NOMENCLATURE ❖ Release...

ENZYMES PBMLS 019 LEC PRELIMS| 2024| PROF. REGINE L. PACE, RMT GENERAL PROPERTIES & DEFINITION ENZYME NOMENCLATURE ❖ Released when there is: 1. The first digit places the enzyme in one of the six a. Damage in an organ or cells classes. b. Impaired removal of enzyme from the 2. The second and third digits represent the subclass plasma and sub subclass of the enzymes c. Increased permeability of cell membrane 3. The final number is the serial number specific to d. Increased production of cells each enzyme in a sub subclass ENZYME KINETICS Simple enzymes Enzyme composed only of protein (amino acid chains) Conjugated Enzyme that has non protein part in enzymes addition to a protein part Apoenzyme Protein part of the conjugated enzyme Cofactor Nonprotein part of the conjugated enzyme, aka as activators Holoenzyme Biochemically active conjugated enzyme produced from an apoenzyme and a cofactor TEMPERATURE & pH Coenzyme Small organic molecule that serves as a ❖ ↑ temperature = ↑ rate of chemical reaction. cofactor in a conjugated enzyme, aka as ❖ For every 10 degree = ↑ two-fold ↑ in enzyme prosthetic group activity. Active site water-free cavity where the substance Optimum temperature 37 °C on which the enzyme acts (substrate) Active temperature 25°C, 30°C, 37°C Allosteric site a cavity other than the active site Inactive temperature 60-65°C Isoenzymes Enzymes with the same catalytic function but different forms in terms of Physiologic pH 7-8 physical properties: 1. Electrophoretic mobility SUBSTRATE CONCENTRATION 2. Solubility 3. Resistance to inactivation First order ❖ Enzyme > Substrate = Reaction kinetics increased as you add substrate ENZYME STRUCTURE Coenzyme ❖ Substrate concentration reached its maximal value Zero order ❖ Reaction rate depends only on kinetics enzyme concentration Michaelis ❖ Constant for a specific enzyme and Menten substrate under defined reaction Constant conditions ❖ An expression of the relationship between the velocity of an enzymatic reaction and substrate concentration PBMLS 019 LEC CRISTOBAL, ALEXIA NICOLE M. (BMLS 3D) 1  ENZYME CONCENTRATION ❖ Enzyme, faster reaction CALCULATION OF ENZYME ACTIVITY ❖ Enzymes are quantified based on their reactivity INHIBITORS rather than absolute values ❖ The units used to report enzyme levels are activity units International Amount of enzyme that will catalyze the unit (IU) reaction of 1u mol of substrate per minute Katal unit 1 mole of substrate per second (KU) CREATININE KINASE ❖ Catalyzes the transfer of a phosphate group between creatine phosphate and adenosine diphosphate. SPECIFICITY ❖ Associated with ATP regeneration ❖ Found in small amounts throughout the body Absolute Enzyme combines with only one ❖ Liver cells and RBCs do not contain this specificity substrate and catalyzes only the one ❖ Involved in the storage of high-energy creatine corresponding reaction phosphate in the muscles ❖ Tissue source: Group specificity Enzyme combine with all substrates containing a particular chemical group, ↑ skeletal muscle, heart muscle and brain tissue such as a phosphate ↓ bladder, placenta, GIT, thyroid, uterus, kidney, Bond specificity Enzymes which are specific to chemical lung, prostate, spleen, liver and pancreas bonds and thereby exhibit bond specificity DIAGNOSTIC SIGNIFICANCE ❖ Elevated in cardiac and skeletal muscle disorders Stereoisomeric Enzymes that predominantly combine (Duchenne Type) specificity with only one optical isomer of a ❖ CK levels are considered as a sensitive indicator of certain compound acute myocardial infarction & muscular dystrophy. ❖ Total serum CK level is also used as an early ENZYME ACTIVITY diagnostic tool to identify patients with vibrio ❖ Photometrically measure vulnificus infections. a. ↑ product concentration ❖ Varies according to: b. ↓ substrate concentration Gender c. ↓coenzyme concentration Race d. ↑ concentration