Clinical Chemistry Lab - 07 Enzymological Methodologies.pdf

• • CLINICAL CHEMISTRY LABORATORY ENZYMOLOGY AND ENZYMOLOGICAL METHODOLOGIES • OUTLINE • Enzymes o What are Enzymes? o Factors that Influence Enzymatic Reactions o Measurement of Enzyme Activity o Liver Enzymes and Liver Function Test o Cardiac Enzymes o Pancreatic Enzymes and o Other Clinically Significant Enzymes • • Fixed time: one time measurement of absorbance Continuous monitoring: multiple readings of absorbance in specific time frame Measurement o High enzyme = high reaction = high production formation o More substrate catalyzed = higher enzyme activity o IU - amount of enzyme that will catalyze 1 umol of substrate o IU/L - measurement of enzyme concentration o 1 IU = 17 nanokatals Enzymes can be measured as analytes or be used as reagents ENZYMES WHAT ARE ENZYMES? • • • • • Proteins o Combination of amino acids in tertiary/quaternary structure Biological catalysts o Enhances rate of reactions o Biological reactions requires energy to occur o Enzymes decreases the energy requirement Binds: substrates and/or cofactors o Substrates - raw materials, converted into functional metabolites/components; target by enzymes o Cofactors - helpers, may open active sites o Active site - binds substrate o Allosteric site - bind cofactors Types of cofactors: activator & coenzymes o Activator - inorganic, ions o Coenzymes - organic cofactors o Apoenzyme + cofactor = holoenzyme Inactive form of enzyme: zymogen/proenzyme o Common among hydrolases LIVER ENZYMES AND LIVER FUNCTION TEST THE LIVER TRANSFERASES • • • • FACTORS THAT INFLUENCE ENZYMATIC REACTIONS • • • • • Substrate concentration o First order kinetics - E > S o Zero order kinetics - E < S (constant) Enzyme concentration pH and Temperature o Most enzymes function in physiologic pH o Most enzymes function in physiologic temp (>/< 37C) o High temp (>40C) denature enzymes/proteins; renders enzyme nonfunctional Cofactors Inhibitors o Competitive - competes for the active site o Noncompetitive - binds on other areas (allosteric reversible inhibition/areas unsuitable for binding - may destroy enzyme) o Uncompetitive - binds in enzyme-substrate complex MEASUREMENT OF ENZYME ACTIVITY • • Enzymes are very low in amount in plasma compared to the concentration of plasma proteins o Challenging to isolate and analyze individually Mode of measurement: fixed-time vs. Kinetic/continuous monitoring • • Used to assess direct liver injury Aspartate & alanine - coupled with alpha-ketoglutarate; converted to glutamic acid Aspartate aminotransferase (AST)/serum glutamic oxaloacetic transaminase (SGOT) o Substrate - aspartate o Product - product o Distribution: heart, liver, skeletal muscle, RBC, kidney, liver, pancreas o Reference value: 5-35 IU/L o Pre-analytical considerations ▪ Avoid hemolysis (false elevation) ▪ Refrigerated serum - preserve activity of enzyme for 3-4 days; repeated freeze-tow cycle may destroy the protein Alanine aminotransferase (ALT)/Serum glutamic pyruvic transaminase (SGPT) o Marker more specific & significant to liver o Distribution: liver, o Reference value: 7-45 IU/L o Pre-analytical considerations ▪ Unaffected by hemolysis ▪ Refrigerated serum - preserve activity of enzyme for 3-4 days; repeated freeze-tow cycle may destroy the protein Coenzyme/cofactor: pyridoxal phosphate/Vit. B6 Measurement/Analysis o AST ▪ Chemical method • Reitmann-Frankel - measure ketoacids • Use dinitrophenylhydrazone • Blue complex (505nm) ▪ CM/Kaarmen method • pH 7.3-7.8 o ALT ▪ Chemical method • Use dinitrophenylhydrazone • Blue complex (505 nm) ▪ CM • pH 7.3-7.8 o • If conversion is from NADH to NAD / with H to without H, decreasing absorbance ▪ Absorbance 1 > A2 > A3 Alkaline phosphatase (ALP) o Hydrolase o Activator: magnesium o Distribution: