MLS Review - CC - Enzymology

MEDICAL LABORATORY SCIENCE REVIEW GENERAL PROPERTIES AND DEFINITIONS CLINICAL CHEMISTRY: ENZYMOLOGY OUTLINE • • • • • Introduction General Properties and Definitions Enzyme Classification and Nomenclature Enzyme Kinetics Enzymes of Clinical Significance INTRODUCTION TO ENZYMOLOGY INTRODUCTION • • • Clinical Enzymology - study of measurement of enzyme activity for the diagnosis and treatment of diseases Enzymes o Biologic protein that catalyzes/accelerate biochemical reactions occurring in the body ▪ Increases rate of chemical reaction o Majority are intracellularly located ▪ When enzymes are increased in serum/plasma, it is associated with cell injury or damage o Produced in the living cells o Affects velocity of chemical reactions in organic matter ▪ Speed up and hasten chemical reactions ▪ Not consumed/changed in composition during rxn ▪ Not participants/involved in actual reaction; they only accelerate the reaction o Specific action with organic substrates o In laboratory, measured in terms of activity instead of their actual value ▪ Activity of enzyme to a substrate is directly proportional to its concentration • Fast activity = Increased enzyme conc. o In the serum, enzymes are normally low o ↑↑ enzyme concentration in serum is indicative of: ▪ Cellular injury or damage • Useful for determining tissue damage and abnormal conditions ▪ Increased membrane permeability • Allow enzymes to pass through & leave cell Functions of Enzymes o Hydration of carbon dioxide (respiration) ▪ Carbonic acid - important in buffering plasma pH ▪ Enzyme hasten formation of carbonic acid o Nerve induction (nerve impulse) ▪ Enzyme hastens nerve impulse transmission o Muscle contractions (locomotion) o Nutrient degradation (digestion) ▪ Digestive juices contain enzymes to facilitate interconversion of complex molecules to promote absorption in the GIT • Macromolecules require enzymatic conversion to simpler molecule to facilitate digestion and absorption process o Growth and reproduction ▪ Enzymes work with hormones in facilitating development of cells and reproduction process o Energy storage and use • Components of an Enzyme o Active Site - waterless cavity of an enzyme where substrates bind and undergo a chemical reaction ▪ Binding site of the substrate ▪ Substrate must fit the active site ▪ Only specific substrates can only bind with active site (Enzyme-Substrate Specificity) o Allosteric Site- waterless cavity other than the active that binds regulatory or effector molecules • Terms associated with enzymes o Substrates: substances acted upon by enzymes; specific for each particular enzyme ▪ Eg. Lactase - disaccharidase enzyme to catabolize lactose to release glucose & galactose o Cofactors: nonprotein substances/entities added in enzyme-substrate complex to manifest enzyme activity; enhances enzymatic reaction ▪ Coenzyme: aka second substrate; organic cofactor that hastens/speeds up enzymatic rxn • Eg. NAD, NADP (specific in reaction of oxidoreductases; oxidized form) o NADH, NADPH - reduced form ▪ Activator: metallic/non-metallic ion added to enhance enzyme-substrate binding • Alters the spatial configuration of the enzyme active site to facilitate substrate binding • Eg. Magnesium, calcium, zinc, iron (metallic); chloride, bromide (nonmetallic) o Isoenzyme: enzymes with similar enzymatic activity but differ in physical, biochemical, and immunologic characteristics ▪ Isoenzyme measurement is preferred than total enzyme measurement 1 • o o Makes the test highly specific to target tissues/organs ▪ Eg. Creatine Kinase (CK) isoenzymes - CKMM, CKMB, CKBB • Total CK = MM+MB+BB • • MB - marker for MI Apoenzyme: protein portion of enzyme subject to denaturation process ▪ When denatured, enzyme activity is lost ▪ Tertiary structure is targeted by denaturation ▪ Heating at 56C promote denaturation Holoenzyme: active substance formed by the combination of coenzyme and apoenzyme ▪ Prosthetic group - coenzyme tightly bound to an apoenzyme ▪ Apo + Co = Holo o 3. 