of an altered coenzyme Degree of physical conditioning ❖ Must be performed in Zero-order kinetics Age ❖ No inhibitors ❖ Reaction rate must be controlled ↑ in CNS disorder such as cerebrovascular accident, seizures, nerve degeneration, and CNS shock METHODS TO MEASURE ENZYME ACTIVITY ↓ in hypothyroidism, malignant hyperpyrexia and Fixed time Reactants are combined, the reaction is Reye's syndrome stopped at a designated time CK ISOENZYMES Continuous Aka kinetic assay. Multiple measurements ❖ Separation of CK isoforms may be visualized by monitoring of absorbance changes are made during high-voltage electrophoretic separation the reaction ❖ Isoforms occur following cleavage of the carboxyl-terminal amino acid from the M subunit by serum carboxypeptidase N PBMLS 019 LEC CRISTOBAL, ALEXIA NICOLE M. (BMLS 3B) 2  CK-MM CK-BB CK-MB Notes 2-6x faster; most commonly used Source Striated muscle CNS, GIT, & uterus Not elevated in & normal during pregnancy. angina SPECIMEN CONSIDERATION serum ❖ Hemolysis ❖ Serum should be stored in a dark place Disease Cardiac , CNS damage, ≥ 6% total CK for skeletal muscle tumors, myocardial damage ❖ Activity can be restored after storage in the dark at 4 injury, & childbirth, C for 7 days or at -20 C for 1 month when the assay hypothyroidism macro-CK is conducted using a sulfhydryl activator. ❖ Reference range: Hours 48 hours after 1-5 hours, rarely Rise: 4-8 hours exercise in serum; 80k Peak: 12-24 hours dalton. Return: 48-72 hours Total CK ❖ Males: 46-171 U/L (37C) ❖ Females: 34-145 U/L (37 C) UNUSUAL CK ISOENZYMES CK-MB 1 Nonviral ❖ Major liver band ❖ Aka, fast liver/alpha 1 liver - Ratio < 1 Viral metastatic carcinoma of the liver, indicator of obstructive liver disease ❖ Chemical inhibition: levamisole SOURCES OF ERROR ❖ ALT is stable for 3 to 4 days at 4°C Bone ALP ❖ Increased due to osteoblastic activity ❖ Unaffected by hemolysis ❖ Least heat stable ❖ Reference range: 7-45 U/L (37°C). ❖ Chemical inhibition: levamisole & 3M urea. ALKALINE PHOSPHATASE ❖ Catalyzes the hydrolysis of various Placental ALP ❖ Most heat stable; 65°C; 30 mins phosphomonoesters at an alkaline pH. ❖ Chemical inhibition: phenylalanine ❖ Optimal pH: 9.0-10.0 ❖ Activator: Magnesium Intestinal ALP ❖ Least anodal ❖ Tissue sources: intestine, liver, bone, spleen, ❖ Increased in digestive tract disease & placenta & bone disorders. cirrhosis ❖ Chemical inhibition: phenylalanine DIAGNOSTIC SIGNIFICANCE ❖ Increased ALP is for the evaluation of hepatobiliary ABNORMAL ALP FRACTIONS ASSOC. WITH NEOPLASMS & bone disorders. ❖ In hepatobiliary disorders, elevations are more Regan ❖ Detected in lung, breast, ovarian and predominant in obstructive conditions than in isoenzyme colon carcinomas hepatocellular disorders. ❖ Migrates to the same position as bone ❖ Highest elevations in ALP occur in Paget’s disease / fraction osteitis deformans. ❖ Most heat stable of all ALP ❖ Increased ALP can also be seen in complications of isoenzymes- pregnancy such as hypertension, preeclampsia. ❖ Inhibited by phenylalanine eclampsia and threatened ❖ Associated with neoplasms ↑ ❖ Hypertension Nagao ❖ Detected in metastatic carcinoma of ❖ Preeclampsia isoenzyme pleural surfaces and adenocarcinoma ❖ Evaluation of hepatobiliary & bone of the pancreas and bile duct. disorders. ❖ Electrophoretic, heat stability and phenylalanine inhibition properties ↓ ❖ Hypophosphatasia are identical with Regan isoenzyme ❖ Can be inhibited by L-leucine and Biliary tract obstruction 3-10x ULN phenylalanine Hepatocellular disorder < 3x ULN METHODS METHODS Substrate Notes Electrophoresis in combination with other separation Shinowara Beta Long incubation time; high blank technique Jones glycerophosphate values PBMLS 019 LEC CRISTOBAL, ALEXIA NICOLE M. (BMLS 3B) 5  Reinhart glycerophosphate King Phenyl Phosphate Endpoint: requires protein Gutman, King Phenyl Phosphate Nonspecific Armstrong