intestine , liver, bone, placenta, with small amounts in kidney and spleen o Reference Range: 53-128 IU/L (male, 49-98 IU/L (female), 54-639 IU/L (children) CARDIAC ENZYMES • Lactate Dehydrogenase (LD/LDH) o Oxidoreductase o Conversion of lactate to pyruvate o Coenzyme: NAD/NADH o Reference range: 125-220 IU/L o Distribution: widely distributed with 5 isoenzymes (LD1 - LD5) ▪ LD1 (HHHH) - heart ▪ LD2 (HHHM) - erythrocytes ▪ LD3 (HHMM) - Lungs ▪ LD4 (HMMM) - Liver ▪ LD5 (MMMM) - Skeletal muscle o Pre-analytical consideration ▪ Avoid hemolysis (false elevation) ▪ Activity is unstable (ideal storage temperature: 25C/room temp, rationale: LD is cold labile/unstable in cold temp) ▪ Never refrigerate LD, LD4 & LD5 are greatly affected by cold o Methods: ▪ Only uses one enzyme system ▪ Wacker (pH: 8.3 - 8.9): forward method; lactate -> pyruvate; increasing absorbance rating ▪ Wroblewksi la Due (pH 7.1-7.4): more preferred due to faster reaction & shorter turnaround time; reverse method; pyruvate --> lactate; decreasing absrobance reading • Creatine Kinase (CK) o Transferase ▪ Kinase - transfer phosphate groups o Function: ATP regeneration and storage (coenzyme: Mg) o 3 isoenzymes ▪ CK-MM - skeletal muscles ▪ CK-BB - widely distributed, majority in organs ▪ CK-MB - heart o Reference range ▪ Total (Male): 46-171 IU/L ▪ Total (Female): 34-145 IU/L ▪ CK-MB: <5% of total CK o Pre-analytical considerations ▪ Avoid hemolysis • Adenylate kinasein the RBC can affect the analysis ▪ Inactivated by light ▪ Restore activity after storage using sulfhydryl activator ▪ Affected also by muscle mass o Analysis/measurement ▪ Tanzer-Gilvarg • Forward method (decreasing absorbance) • pH: 9 o • Preanalytical consideration ▪ Avoid hemolysis ▪ Avoid prolonged standing - may inactivate enzyme ▪ Inhibited by fluoride, oxalate and heparin additive ▪ Affected by high fat meal (by 3-10%) intestinal ALP • Note: applicable for type B, O secretors o Measurement/analysis Methods Substrate Measurement End Product Bessy, Lowery, PP-nitrophenol and Brock nitrophenylphosphate (405nm) (most common) Shinowara B-glycerophosphate Glycerol King armstrong Phenylphosphate Phenol Huggins and Phenolphthalein Red Talalay diphosphate phenolphthalein Moss AlphaAlpha-naphthol naphthylphosphate Klein, Babson, Buffered phenolphthalein and Reed phenolphthalein phosphate Gamma-glutamyl Transferase (GGT) o Transferase o Transfer gamma glutamyl amino acid residues (gluthathione) o Functions ▪ Protein synthesis ▪ Regulation of tissue gluthatione levels ▪ Amino acid transport o Marker of chronic alcoholism o Tested with ALP o Marker of biliary obstruction ▪ Reference range: 6-55 IU/L (male), 5-38 IU/L (female) o Pre-analytical considerations and analysis/measurement ▪ GGT is stable up to 1 week seum is refrigerated, not affected by hemolysis ▪ Szasz Method • Gamma glutamyl-p-nitroanilide + glycylglycine --> gamma-glutamylglycylglycine + p-nitroanilide (420nm) ▪ ▪ Oliver-Rosalki • Reverse method • pH: 6.8 PANCREATIC ENZYMES AND OTHER CLINICALLY SIGNIFICANT ENZYMES ▪ PANCREATIC ENZYMES • Amylase (AMY) o Hydrolase o Produced by the pancreas; also found in salivary gland o Smallest enzyme ▪ Easily pass though the fenestration of the kidneys ▪ One of the enzymes detected in the urine o Breaks down carbohydrates: glycogen (animals) & starch (plants source) o Starch structure: α 1-4 glycosidic bonds (main chain) and α 1-6 glycosidic bonds (side chain) ▪ Amylase is specific to the 1-4 ▪ Glycogen debranching enzyme (GDE) - for 1-6 o Targets glycosidic bonds of carbohydrates in the presence of water o Coenzyme: Calcium, chloride ions (activators) o Reference range: 29-100 IU/L o o Pre-analytical Considerations ▪ Triglycerides inhibits amylase activity causing false decrease • Patients should fast prior to testing so triglycerides are in normal range ▪ Opiates & morphine can cause falsely elevated results • Opium/morphine-based painkillers may affect test • Constriction of the sphincter of Oddi which may cause leakage of AMY in the blood Measurement/Analysis ▪ Substrate is always used; • Starch - most common substrate o High affinity to iodine • Converted to simpler carbohydrates (glucose) ▪ Amyloclastic - Measures the disappearance of starch substrate • Starch is coupled w/ iodine (purple complex) • Purple complex lightens upon the axn of amylase when starch is converted to glucose • Result: light purple/colorless • Decreasing absorbance reading ▪ Saccharogenic - Measures the appearance of the product • Simple carbohydrates are measured • Glucose - easiest to measure • Chemical methods (Folin Wu, Schiff’s) • Enzymatic method (glucose oxidase, hexokinase) Chromogenic - Measures the increasing color from production of product coupled with a chromogenic dye • End color is required • Hexokinase - do not yield visible color o Any method involving NAD/NADP is measured at 340 nm o End color - not visible to the naked eye • Only methods with visible end color are used o Chemical method & glucose oxidase o Glucose oxidase - red complex Continuous monitoring - Coupling of several enzyme systems to monitor amylase activity • Usually practiced • • Hexokinase (HK) - transfers phosphate group • NAD → NADH - increasing absorbance (UV) • NADH → NAD - decreasing absorbance (UV) Lipase (LPS) o Hydrolase o Brakes ester bonds in lipids (produce fatty acids & glycerol) o Coenzymes: organic substances; colipase & bile salt o Primarily found in the pancreas ▪ No isoenzymes & salivary counterpart o Reference range: <38 IU/L o Pre-analytical Considerations ▪ Triglyceride is the substrate ▪ Hemoglobin inhibits lipase activity • Hemolyzed sample is rejected o Measurement/Analysis ▪ Method of analysis: Cherry Crandall Method • Triglyceride substrate: olive oil or triolein • • Measurement o Turbidimetric - measures decreasing pattern of absorbance ▪ Solution become less turbid when triglyceride is broken down ▪ Decreasing turbidity/absorbance o Colorimetric/Enzymatic: peroxidase & glycerokinase enzyme (coupling) ▪ Reddish-purple complex product ▪ Increasing absorbance ▪ Measurable at 540-550nm Summary of Pancreatic Markers Amylase Lipase Rise 5-8 hrs 4-8 hrs Peak 24 hrs 24 hrs Decline 3-5 days 8-14 days OTHER CLINICALLY SIGNIFICANT ENZYME • Acid Phosphatase (ACP) o Similar function with ALP but works at different pH and environment o Distribution: protate, liver, spleen, kidney, RBCs, platelets ▪ Highest in prostate and RBCs o Clinical significance: used in prostate disorders (enlargement, cancer), used in investigating rape (due to ACP presence in seminal fluid) o Reference range: PAP = 0-3.5 ng/mL, Adult = 1.5-4.5 IU/L, children = 3.59 IU/L o o o Pre-analytical considerations ▪ Avoid hemolysis (false increase) • Hemolysis releases ACP from RBCs ▪ Separate serum from RBCs immediately ▪ Loss of activity in prolonged standing of serum samples (false decrease) • Loss of partial pressure of CO2 (PCO2) attributed to the acidity of the blood • Blood pH may be imbalanced (alkaline) o Decreases ACP • Blood pH maintained at 7.35-7.45 Measurement/Analysis ▪ Same with ALP (Bessy Lowry Brock) but the pH of the buffer is acidic (pH 5) • Substrate: PNP • ▪ • p-Nitrophenolphosphate p-Nitrophenol + phosphate ion • Para-nitrophenol - colorless at acidic pH • NaOH - color developer; makes sample alkaline for • p-np + NaOH = yellow complex product Measurement of Prostatic isoenzyme • PACP - prostatic acid phsophatase • Direct o Kinetic - α-naphthylphosphate o Endpoint - thymolphthalein monophosphate as substrate • Indirect - inhibit prostatic isoenzyme by Ltartrate o Total - Inhibited = PACP