4. ENZYME CLASSIFICATION AND NOMENCLATURE ENZYME NOMENCLATURE • • Enzyme Commission (EC) of International Union of Biochemistry (IUB) o Responsible for naming and classifying enzymes o Systematic name (long name): defines substrate acted on, reaction catalyzed, and possibly coenzyme involved o Recommended name: trivial/usable name; commonly used names ▪ Eg. Lactase, amylase o EC numerical code: composed of 4 digits ▪ E.C.__.__.__.__ • 1st digit - enzyme class • 2nd digit - enzyme subclass • 3rd digit - enzyme sub-subclass • 4th digit - enzyme serial number specific to the enzyme sub-subclass Examples Of Enyzmes (w/ Nomenclature) o Acid phosphatase ▪ Orthophosporic monoester phosphohydrolase (acid optimum) ▪ E.C.3.1.3.2 o Amylase ▪ 1-4-D-Glucan-Glucanohydrolase ▪ E.C.3.2.1.1 o Alanine aminotransferase ▪ L-alanine: 2 oxoglutarate aminotransferase ▪ E.C.2.6.1.2 5. 6. E.g: ▪ Transferases (ALT - alanine aminotransferase; AST - aspartate aminotransferase; GGT - gamma glutamyl transferase) ▪ Kinase - specific type that catalyze the transfer of phosphate form one substrate to another • Eg. Creatine Kinase (CK) Hydrolases o Catalyzes the hydrolysis of various chemical bonds o Requires water to function o A–B + H2O → A–OH + B–H o E.g.: ▪ Amylase - targets glycosidic bonds in carbohydrates ▪ Lipase - targets ester bonds in lipids ▪ Phosphatase - targets phospho-monoester bonds; eg. acid & alkaline phosphatase Lyases o Catalyzes removal of groups from substrates w/o hydrolysis o Do not require water o Product retains its double bonds o ATP → cAMP + PPi ▪ cAMP - cyclic adenine monophosphate ▪ PPi - immediate diphosphate o E.g. : fructose biphosphate aldolase (ALS) Isomerases o Catalyzes interconversion of geometric, optical, and positional isomers o A→B o E.g.: triphosphate isomerase (TPI) Ligases o Catalyzes the joining of two substrate molecules coupled with breaking of pyrophosphate bond in ATP o Ab + C → A–C + b o E.g. : glutathione synthetase (GSH-S) ENZYME KINETICS CATALYTIC MECHANISM OF ENZYMES • • • • Enzymes catalyze physiologic reactions by lowering the activation energy level that the reactants must reach Activation energy level o Required to proceed with reaction o Important in product formation Greater required activation energy = longer the reaction will proceed (longer product formation) Lower required activation energy = faster product formation CLASSES OF ENZYMES 1. 2. Oxidoreductases o Catalyzes redox reaction between two substrates ▪ Redox - Transfer of electrons/charged ions o Hydrogen ion is commonly transferred o Utilizes coenzymes ▪ NAD, NADP o A- + B → A + Bo E.g: Dehydrogenases (LDH - lactate dehydrogenase; G6PD - glucose-6-phosphate dehydrogenase) Transferases o Catalyzes the transfer of chemical groupX (phosphate, methyl, etc. other than H+ ions) between two substrates o A-X + B → A + B-X 2 • Relationship between enzyme, substrate and product o A substrate complex is formed o E + S ⇌ ES → E + P • Absolute specificity: enzyme combine with one substrate and catalyze only one corresponding reaction o Eg. Lactase catalyze lactose catabolism to release glucose Group specificity: enzyme combine with all substrates containing a particular chemical group o Eg. Kinases targets all with phosphate group Bond specificity: enzyme specific to chemical bonds o