removal Armstrong Bessey Lowry P nitrophenyl Endpoint or kinetic, rapid Hudson P nitrophenyl Rapid, nonspecific Brock phosphate phosphate Bowers P nitrophenyl Uses phosphate-accepting buffer, Babson & A naphthyl Complicated, less sensitive McCromb phosphate reference method Reed phosphate Roy Thymolphthalein More specific for prostatic form ASSAY FOR ENZYME ACTIVITY monophosphate ❖ Continuous monitoring technique based on Bowers and McComb allows calculation of ALP activity based Rietz, Guil 4-methylumbellifer Fluorescent, some improved balt one sensitivity on the molar absorptivity of p-nitrophenol. ❖ p-Nitropheny|phosphate (colorless) is hydrolyzed to p-nitrophenol (yellow) and the increase in ASSAY FOR ENZYME ACTIVITY absorbance at 405 nm, which is directly proportional ❖ Assay procedures for total ACP use the same to ALP activity, is measured techniques as in ALP assays but are performed at an acid pH SOURCES OF ERROR ❖ Immunochemical techniques for prostatic ACP use ❖ Hemolysis may cause slight elevations because ALP is several approaches, including immunoprecipitation, approximately six times more concentrated in RIA and counterimmunoelectrophoresis erythrocytes than in serum ❖ Activity in serum increases approximately 3% to 10% SOURCES OF ERROR on standing at 25°C or 4°C for several hours. ❖ Serum should be separated from the red cells as ❖ Reference range: soon as the blood has clotted to prevent leakage of erythrocyte and platelet ACP ACID PHOSPHATASE ❖ Serum activity decreases within 1-2 hours if the ❖ Catalyzes the same reaction made by ALP, except sample is left at room temperature without the that it is active at pH 5.0. addition of a preservative ❖ Tissue source prostate, bone, liver, spleen, kidney, ❖ If not assayed immediately, serum should be frozen erythrocytes, and platelets or acidified to a pH lower than 6.5. With acidification, ACP is stable for 2 days at room DIAGNOSTIC SIGNIFICANCE temperature ❖ Detection of prostate carcinoma (metastatic ❖ Hemolysis should be avoided because of carcinoma of the prostate) contamination from erythrocyte ACP. ❖ Useful in forensic clinical chemistry in the ❖ Reference range: investigation of rape-cases ❖ Elevated in bone diseases (Paget’s disease), breast Prostatic ACP 0-3.5 ng/mL cancer with bone metastases and in Gaucher’s disease, Niemann pick. Tartrate resistant ATC ❖ Adult: 1.5-4.5 U/L ❖ Useful in forensic clinical chemistry in the ❖ Children: 3.5-9.0 U/L investigation of rape-cases. ❖ ALP + PSA = recurrence of prostatic carcinoma GAMMA GLUTAMYLTRANSFERASE monitoring. ❖ Involved in the transfer of the y-glutamyl residue from y-glutamyl peptides to amino acids, water, and Present in Seminal fluid other small peptides ❖ Tissue sources: kidneys, brain, prostate, pancreas, Sample Vaginal washings and liver Up to 5 days DIAGNOSTIC SIGNIFICANCE ❖ GGT is located in the canaliculi of the cells hepatic METHODS cells and particularly in the epithelial cells lining the biliary ductules Substrate Notes ❖ ALP levels: skeletal disorder or pregnancy. ❖ Clinical applications are confined mainly in the Bodansky Beta Lengthy assay, nonspecific evaluation of liver and biliary system disorders PBMLS 019 LEC CRISTOBAL, ALEXIA NICOLE M. (BMLS 3B) 6 ❖ GGT levels will be increased in patients receiving peptic intestinal obstruction, cholecystitis, ruptured enzyme-inducing drugs (up to 4x ULN (upper limit of ectopic pregnancy, mesenteric infarction, acute normal) such as appendicitis, renal insufficiency and diabetic Warfarin ketoacidosis Phenobarbital Phenytoin AMYLASE ISOENZYMES ❖ Most sensitive indicator of chronic alcoholism ❖ Electrophoresis ❖ Elevated GGT levels may indicate alcoholism, ❖ Chromatography particularly chronic alcoholism (2-3x ULN) ❖ Isoelectric focusing ❖ GGT levels are also elevated in acute pancreatitis, P-type ❖ Derived from pancreatic tissue DM, and myocardial infarction (amylopsin) ❖ Predominates normal urine METHODS ❖ Seein in acute pancreatitis, predominantly P3 ❖ Continuous Monitoring ❖ P3 can also be detected in renal Absorbance failure. Chromogenic product Measured at 405 - 420 nm S-type ❖ Derived from salivary gland, fallopian Fixed point assay or endpoint assay tube & lung ASSAY FOR ENZYME ACTIVITY ❖ Represents approximately ⅔ of AMS activity in normal serum ❖ Szasz ❖ Rosalki & Tarrow ❖ Substrate: Gamma glutamyl p nitroanilide ASSAY FOR ENZYME ACTIVITY SOURCES OF ERROR Amyloclastic ❖ Amylase + Starch with iodine (dark ❖ GGT that is stable and with no loss of activity for a method blue) week when it is being stored in 4°C ❖ __ Amylase activity = ↓ starch size ❖ Won't be affected if tested with hemolysis ❖ ↓ starch size -iodine detaches __ color ❖ GGT enzyme not present in erythrocytes ❖ ↓ color = ↓ Amylase activity ❖ Values are lower in females, presumably because of suppression of enzyme activity resulting from Saccharogeni ❖ Amylase + Starch = ____ estrogenic or progestational hormones. c method ❖ ↑ Reducing Sugars = ↑Amylase Activity ❖ Reference values Male: 6-55 U/L 1 ❖ Reference Method Female: 5-38 U/L Chromogenic ❖ Amylase + Starch with insoluble method chromogenic dye b AMYLASE ❖ ↑ Amylase activity = smaller starch ❖ Catalyze the breakdown of starch & glycogen. with dye (soluble) ❖ Earliest pancreatic marker ❖ ↑ soluble dye substrate= ↑Amylase ❖ Smallest enzyme (MW 50,000-55,000) activity ❖ Activators: Calcium & chloride ❖ Tissue sources: Coupled ❖ Continuous-monitoring technique enzyme ❖ Change in the absorbance of NAD at ↑ Acinar cells of the pancreas & salivary glands method 340 nm (pH 6.9) ↓ Skeletal muscle, small intestine & fallopian tube SOURCES OF ERROR ❖ Inhibitors of amylase activity DIAGNOSTIC SIGNIFICANCE Triglycerides ❖ Diagnosis of acute pancreatitis TAG with germ lectin ❖ Morphine = falsely elevated amylase Rise 2-12 hours ❖ Reference range Serum: 30-220 U/L Peak 24 hours Urine: 1-15 U/h Return 3-5 days LIPASE ❖ Increased in salivary gland lesions (mumps and parotitis), intra-abdominal diseases (perforated 0 ❖ Most specific pancreatic marker PBMLS 019 LEC CRISTOBAL, ALEXIA NICOLE M. (BMLS 3B) 7  ❖ Hydrolyzes the ester linkages of fats to produce Primaquine alcohols & fatty acids. ❖ Increased levels: myocardial infarction & ❖ Tissue source: pancreas, stomach, & small intestine megaloblastic anemia. DIAGNOSTIC SIGNIFICANCE ASSAY FOR ENZYME ACTIVITY ❖ Confined almost exclusively to the diagnosis of acute ❖ A red cell hemolysate is used to assay for deficiency pancreatitis of the enzyme, serum is used for evaluation of ❖ In contrast with AMS levels, LPS levels are normal in enzyme elevations condition of salivary gland involvement. ❖ Reference range: 7.9-16.3 U/G HgB ❖ In acute pancreatitis: OTHER CLINICALLY SIGNIFICANT ENZYMES Rise 4-8 hours after onset of attack 5 ‘ NUCLEOTIDASE Peak 24 hours ❖ Phosphoric monoester hydrolase ❖ Predominantly secreted by the liver Remain 7 days 10 ❖ Marker for hepatobiliary disease & infiltrative lesion of the liver Normalize 8-14 days ❖ Methods: Dixon & Purdon, Campbell, Belfield & Goldberg ASSAY FOR ENZYME ACTIVITY ❖ Reference value: 3-9 U/L CHOLINESTERASE Cherry ❖ Principle: hydrolysis of olive oil after Crandall incubation for 24 hours at 37°C and ❖ Index of parenchymal function method titration of fatty acids using NaOH ❖ Secreted by the liver ❖ Substrate: Olive oil 50% / triolein ❖ Used to monitor the effect of muscle relaxants after ❖ End product: Fatty acid surgery ❖ Reference Method 1 0 ❖ Marker for insecticide / pesticide poisoning ❖ Methods: Ellman technique potentiometric Peroxidase ❖ Most commonly used method coupling ❖ Does not use 50% olive oil Tietz & Friereck SOURCES OF ERROR ❖ LPS is stable in serum, with negligible loss in activity at room temperature for 1 week or for 3 weeks at 4°C. ❖ Hemolysis should be avoided ❖ Reference range: