Clinical Chemistry 2 - Lec PDF

CLINICAL ENZYMOLOGY DEFINITION OF TERMS ✓ Group of related enzymes catalyzing the same reaction but having different ✓ The protei...

CLINICAL ENZYMOLOGY DEFINITION OF TERMS ✓ Group of related enzymes catalyzing the same reaction but having different ✓ The protein part without the cofactor molecular structures and physical, Apoenzyme: (activator) necessary for catalyst biochemical, and immunological properties. ✓ An effector molecule that increases ✓ Example: Lactose Dehydrogenase the catalytic activity of an enzyme, e Isoenzyme ✓ Isoenzyme can be differentiate or when it binds to a specific site separate from one another by Activator: ✓ Can be Organic (NAD/NADH), or immunologic or physical separation. Inorganic (electrolyte) - Physical: Electrophoresis and - All ionic (contain charge) are Chromatography inorganic - Immunologic: based on affinity of ✓ Substance that diminishes the rate of a H or M unit chemical reaction (inhibition) ✓ Limit the capabilities of enzyme International ✓ Standard unit of measurement Inhibitor: ✓ It binds to Active site and Allosteric site unit (IU) ✓ Used to denote - Active site: where substrate will ✓ Express by IU or Katal bind - Allosteric site: where inhibitor will Substrate ✓ Reactant that we use in biochemical bind reaction Product ✓ Endpoint of measurement ✓ Ability of enzyme to enhance the ✓ Substance produced by the enzyme- biochemical reaction catalyzed conversion of a substance ✓ Rate of conversion Catalytic ✓ Property of a catalyst that is measured activity: by the catalyzed rate of the conversion GENERAL PROPERTIES OF ENZYMES of specified chemical reaction 1. ACTIVE SITE produced in a specific assay system 3 ▪ Water-free cavity ✓ Sometimes measured in the lab ▪ Specific portion of enzyme that binds on the substrate - Endpoint→ final product - Kinetic→ continuous monitoring 2. ALLOSTERIC SITE or change overtime ✓ Similar function to activator, but only ▪ A cavity other than the active site which may bind with different to nature regulatory molecules Coenzyme: ✓ LMW diffusible, heat-stable substance, ▪ Regulatory molecules are the activator and coenzymes when combined with an inactive which activates into functional form of enzyme into protein called apoenzyme, forms an holoenzyme active compound (complete enzyme) called a holoenzyme - Enzyme + Activator = Holoenzyme ✓ A protein molecule that catalyzes chemical reactions without itself being destroyed, altered, consumed ✓ Enzyme is measured to assess extend Enzyme: damage to organs ✓ Specific for a substrate that it covert ✓ Large molecules that are normally confined within cells unless increase membrane permeability allows them to enter the blood ✓ Appear in serum after cellular injury and degradation of cells CARMEN, M.C.A | MED228 1  ENZYME THEORIES MICHAELIS MENTEN KINETICS 1. Emil Fisher’s/ Lock & Key Theory ▪ Maximum velocity Vmax – the point where all the active ▪ Enzymes have RIGID active site sites are bound to substrate (enzyme is saturated/ ▪ Shape of the key (substrate) must and specifically fit into saturation point) the lock (enzyme) ▪ Machaelis constant Km- Concentration of the substrate at which half of Vmax (maximum rate is reach) 2. Kochland/ Induced Fit Theory ▪ Enzyme has a FLEXIBLE active site ▪ Based on the substrate binding to the active site of the enzyme ENZYME KINETICS ▪ Enzymes work by lowering activation energy Activation energy - the minimum energy input needed for a reaction to occur and convert the substrates into products. Transition state- intermediate binding between substrate and enzyme. ENZYMATIC REACTION ORDERS Notes: To convert substance to different substance one of the 1. Zero-order reaction best methods is heating (energy). Enzyme function is reducing ▪ Reaction rate depends only on enzyme concentration the required activation energy to convert substance into different ▪ Applicable during the period there is increase in velocity product. ▪ Substrate = Enzyme | Substrate > Enzyme Low energy requirement = less time to gain energy 2. First-order reaction Substrate binds with enzyme → enters transition state→ ▪ Reaction rate is directly proportional to the substrate substrate convert to different product. concentration ▪ Directly proportional to substrate concentration - Substrate concentration is less than enzyme concertation (as long as enzyme is greater/ more than the substrate) ▪ Substrate < Enzyme Note: First order and zero order is applicable to enzymatic reaction. The second order is only applicable when there is no enzyme involve. Second order states that “reaction is highly dependent to amount of substrate” (exponential relationship) TYPES OF SPECIFICTY ENZYME BASED ON TYPE OF REACTION Enzymes are very specific and only work with certain substrate. 