Eg, amylase, lipases, phosphatases Stereoisometric specificity: enzymes combine with only one optical isomer • • • o • FACTORS THAT INFLUENCE ENZYMATIC REACTIONS • • • • Substrate Concentration: increased substrate conc. = higher enzyme concentration = faster reaction rate o Saturation kinetics - when a substrate concentration reaches a maximal value, higher concentration of substrate can no longer result to increased rate of reaction ▪ Enzymes available are already exhausted o First-order kinetics - reaction rate is directly proportional to the substrate concentration o Zero-order kinetics - only a fixed number of substrate (in excess) is converted to product per second ▪ Utilized in the clinical laboratory Enzyme Concentration: higher enzyme concentration = faster reaction rate; more enzymes mean more active sites for binding o First order kinetics o Zero-order kinetics pH: most enzymes are able to perform physiologic reaction occur in range of physiologic pH (7.0 - 8.0; near plasma pH 7.35-7.45) o Extreme pH may cause protein denaturation leading to loss of function/catalytic activity o Phosphatases - able to perform at very high/low pH ▪ ALP Temperature: active at 25C - 37C o Assay temperature should be constant withing +/0.1C at which the enzyme is active at 25C, 30C, 37C o Body temp (37C) - optimal temp for enzyme activity o Increased temp = increases and hasten activity (de to increase movement of molecules) ▪ In the lab • Body temp - incubtation time is shorter • Room temp - incubation time is longer • Warm temp - • • • Temperature coefficient (Q10) - as temperature increases by 10C, there is a characteristic two-fold increase in enzyme activity o If temperature exceeds body temp (37C), it will cause protein denaturation o When temperature reaches 40C-50C, denaturation of protein increases ▪ In denaturation, enzyme activity decreases o 56C - denaturation of protein is at peak o 60C-65C - enzymes are completely inactivated o Most enzymes are thermolabile (inactive in high temp) ▪ Azoenzyme (protein part) cause denaturation rxn ▪ Placental ALP (Regan ALP) - thermostable o At low temperatures, enzymes are reversibly inactive Cofactors: nonprotein entities that enhances enzyme activity o Activators - Alter spatial configuration of enzymes for proper substrate binding (active site binding) ▪ Metallic (Ca2+, Fe2+, Mg2+, Mn2+, Zn2+, etc.) and Non-Metallic (Br- and Cl-) o Coenzymes (prosthetic groups) - serve as second substrates for enzymatic reactions (NAD) ▪ Velocity of enzymatic reaction increases o Presence of cofactors enhance enzymatic reactions Inhibitors: agents/substances that can interfere with enzyme-substrate reaction; prevent enzymatic reactions o 3 types: competitive, noncompetitive, uncompetitive o Competitive inhibitor - competes with the substrate on the active site of the enzyme ▪ Can physically bind to an active site ▪ Can directly alter the enzyme o Noncompetitive inhibitor - binds to a place other than active site of the enzyme; binds to allosteric site o Uncompetitive inhibitor - binds to the enzymesubstrate complex o Inhibitor > substrate = enzymatic activity is decreased Storage of Serum/Plasma o Freezer temperature (-20C or lower) - could make enzymes reversibly inactive for long period of time ▪ Ideal temperature to maintain enzyme ▪ Frozen sample should be thawed prior to testing which is only allowed once • Multiple freezing and thawing of sample could destroy proteins including enzymes • Once thawed & tested, sample should be discarded o Refrigerator temperature (2C-8C) - storage for substrate, coenzyme and reagents for testing o Room temperature (20C-24C) - ideal storage for enzymes not requiring