1. Oxidoreductases ▪ Absolute specificity - combines with only one substrate and catalyzes only one reaction. Specific chemical can bind ▪ Oxidase, Dehydrogenase the substrate ▪ Reversible and requires cofactor NADPH/ NAD ▪ Group specificity - combine with all the substrates in a - All enzymatic reactions are reversible chemical group, specific characteristics. (e.g. all alcohol is - NAD/NADPH receives hydrogen target) ▪ Oxidation: Loses of electrons or addition of oxygen ▪ Bond specificity – react with specific chemical bonds. ▪ Reduction: Gain of electrons or no oxygen or hydrogen Enzymes capable of targeting specific substances that can addition perform specific chemical bonds (e.g. Ionic, covalent, ester ▪ Catalyze the transfer of electrons from 1 molecule (oxidant) bonds) to another molecule (reductant) ▪ Stereoisomers - Types of enzymes target different ▪ Eg. Lactate dehydrogenase, succinate dehydrogenase, Isoenzymes/ Isoforms. (e.g. D and L glucose. glucose oxidase CARMEN, M.C.A | MED228 2  6. Ligase ▪ DNA ligase ▪ Joining enzymes ▪ Catalyzes ligation of 2 molecules with the hydrolysis of a diphosphate bond in ▪ ATP or any triphosphate ▪ Joining 2 or more functional groups ENZYME NOMENCLATURE ▪ International Union of Biochemistry- developed a systematic and trivial name for enzymes. 2. Transferases A. Systematic name - describes the nature of the reaction ▪ Transferase, Kinase catalyzed ▪ Catalyzes the movement of a functional group from one Ex. L-Lactate:NAD+ oxidoreductase molecule to another B. Enzyme Commission Numerical nomenclature - ▪ Functional groups: phosphate, methyl, glycosyl associated with a unique numerical code designation - 4 Kinases- catalyzes the transfer of phosphate groups in numbers separated by periods + EC prefix a process called phosphorylation ✓ 1st number-class of reaction (1-6 | OTHIL) o Eg. Creatine Kinase or Creatine ✓ next 2- subclass Ex. EC1.1.1.27 phosphokinase ✓ Last 2 – sub subclass Deaminases- catalyzes the transfer of an amine group o Eg. Alanine Aminotransferase or Glutamate C. Trivial or practical name – may be identical with the Pyruvate Transaminase systematic name but is simplified suitable for everyday use Ex. Lactate dehydrogenase 3. Hydrolases ▪ Nuclease, protease ▪ The use capital letter abbreviations for the names of ▪ Split or destroy bonds certain enzymes ▪ Involves water Example: ▪ Catalyzes hydrolysis (breaking of single bonds through the ALT = Alanine aminotransferase addition of water) AST = Aspartate aminotransferase ▪ E.g. lipase, protease, nuclease, amylase, phosphatase LD = Lactate dehydrogenase CK = Creatine kinase 4. Lyases ▪ Decarboxylase, aldolase CHARACTERISTICS OF ENZYMES ▪ Doesn’t need water ▪ Catalyzes lysis reactions that generate a double bond ▪ speed up chemical reactions ▪ Elimination reaction but not hydrolytic or oxidative ▪ required in minute amounts ▪ Eg. Carboxylase, Aldolase ▪ highly specific in their action ▪ Reverse reaction = catalyzes addition reaction (substrate ▪ affected by temperature is added to a double bond) - most enzymes are only capable up to 40c, - 56c-60c = Nonfunctional 5. Isomerases ▪ affected by pH ▪ Isomerase - 7.35-7.45 is the optimal pH of the body ▪ Conversion of stereoisomers or positional isomers ▪ some catalyze reversible reactions ▪ Catalyze structural changes within a molecule - only one ▪ some require coenzymes substrate and one product with nothing gained or lost (only ▪ inhibited by inhibitors a change in shape) ▪ E.g D,L, Cis, Trans CARMEN, M.C.A | MED228 3  ENZYMES NOTES: ▪ Most enzymes are INACTIVATED at 65C ▪ Low temperature or FREEZING does NOT usually destroy Enzymes speed ✓ by lowering the activation energy enzymes up chemical needed to start the reaction Except: reactions a. LD – Freezing at -20C b. CK – Storing at 4C ✓ Measurement of enzyme activity, we measure the product not the concentration due to small amount ENZYMES DIFFERNET OPTIMUM pH ✓ Enzymes remain chemically Enzymes are unchanged after catalysing the ▪ Different enzymes have different optimum pH (M) required in reactions ▪ Enzymes are affected by the acidity or alkalinity of the minute amounts ✓ enzyme molecules can be reused solutions over again ▪ Enzyme activity reduces when the conditions are slightly ✓ only a small amount of enzyme is more acidic or alkaline than the optimum pH required to catalyze a large number - reversible changes of reactions - restored by bringing the enzyme back to the optimum pH ✓ Only bind to specific group or type ▪ Examples: ✓ Each chemical reaction is Enzymes that work best at ACIDIC conditions catalysed by its own specific, o Renin and Pepsin (stomach) Enzymes are unique enzyme. highly specific ✓ due to enzyme’s specific 3D Enzymes that work best at ALKALINE conditions configuration o Intestinal Enzymes ✓ Highly depend to structure Enzymes that work best at NEUTRAL conditions o Amylase Active sites ✓ Binding sites of substrate ✓ Enzyme + Substrate = Transition Note: When extreme pH conditions Enzymes are DENATURED. It state/ enzyme substrate complex changes in the charges at the active sites → repels the substrate Proteins are held ✓ By hydrogen bonds molecules. Irreversible alteration to the bonds that hold the together ✓ And responsible for secondary shape of the enzyme. And loses its original 3D structure (active structure site loses its shape) OPTIMUM TEMPERATURE ENZYMES CATALYZE REVERSIBLE REACTION ▪ Enzymes can function over a range of temperatures ▪ All enzymes have their own optimum temperature ▪ The temperature at which the enzyme is most active, catalyzing the largest number of reactions per second ▪ Example: most enzymes in the human body function best at about 37C Enzymes of thermophilic bacteria that live in hot springs will have very high optimum temperature. ▪ Some enzymes catalyze both reactions until equilibrium is reached ▪ Increase in temperature = increases the vibrations of the ▪ Reactions will proceed in the direction where the products atoms in the enzymes → breaking of the hydrogen bonds that are constantly being removed hold the 3D structure in place ▪ When BOUND tightly to ENZYME, the co-enzyme called ▪ The enzyme loses its shape and active site (Denaturation) prosthetic group ▪ Beyond the OT = enzyme activity decreases ▪ The enzymes (apoenzyme) + coenzyme = HOLOENZYME Note: Incubation of enzyme should be 37c and not in boiling bath. Increase temperature result to protein denaturation CARMEN, M.C.A | MED228 4  ENZYMES ARE INHIBITION (MECHANISM) LIVER ENZYMES AND LIVER FUNCTION TEST The Liver Transferases ▪ Used to assess extend of Liver Injury ▪ Two Liver Transferases: AST and ALT ASPARTATE AMINOTRANSFERASE (AST)/ SERUM GLUTAMIC OXALOACETIC TRANSAMINASE (SGOT) ▪ New name Indicated the name of the substrate ▪ Old names indicate the second substrate (glutamic acid or ketoglutamate ▪ Two products: Oxaloacetic acid and Glutamic acid ▪ Distribution: Liver, Heart, Skeletal muscle, kidney, pancreas, RBCs ▪ Reference value: 5-35 IU/L Enzyme Reaction: EC2.6.1.1 A. Competitive - inhibitor competes with the substrate in the ▪ Substrate: Aspartate together with Alpha keto glutamate active site. ▪ Product: Oxaloacetic and Glutamic Acid B. Non-Competitive- Binds to allosteric, competes with the Note: There are two types of liver enzyme: (1) those use to assess activator liver injury; (2) those used to assess biliary obstruction→ The bile C. Uncompetitive- Bins the ES complex, Will wait for enzyme duct or linkage of liver to gall bladder is part of liver. Enzyme is much and substrate bind then binds after them. Irreversible more clinically significant if its elevated = Injury to organ Pre-Analytical Considerations TWO GENERAL METHODS IN ▪ Avoid hemolysis MEASURING ENZYMATIC REACTIONS ▪ Refrigerated SERUM: Must sperate serum from the red cells 1. Fixed time (End point Method) ▪ Isoenzymes: a. Mitochondrial (only found if the cell is destroyed) ▪ Reaction takes place at a certain period b. Cytoplasmic (can be found in normal serum sample ▪ The reactants are combined ▪ The reaction proceeds for a designated time Measurement/ Analysis ▪ The reaction is stopped, and measurement is made ▪ Substrate use: L-Aspartate 2. Continuous Monitoring (Kinetic Assay) 1. Chemical Method: (Reitman Frankel) → Endpoint Method ▪ Measured absorbances at a certain period ✓ Use dinitrophenylhydrazone ▪ Multiple measurements of change in absorbance are ✓ Redish-Brown (505nm) made during the reaction 2. CM/Karmen Method: Continuous Monitoring ▪ More advantageous than Fixed time ✓ pH: 7.3 - 7.8 - allows monitoring of linearity UNITS FOR EXPRESSIGN ENZYMATIC ACTIVITY 1. International Unit (IU/U) - Conversion unit, Amount of enzyme that catalyze 1 First part of reaction is similar to enzymatic (continuous monitoring: micromole of substate/ min Aspartate + a-ketoglutarate produces → keto acids (oxaloacetic acid 2. Katal Unit (KUI) and glutamic acid) → ketoacid will be added to dinitrophenyl - 1 mole of substrate/ seconds hydrosol → d.hydrasol will react to oxaloacetic acid in alkaline environment (AST and ALT use sodium hydroxide buffer for color Conversion factor: intensifier) → Redish brown product 1 IU = 16.7 nkat/ 17nkat 1 kat/L = 0.06 IU/L CARMEN, M.C.A | MED228 5 Diagnostic Significance Chemical Method of Continuous Monitoring: KARMEN METHOD ▪ Hepatocellular injury→ Marker of Liver Injury First part of reaction: is similar to Reitman Frankel, we need to ✓ Hepatitis - 100x elevation (upper limit: 35IU) produce keto acid from the Aspartate/ Alanin substrate: ✓ Cirrhosis - 4x elevation (upper limit: 35IU) AST: Reacts with a-glutarate to produce Oxaloacetate ▪ Skeletal muscle injury – 4-8x elevation and glutamate ✓ Dermatomyositis ALT reacts with alanine to produce pyruvate and ▪ Heart conditions glutamate. ✓ AMI (Acute Myocardial Infraction) Second part: Product of the first part is different keto acid (AST: ✓ CHF (Congestive Heart Failure | Slow pump of heart) and Oxaloacetic, ALT: Pyruvate Acid) Pulmonary Embolism (Complication of CHF) AST: use the enzyme Malate Dehydrogenate: converts Oxaloacetic acid into→ Malate with the presence of NAD+ (NADH→NAD | Reduce→ Oxidize) → Check the DECREASE of ABORBANCE READING ALANINE AMINOTRANSFERASE (ALT) ALT: use the enzyme Lactate Dehydrogenate (LD): SERUM GLUTAMIC PYRUVIC TRANSAMINASE (SGPT) converts Pyruvic acid into→ Lactate/ Lactic acid with the ▪ Substrate: Alanine and Alpha Keto Glutamate presence of NAD+ (NADH→NAD | Reduce→ Oxidize) ▪ Product: Glutamate or Glutamic acid and Pyruvic acid → Check the DECREASE of ABORBANCE READING ▪ Distribution: Liver ▪ Reference value: 7-45 IU/L Absorbance reading is high INITIALLY and as go a long to conversion of pyruvate to lactate the reading gradually decreases during the ▪ Coenzyme/cofactor: Vitamin B6 (Pyridoxal Phosphate) process. UV wavelength is use (340 nm) Enzymes Reaction: EC2.6.1.2 Diagnostic Significance ▪ Substrate: Alanine ▪ Hepatocellular injury→ Marker of Liver Injury ▪ Product: Alpha keto glutarate: Pyruvic and glutamic ✓ Higher half life than AST Note: ✓ Slight elevation in obstructive jaundice There are two types of liver enzyme: ✓ More specific (1) those use to assess liver injury; ▪ Viral/Toxic hepatitis - 20x elevation (upper limit) (2) those used to assess biliary obstruction→ The bile duct or ▪ Hepatocellular carcinoma (liver Cancer) linkage of liver to gall bladder is part of liver ▪ IM (Intramuscular Injection) Enzyme is much more clinically significant if its elevated = ▪ Obstructive jaundice - Slight elevation Injury to organ ▪ De Ritis Ratio: Pre-Analytical Considerations ✓ Acute hepatitis - ALT:AST = >1.0 ✓ Alcoholic hepatitis – ALT:AST = (1:3-4) ▪ Unaffected by hemolysis ▪ Prolonged or lifespan for 3-8 ▪ Refrigerate the serum: 3-4 days DIAGNOSTIC SIGNIFICANCE (AST and ALT) Measurement/ Analysis 1. Liver disease - ALT activity >AST Exceptions: ▪ Substrate use: L- Alanine ✓ Alcoholic hepatitis ▪ Chemical Method: (Reitman Frankel) ✓ hepatic cirrhosis ✓ Use dinitrophenylhydrazone ✓ liver neoplasia (liver cancer) ✓ Redish-Brown (505nm) 2. Viral hepatitis and acute hepatic necrosis- serum AST ▪ CM: and ALT concentrations are elevated even before the ✓ pH: 7.3 - 7.8 clinical signs and symptoms of disease (jaundice) appear ✓ Hepatitis may reach 100 times the upper reference limit ✓ Peak values of aminotransferase activity occur between the seventh and twelfth days. ✓ Gradually decrease by the 3rd -4th week CARMEN, M.C.A | MED228 6 ▪ If persistence of increased ALT for more than 6 months - Note: Remember the Substrate then remove the -phosphate diagnose chronic hepatitis and that’s the product ▪ Chronic hepatitis - maximum ALT < 7x times the upper reference limit Analysis of ALP Isoenzymes ▪ ALT - may be NORMAL in 15%-50% of chronic Hep C ▪ Electrophoresis: migration from cathode(-) to anode (+) patients (IPBL) ▪ Twofold to fivefold elevations of both enzymes - primary or ✓ I Promise to Be Loved (IPBL) metastatic carcinoma of the liver (AST > ALT) ✓ Pattern: Intestinal, Placenta, Bone, Liver ▪ Heat stability test: Which is stable 56 degrees Celsius for ALKALINE PHOSPHATASE (ALP) 10 minutes: ✓ Pattern: Placenta > Intestinal > Liver >Bone ▪ For Hepatobiliary Obstruction → Obstructive jaundice ✓ Placenta is stable to 65 degrees for 30 mins (stability of abnormal ▪ EC3.1.3.1 isoenzymes) ▪ Hydrolase enzyme that breaks down phosphomonoester ▪ Chemical inhibition: groups to become alcohol and phosphate ion in an ✓ Phenylalanine: Intestinal, Placental ALKALINE ENVIRONMENT ✓ Levamisole: Bone, Liver ▪ Activator: Magnesium Ion/ Mg2+ ✓.03 molar urea: Bone ▪ Distribution: Intestine, Liver (bile duct), Bone (Osteoblast), Placenta with small amounts in kidney and spleen Abnormal Isoenzymes (carcinoplacental) Indicator ▪ Reference Range: 53-128 IU/L (male), 49-98 IU/L (female), Regan – Phenylalanine Lung Cancer and Breast, Ovarian cancer 54-369 IU/L (children) Nagao- Phenylalanine and L- leucine Adenocarcinoma and Bile Duct Kasahara - none Hepatoma (Liver Cancer) Note: Migrate in the same area Bone is migrated, Heat stability is similar to placenta. Inhibitors: Phenylalanine and L-leucine Clinical Significance Pre-Analytical Considerations ▪ Avoid hemolysis ▪ Avoid prolonged standing: 3-10 x → Falsely elevate ▪ Inhibited by fluoride, oxalate and heparin additives ▪ Affected by high fat meal (by 25 %) ✓ Note: Not applicable to all people, this is due to intestinal isoenzyme of blood type B and O secretors individuals Measurement/ Analysis ▪ Bessy, Lowry and Brock/Bowers-McComb is the most common method for ALP determination that uses Para- Nitrophenyl phosphate, when expose to alkaline phosphatase the phosphate group will be remove → P-nitrophenol which is a yellow solution CARMEN, M.C.A | MED228 7  GAMMA-GLUTAMYL TRANSFERASE (GGT) PANCREATIC ENZYMES AND OTHER CLINICALLY SIGNIFICANT ENZYMES ▪ Transferase (EC2.3.2.1) ▪ Associated with Biliary Obstruction AMYLASE (AMY) ▪ Transfer gamma glutamyl amino acid residues to produce glutathione ▪ Hydrolase (EC3.2.1.1) ▪ Glutathione is antioxidant, side effect includes lighting ▪ Smallest enzyme: Only enzyme can be detected in the skin color URINE ▪ Functions: ▪ Breaks down carbohydrates: starch (plant) & glycogen ✓ Protein synthesis (animal) ✓ Regulation of tissue glutathione levels ▪ Starch structure: α 1-4 glycosidic bonds and α 1-6 glycosidic ✓ Amino acid transport bonds ▪ Marker of: CHRONIC ALCOHOLISM and for ▪ Coenzyme: Calcium and Chloride Obstructive Jaundice ▪ Reference range: 28-100 IU/L ▪ Tested with ALP Analytical Considerations and Measurement/Analysis ▪ Reference range: 6-55 IU/L (male), 5-38 IU/L (female) ▪ Triglycerides inhibits amylase activity Pre-Analytical Considerations ▪ Administration of morphine and other opium-based ▪ GGT is stable up to 1 week in serum is refrigerated, not painkillers can cause falsely elevated results affected by hemolysis ✓ Constriction of the sphincter of Oddi ▪ Increased in patients receiving enzyme-inducing drugs ▪ Heparin used when additive tube used such as warfarin, phenobarbital, and phenytoin Amyloclastic Measures the disappearance of starch substrate ▪ Szasz Method: (use iodine that has high affinity to starch, as starch disappear in the sample the iodine also disappears) → Gamma-glutamyl-p-nitroanilide + glycylglycine → gamma- decrease absorbance reading glutamyl-glycylglycine + p-nitroanilide (420nm) Saccharogenic Measures the appearance of the product (simple carbohydrates, use old method of carbohydrates Note: Gamma-glutamyl transfers to a receiver glycineglycine measurement) → in somogi units Chromogenic Measures the increase color from production of Clinical Significance of GGT product coupled with a chromogenic dye (only measures specific carbohydrates by colorimetric assay | ▪ Renal tissue – highest GGT but serum has liver GGT Glucose) ▪ Elevated - alcoholic hepatitis, heavy drinkers Continue Coupling of several enzymes system to monitor ▪ Indicator of hepatobiliary disease monitoring amylase activity ▪ Primary/metastatic liver neoplasms Enzymatic Method ▪ Moderate elevations - Infectious hepatitis ▪ Differentiates INCREASE in ALP ▪ Substrate (ethylidene-pNP-G7) - cleaved (hydrolyze) by the amylase, a synthetic starch ▪ Final Product: P-Nitrophenol ▪ One unit is the amount of amylase that cleaves ethylidene- pNP-G7 to generate 1.0 µmole of p-nitrophenol per minute at 25C ▪ Alternative Substrate: 2-chloro-p-nitrophenyl-alpha-d- maltorioside (CNP-G3) *assay does not require glucosidase (direct" assay) CARMEN, M.C.A | MED228 8 Note: LIPASE (LP) Use alternative/ synthetic substance: G7 and G3. For G7 uses a-glucosidase to simply the substrate to be able to release the ▪ Hydrolase (EC3.1.3.3) P-Nitrophenol (yellow| 405nm). ▪ Breaks ESTER BONDS in lipids (produces Fatty acids and Glycerol) ▪ Coenzymes: BILE SALTS and COLIPASE ENZYME Isoenzyme Analysis of AMY ▪ Primarily found in the PANCREASE → Pancreatitis ▪ Ref range: < 38 IU/L ▪ Also known as Salivary Amylase ▪ Present in the fallopian tubes and lungs Pre-Analytical Considerations and and sometimes in gastrointestinal tract Measurement/ Analysis ▪ Predominant in the Serum Ptyalin ▪ Migrates faster in electrophoresis ▪ Avoid Hemolysis→ Hemoglobin inhibits lipase activity (F↓) ▪ Selective inhibition of the S-AMY by wheat ▪ Method of Analysis germ inhibitor→ Used as inhibitor for 1. Cherty Crandall Method Salivary amylase - Triglyceride substance: Olive oil or Triolein Ex: Test for total then add inhibitor then test - Product: Glycerol and Fatty Acids again (Total – Amylopsin total= conc ptyalin) - Measurement: Turbidimetric Amylopsin ▪ Also known as Pancreatic Amylase - End Color: PINK (P1, P2,P3) ▪ Predominant in Urine ▪ Predominant amylopsin in acute 2. Colorimetric/ Enzymatic: Peroxidase and Glycerokinase pancreatitis: P3 Enzyme Clinical Significance - Use the method for TG determination (TG determination is a Reagent) ▪ Acute Pancreatitis: amylase is less specific than pancreas - Peroxidase: Oxidation of Dye ▪ Parotitis→ Inflammation of salivary/ parotid gland ▪ Ectopic pregnancy → elevated of Ptyalin ▪ Other Methodologies: ▪ Peptic Ulcers Titrimetric- Measures DIRECTLY free Fatty Acids by ▪ Alcoholism titrating with dilute alkali ▪ Appendicitis - The amount of alkali used- recorded as a function of ▪ Mesenteric infraction → Problem in colon (insufficiency of time and serves as a measure of fatty acid produces supply) the reaction ▪ Macroamilocemia → No CS but must take remember that there is immunoglobulin attach to enzyme. Type of amylase that cannot be detected due to not being filter by Summary of PANCREATIC MARKERS glomerulus (due to small size) Amylase Lipase ▪ Biliary tract disease Rise 2-12 hrs 6 hrs Peak 24 hrs 24 hrs Decline 3-5 days 7 days CARMEN, M.C.A | MED228 9  CARDIAC PROFLE AND CARIAC MARKERS CARDIOVASCUAR DISEASES ▪ One type of most common odase that affect the general population Angina (Chest pain) and Doc Dacanay ▪ Types: ❖ Coronary Heart Disease o Manifest as Chest pain (Angina) & Acute Myocardial Infraction ❖ Cerebrovascular Disease LACTATE DEHYDROGENASE (LD/ LDH) ❖ Heat is already effect in the nervous system (Peripheral Arterial Disease) ▪ Oxidoreductase (EC.1.1.1.27) o Localized usually transient or temporary ▪ Conversion of lactate to Pyruvate ❖ Aortic Atherosclerotic Disease ▪ Coenzyme: NAD/ NADH o Abnormal weaking of the artery (aka. ▪ Reference range: 125-220 IU/L Aneurysm) ▪ Distribution: widely distributed with 5 izonezymes o Continuous elevated blood pressure → weaking of artery) Isoenzyme Subunit Location Serum Associated Disorders LD1 H-H-H-H Heart 2nd Myocardial Atherosclerosis Infarction RBC Hemolytic disorder ▪ Increase the likelihood of ischemia (lack of blood supply) LD2 H-H-H-M RBC (1st) Myocardial localized to different parts of the body Heart Infarction Hemolytic disorder LD3 H-H-M-M Lung 3rd Pulmonary Atherosclerosis Arteriosclerosis embolism Disorder affecting the lumen Disease affecting the walls Lymphocytosis Acute pancreatitis of the arteries due to plaque of the arteries due to ageing Extensive buildup pulmonary Due to obstruction of blood Blood vessels become stiff → pneumonia LD4 H-M-M-M Liver 4th Hepatitis vessel due to accumulation incapable of adjusting and LD5 M-M-M-M Skeletal 5th Skeletal muscle of FAT and will turn to expanding Muscle injury BLOOD CLOTS → go to brain *LD-FLIP → Indicates Myocardial Infraction (stoke) Result of high cholesterol A genetic trait, that cannot Pre/Analytical Considerations diets and poor exercise be changed ▪ Avoid hemolysis CARDIOVASCUAR MARKERS ▪ Activity is unstable (ideal storage temperature: Ref Temperature, ▪ Angina aka. Chest Pain Rationale: Freezing is not recommended to LD4 and LD5) ▪ 2 types: Stable Angina (activity-related) Analysis/ Method - Chest pain usually experience after performing ▪ Chemical Inhibition: a-hydroxyurea (H-subgrroup strenuous activity inhibitor) - Transient→ once rest it will be okay - When added to sample LD 3, LD4, LD5would be Unstable angina (manifestation of Heart Attack) affected - Usually there is no trigger 1. Wacker - Can be trigger by strenuous activity or stress - Reference method ▪ Classic manifestation o Angina pectoris: - Most commonly used method ✓ Chest pain (“squeezing” pain) - Measures the enzymatic activity as lactate → pyruvate ✓ Burning feeling - pH 8.3-8.9 ✓ Difficulty in breathing - Preferred for LD-1 - Measurement of activity can be through kinetic or colorimetric CARMEN, M.C.A | MED228 10 2. Wroblewski CREATINE KINASE (CK) - Fast Method - Measures enzymatic activity as pyruvate → lactate ▪ Transferase (EC 2.7.3.2) (becomes acidic since pinasahan ng H → increase in H, ▪ Function: ATP regeneration and storage (Creatine lowers pH) Phosphate) - Measure the DECREASE in the absorbance at 340nm - ATP binds to Creatine as NADH is converted to NAD ▪ 3 isoenzymes: - 3x faster than the Wacker reaction CK-MM: CK-3 | Muscle - pH 7.1-7.4 CK-MB: CK-2 | Heart - Preferred for LD-5 CK-BB: CK-1 | Brain ▪ Reference: ✓ Total (Male): 46-171 IU/L WACKER WROBLEUSKI-LA DUE ✓ Total (female): 34-145 IU/L Type of assay FORWARD / Direct REVERSE / Indirect ✓ CK-MB: < 6% of total CK Product Lactate → Pyruvate Pyruvate → Lactate pH pH 8.8 pH 7.2 Cofactor NAD → NADH NADH → NAD Isoenzyme Tissue Condition Preferred Isoenzymes LDH-1 LDH-5 Myocardial Infraction Skeletal Muscle Disorder Muscular dystrophy Isoenzyme Analysis CK-MM Heart Polymyositis Skeletal Muscle Hypothyroidism Malignant hyperthermia Physical activity Intramuscular injection Myocardial Infraction Myocardial Injury Ischemia CK-MB Heart Angina Skeletal Muscle Inflammatory Heart disease Cardiac surgery Duchenne-type muscular dystrop. Malignant hyperthermia Reyes syndrome Rocky mountain spotted fever Carbon monoxide poisoning Central Nervous system shock Other Clinical Significance Brain Anoxic encephalopathy Bladder Cerebrovascular accident ▪ Highest total LD: Pernicious Anemia CK-BB Lung Seizure ▪ Liver disorders, AMI and Pulmonary infraction: Prostate Placental or uterine trauma Uterus Carcinoma - Increase in LD5-hepatocellular necrosis and Colon Reyes syndrome hepatocellular carcinoma Stomach Carbin monoxide poisoning ▪ Skeletal muscle disorders Thyroid Malignant hyperthermia Acute and chronic renal failure ▪ Neoplasm -Acute lymphoblastic leukemia Note: If not in in RBC (LD1/2) if not in Liver (LD4), Not in Pre-Analytical Considerations muscle(LD5)…most likely its in LD3 ▪ Avoid Hemolysis - Not because there is CK in RB. This is due because enzyme similar to CK is Adenylate kinase. Causes Falsely Elevated F↓ ▪ Inactivated by light ▪ CK is also affected by Muscle Mass ▪ Restore activity after storage using: Uses CK activator: Sulfhydryl activator (incorporated in the buffer) ▪ Most prevalent isoenzyme in the serum: CK-MM ▪ Most routine methods cannot measure: CK-BB (seen in CSF due to Blood brain barrier) CARMEN, M.C.A | MED228 11  Analysis/ Measurement CARDIAC TROPONIN (Tpn) 1. Tanzer-Gilvarg ▪ Early marker of AMI - Forward method/ Direct ▪ Regulate the Calcium - pH: 9.0 at 340nm dependent interaction of - Creatine → Creatine Phosphate actin and myosin - Calcium will bind to 2. Oliver-Rosalki ACTIN and twist→ expose the binding site of myosin - Reverse Method ▪ Attached to Actin filament via tropomyosin filament - pH: 6.8 at 340nm ▪ 3-protein complex - Creatine Phosphate → Creatine ✓ TnT: Attachment point to tropomyosin ✓ TnI: Inhibitor Isoenzyme Analysis ✓ TnC: Binding site of calcium ▪ Major Isoenzyme can be seen: CK-MM ▪ Reference range: 0-10 ng/mL ▪ Sensitive marker of AMI and Duchennes muscular ▪ Preferred sample: Heparinized plasma and Red Top dystrophy ▪ High sensitivity troponins ▪ Ectopic Pregnancy: CK-BB - Tool for detect (higher sensitivity) - Uses CK progesterone ratio (hormone testing) - As low as 2-3 nanograms ▪ 2 Abnormal Isoenzyme ▪ Cardiac troponin in Blood (Tpn) = Damage in Cardiac  Macro CK: between MM and MB muscle tissue  Mitochondrial CK (CK-Mi): IgA and IgG Troponin T CK-BB (CK-1) – FASTEST and labile ▪ Useful for assessment of early and late AMI CK-MB (CK-2) -macro- ▪ Elevated also in renal disease and muscle dystrophy CK-MM (CK-3) – SLOWEST ▪ Sensitive marker for diagnosis of unstable angina (angina at -mitochondrial- rest)→ when patient experience chest pain ▪ Useful in monitoring the effectiveness of thrombolytic therapy in AMI patients → can be seen for 1 week Troponin I ▪ Inhibitory subunit of Actin-binding unit ▪ Gold standard for AMI Diagnosis ▪ TpI is predominant in cardiac muscle Other Methods ▪ It is only found in myocardium- grater cardiac specificity than TnT ▪ Immunochemical methods - applicable to the direct ▪ Very sensitive indicator even a minor amount of cardiac measurement of CK-MB necrosis - Test total CK (use Antigen-Antibody) ▪ Immuno-inhibition technique Myoglobin (Myg) - Use Anti-CK-M subunit antiserum- inhibits both/all M- subunits of CK-MM and CK-MB ▪ Binds Carbon Dioxide and oxygen (same as hemoglobin) - allow determination of the enzyme activity of the B in the muscle subunit of CK-MB and CK-BB ▪ General marker of muscle injury - Test for total → immunochemical for CK-MB→ Perform - Earliest cardiac marker Anti CK-MB and CK-BB for indirect measurement of ▪ Problem: CK-MM - Low Specificity → Not specific marker to heart ▪ Mass immunoassays - measure the concentration of CK- - It is measured in serum by immunoassay MB using the "sandwich" technique - At high levels in urine (myoglobinuria) – (+) dipstick - one antibody specifically recognizes ONLY the MB reaction for occult blood due to peroxidase activity dimer CARMEN, M.C.A | MED228 12  Summary of Cardiac Markers ACID PHOSPHATASE (ACP) RISE (Onset) PEAK DECLINE (normalize) ▪ EC 3.1.3.2 LD 12-24 hrs 48-72 hrs 10-14 days ▪ Similar function with ALP but works at different pH and CK-MB 4-6 hrs 12-24 hrs 2-3 days environment Troponin T 3-4 hrs 10-24 hrs 10-14 days - separate alcohol from phosphate group but diff pH and Troponin I 3-6 hrs 12-18 hrs 5-10 days location Myoglobin 1-3 hrs 5-12 hrs 18-30 hrs ▪ Distribution: PROSTATE, liver, bone, spleen, kidney, AST 6-8 hrs 18-24 hrs 4-5 days ERYTHROCYTES and platelets ▪ Reference range: Other Cardiac Markers ✓ PAP = 0 – 3.5 ng/ml ✓ Adult = 1.5 – 4.5 IU/L ▪ Hearth Type Fatty Acid Binding Protein (H-FABP) ✓ Children = 3.5 – 9 IU/L ✓ Similar to Myoglobin but more SPECIFIC to the Heart o Due to active ✓ Higher SENSITIVITY but lower SPECIFICITY compared to Troponin ▪ Ischemia-Modified Albumin (IMA) ✓ Predictor of AMI ✓ Starts to elevate when high risk of AMI ✓ Elevated free radical formation on the system, Pre-Analytical Considerations and (accumulation) can cause medication in albumin Measurement/Analysis during ischemia o Albumin tends to bind to Cobalt ▪ Avoid hemolysis→ release ACP in the plasma o ▪ Separate serum from the red cells immediately→ remove the ▪ Markers of plaque instability serum and move to another container ✓ Similar with Troponin in early detection of AMI - ACP has platelet distribution o Myeloperoxidase (MPO) - Platelet left in RBC will leak the ACP o C-reactive protein (CRP_ ▪ Loss of activity in prolonged standing of serum sample o Myeloid rlated protein 18/4 (MRP- 18/4) (rationale: Loss of gasses (CO2) → Alkalinization) o Pregnancuy associated plasma protein A - In long run Accumulation of Carbon dioxide in the sample and (PAPP-A) loss of significant gasses which leads to ALKALINITY OF THE SAMPLE→ Loss of Activity ▪ Measurement: Same with ALP (Bessy Lowry Brock) but the ▪ B-Type Natriuretic Peptide and C-Reactive protein pH of the buffer is acidic p-nitrophenyl phosphate (uses ✓ Rule out congestive heart failure pH 5 as buffer) ▪ Homocysteine ▪ Inhibitors: ✓ Atherosclerosis and thrombosis ✓ L -tartrate - prostatic ACP (indirect) ✓ 2% Formaldehyde – RBC ACP (direct method) ✓ 0.001M Cupric sulfate- RBC ACP (direct method) Methodology CARMEN, M.C.A | MED228 13  Note: Clinical Significance Remove the phosphate in the substrate to obtain the measurable product. Hudson uses pH 5.0. ▪ Deficiencies ✓ Sex-linked trait Roy method is an end method ✓ Drug-induced hemolytic anemia (primaquine) Clinical Significance ✓ Favism- fava beans ingestion ✓ Neonatal jaundice ▪ USE TO CHECK OR ASSESS THE PROSTATE ✓ Chronic non-spherocytic hemolytic leukemia ▪ Metastatic Prostatic Carcinoma- Increased serum ACP ▪ Elevated: ▪ Forensic clinical chemistry –Rape investigation ✓ Myocardial Infarction ▪ Benign prostatic hypertrophy (BPH)- slight elevations after ✓ Megaloblastic anemia prostatic massage ▪ Paget’s disease CHOLINESTERASE ▪ Cancers of breast with BONE metastases ▪ Gaucher’s disease (Blood disorder) ▪ Not routinely performed ▪ Niemann Pick disease (Blood disorder) ▪ Two related Enzymes: 1. Acetylcholinesterase (AChE)- true cholinesterase - Tissue location: Red Blood cells, Lungs, Spleen, nerve endings, gray matter of brain GLUCOSE-6-PHOSPHATE DEHYDROGENASE 2. Aclycholine acylhydrolase (PChe)- pseudocholinesterase ▪ ERYTHROCYTES, Adrenal cortex, Spleen, Thymus, Lymph - Tissue Location: Liver, pancreas, heart, white matter of nodes and Lactating mammary gland brain, serum ▪ Serves as ANTIOXIDANT, and prevents oxidation of other Methodology substances to prevent hemolysis ▪ Hexose Monophosphate Shunt of glucose metabolism ▪ Butyrylthiocholine (substate) is hydrolyzed to butyrate and ▪ Not routinely performed as diagnostic test thiocholine ▪ Maintain NAD to NADH ▪ Thiocholine + m5’disthiobis or 2-nitrobenzoate (DTNB) → ▪ NADH is important to prevent free radical formation colored - Free radicals are harmful to red cells that causes ▪ Final Product: 5- mercapto- 2 nitrobenzoic acid hemolytic disorders (measured spectrophotometrically at 410nm) (Yellow) ▪ preferred sample: SERUM Specific function of G-6-PD in the Erythrocytes ▪ NV: 33-76 U/L ▪ Maintains NADPH in REDUCED form ▪ NADPH regenerate sulfhydryl-containing proteins like GLUTATHIONE ▪ Glutathione PROTECTS hemoglobin from oxidation Clinical Significance (denaturation) ▪ Test of liver function ▪ Indicator of possible insecticide poisoning Analysis ▪ Detection of patients with atypical forms of enzymes who are at risk of prolonged response to certain muscle ▪ Deficiencies: red cell hemolysate relaxants used in surgical procures ▪ Elevations: serum 5-Nucleotidase Note: When expected elevated G6PD measures the serum ▪ Main source: Biliary epithelium sample. When expected deficiency measures the hemolysate ▪ Highest level in cholestatic conditions (whole blood with hemolyze agent) ▪ Test to confirm that an elevated ALP is due to hepatobiliary disease ▪ Substrate: adenosine 5’ monophosphate (5’AMP) - Product: Converted to adenosine and inorganic phosphorus CARMEN, M.C.A | MED228 14 All information and notes are from Enzymology PDF of Mr. John Laurence A. Bautista, RMT.