cold storage (eg. LDH) Hemolysis o Hemolyzed sample may cause false increase in enzyme concentration (erroneous reporting) o Enzymes in lysed RBCs are released in the plasma increasing enzyme concentration o Mild hemolysis is also unacceptable 3 • Lactascence o Milky & lipemic specimens ▪ Maybe patient did not follow fasting protocol ▪ Maybe due to lipid metabolism disorders o Increased chylomicrons in blood may affect enzyme measurement causing false decreased enzyme conc. MEASUREMENT OF ENZYME ACTIVITY • • • Measurement of Catalytic Activity o Increase in product concentration ▪ ↑ product = ↑ enzyme o Decrease in substrate concentration ▪ Substrates are converted to products ▪ ↓ substrate = ↑ products o Decrease in coenzyme concentration (NADH) ▪ Coenzymes are also converted o Increase in altered coenzyme concentration ▪ Oxidized (NAD) ↔ reduced (NADH) • Indirectly proportional concentrations • NADH - increased absorbance of coenzyme 340nm • NAD - decreased absorbance ▪ Oxidoreductases alter coenzymes o Dependent on enzyme concentration o Always performed in zero-order kinetics ▪ Fixed concentration used for baseline o Performed during the linear phase of reaction General Methods of Measuring Enzymatic Reaction o Fixed-time (two point) assay ▪ The reagents are combines, the reaction proceeds, the reaction is stopped and the amount of reaction is measured ▪ One specific time for measurement o Continuous-monitoring or kinetic assays ▪ Multiple enzyme activity is included ▪ Involves • Primary enzyme - target enzyme, enzyme of interest • Secondary enzyme (coupling/indicator enzyme) - rely on primary enzyme activity to proceed to function ▪ Multiple measurements at specific time intervals (30s - 60s) or continuous measurement as absorbance changes ▪ Absorbance should be increasing from the first reading to the final reading ▪ Absorbance is directly proportional to enzyme concentration/activity ▪ Deviation from zero kinetics can be observed Michaelis-Menten Equation - mathematical expression of relationship between the velocity of enzymatic reaction and substrate concentration CALCULATION OF ENZYME ACTIVITY • • IU (EC) - the amount of enzyme that will catalyze the reaction of 1 μmol of substrate per minute. (μmol/min) Kat/Katal (SI) - the amount of enzyme that will catalyze the reaction of 1 mol of substrate per second (mol/s) MEASUREMENT OF ENZYME MASS • • Quantified by electrophoretic techniques that provides resolution of isoenzymes and isoforms o Isoenzymes - have different electrophoretic migration Grading o 1 - fastest; nearest to anode o 5 - slowest; farthest from the anode ENZYME THEORIES • • Emil Fisher's/ Lock and Key theory - Shape of key (substrate) must fit into the lock (enzyme’s active site) Kochland's Induced Fit Theory - based on substrate binding to the active site of the enzyme; fit on the configuration of substrate to the active site ENZYMES OF CLINICAL SIGNIFICANCE LIVER ENZYMES • Aspartate Aminotransferase (AST) o Serum glutamic-oxaloacetic/glutamate-oxaloacetate transaminase (SGOT) (AST = SGOT) o A transferase that transfers an amino group between aspartate and α-keto acids o Involved in the synthesis and degradation of amino acids in the liver o Not liver-specific but widely distributed, highest activities in cardiac, liver and skeletal muscle. o 2 Isoenzymes: ▪ Cytoplasmic AST - originated from cytoplasm • Predominant AST isoenzyme in serum ▪ Mitochondrial AST - originated from mitochondrial membrane • Elevated in severe injury/damage to cell causing release of AST from damaged mitochondrial membrane o o Where S = substrate; E = enzyme; ES = enzymesubstrate complex; K1, k-1,k2 = rate constants Diagnostic Significance ▪ After MI, AST levels begin to rise in 6-8 hours, peak at 24 hours, and return to normal in 5 days. • Associated with acute MI ▪ Also ↑ in hepatocellular and skeletal muscle dis. • AST is released to a greater degree in chronic disorders of the liver with progressive damage to hepatocytes 4 SGOT/AST SGPT/ALT Heart, muscle, Liver liver Aspartate alpha Alanine alpha ketoglutaric acid ketoglutaric acid End Products Glutamic acid + Glutamic acid + oxaloacetic acid pyruvic acid Color Developer 2,4 DNPH 2,4 DNPH Color Intensifier 0.4 N NaOH 0.4 N NaOH Methods Reitman & Frankel Reitman & Frankel o Reitman and Frankel - measurement for transaminase o Aminotransferases require coenzyme - pyridoxal phosphate (Vit. B6) Conditions of Increased Transaminases o Toxic hepatitis - ↑ AST & ALT; ▪ Chronic - ↑ AST; Acute - ↑ ALT o AMI - ↑ AST o Wolff-Parkinson White syndrome (involuntary muscle contractions) - ↑ AST o Trichinosis (parasitic infection T. spiralis) - ↑ AST o Chronic alcoholism - ↑ AST ▪ Acute = ↑ ALT o Dermatomyositis - ↑ AST o Hepatic cancer- ↑ AST & ALT ▪ Chronic - ↑ AST; Acute - ↑ ALT o Reye’s syndrome (liver syndrome) - ↑ AST & ALT o Viral hepatitis - ↑ AST & ALT; higher ALT o Muscular dystrophy - ↑ AST o Acute pancreatitis - ↑ AST Gamma-Glutamyltransferase (GGT) o Catalyze the transfer of the γ-glutamyl residue from γglutamyl peptides to amino acids, H2O, etc. o Most common donor of gamma glutamyl peptide is glutathione o Used for diagnosis hepatobiliary disorders and chronic alcoholism o Sensitive marker for determination of ethanol intoxication o Marker for occult alcoholism (hidden type) Major Organ Affected Substrate o Assay for Enzyme Activity ▪ Karmen Method • Uses malate dehydrogenase and monitors decrease in absorbance at 340 nm o Reduced (H) = high absorbance (X → H) o Oxidized = decreased absorbance o Oxidized NAD = colorless result ▪ 400 - 700 nm = colored product ▪ <400 & >700 nm = colorless product • Malate dehydrogenase (MD) - secondary, coupling, indicator enzyme; part of reagent o Part of oxidoreductase class • AST activity is directly proportional to the secondary reaction; product of AST activity serve as substrate for MD • Falsely ↑ in hemolyzed sample • Reference Range: 5 – 30 U/L ▪ Hemolyzed sample is not accepted; may cause false elevation • • • Alanine Aminotransferase (ALT) o Aka serum glutamic-pyruvic transaminase (SGPT) o Alanine is substrate o Transfer of an amino group between alanine and αketoglutarate o Increased in hepatocellular disorders; considered as liver specific enzyme o ALT is a significant marker of evaluation of hepatic disorders specifically in acute inflammatory conditions o Used to review course of hepatitis treatment response o Used to monitor effects of drug therapy (chronic diseases) o Considered as more specific & sensitive screening test for post-transfusion hepatitis and occupational toxic exposure o Used to screen blood donors o • Assay for Enzyme Activity ▪ Uses Lactate Dehydrogenase (LD) and monitors decrease in absorbance at 340 nm • Lactase dehydrogenase - secondary, coupling, indicator enzyme • Oxidized coenzyme • Colorless product ▪ Reference Range: 6-37 U/L De Ritis Ratio o The AST/ALT Ratio o Differentiates the cause of hepatic disorder o Ratio > 1 → no viral origin o Ratio < 1 → viral in origin o Assay for Enzyme Activity ▪ Szaz Assay • The absorbance of p-Nitroaniline is measured at 405-420 nm • Colored product M.I. PROFILE • • Aspartate Aminotransferase (AST) Creatine Kinase (CK) o Dimeric enzyme composed of two monomer subunits o Involved in the storage of high-energy creatine phosphate in muscle cells ▪ Creatine phosphate - reservoir of energy in muscle; used when there is a need for energy o Widely distributed, highest activities in skeletal muscle, heart, and brain 5 o Methods of Determination ▪ Forward Reaction (Tanzer-Gilvarg) • ↓ in absorbance at 340 nm is determined o Oxidized NAD product (colorless) • Optimum pH is 9.0 • Pyruvate kinase (PK) & LD - reagents ▪ ▪ o o o Reverse Reaction (Oliver-Rosalki) • ↑ in absorbance at 340 nm is determined o Reduced NAD product • Optimum pH: 6.8 • Hexokinase (HK) & glucose-6-phosphate dehydrogenase (G6PD) Source of Error ▪ Hemolysis can cause false ↑ CK activity • Blood has adenylate kinase (AK) • AK & CK - has same activity • Lysed RBC releases AK causing false elevation due to similar activity with CK ▪ CK is inactivated by light (photosensitive) ▪ Physical activity and IM injections cause ↑ CK o Reference Range ▪ M → 15-160 U/L : F → 15-130 U/L ▪ CK-MB: <6% of total CK CK Isoenzymes CK-3/CK-MM/ CK-2/CK-MB/ CK-1/CK-BB/ Muscle type Hybrid Type Brain Type Slowest mobility 2nd fastest to Migrate fastest toward the anode migrate toward toward the anode the anode Major isoenzyme Significant Highest conc. in in striated muscle quantities are CNS (CSF), GI and normal serum found in heart tract and uterus tissues (pregnancy) ▪ M (muscle) or B (brain) subunits • o Diagnostic Significance of CK Isoenzymes ▪ After MI, CK-MB (>6%) levels begin to rise within 4-8 hours, peak at 12-24 hours, and returns to normal levels (6%) within 48-72 hours. ▪ CK-MB - 1st enzyme to elevate in AMI patients • Myoglobin - 1st marker to elevate in AMI ▪ Total CK - measures all CK isoenzyme ▪ CK Isoenzyme - measures single isoenzyme; more diagnostic due to organ specificity Reference values: • CK-MB - 2-6% • CK-MM - 94-98% Other CK Isoenzymes ▪ Macro-CK - CK is coupled/complexed with antibodies or lipoproteins • CKBB complexed with antibodies (IgG/IgA) • CKMM with LPP • Migrate between CKMM and CKMB ▪ Mitochondrial CK (CK-Mi) • Bound to the mitochondrial membrane • Elevated levels suggest injury/damage to cells • Migrates cathodal to CKMM Macro CK-Mi Lactate Dehydrogenase (LD) o Type of oxidoreductase o Catalyzes the interconversion of lactic and pyruvic acids ▪ Also catalyze NAD o Not tissue specific, widely distributed, highest activities in heart, hepatic, skeletal muscle and RBC o Reaction is reversible o Assay of Enzyme Activity ▪ Wacker method • Forward Reaction (Lactate → Pyruvate) • Increase in absorbance monitored at 340 nm • Colorless product • Optimal pH is 8.3 – 8.9 (basic) 6 ▪ Wrobleuski La Due • Reverse Reaction (Pyruvate → Lactate) • Decrease in absorbance monitored at 340 nm o Measures oxidized NAD • Colorless product • Three times faster but more susceptible to substrate exhaustion o Testing must be done immediately o Delayed testing will cause error; false ↓ • Optimal pH is 7.1 to 7.4 ▪ ▪ o o o α-hydroxybutyrate dehydrogenase (α-HBD) • Has greater affinity of H subunits of LDH • More H subunit has more binding of α-HBD • Represent LDH-1 After MI, LD begin to rise within 10-24 hours, peak at 48-72 hours, and remains elevated for 10 days. ▪ Last to rise ▪ LDH is not a preferred immediate marker for AMI in managing and treating emergency cases • After onset of AMI, it is important to immediately identify AMI ▪ LDH is used for monitoring patient response to treatment due to prolonged elevation Reference Range: 100-225 U/L Lactate Dehydrogenase (LDH) Isoenzyme ▪ Tetramer (4) containing two active subunits • H - heart • M - muscle Isoenzyme Tissue Disorder (↑) LDH-1 (HHHH) Heart, RBC MI, Hemolytic anemia LDH-2 (HHHM) Heart, RBC RI, Megaloblastic anemia LDH-3 (HHMM) Lung, Spleen, Pulmonary Pancreas embolism LDH-4 (HMMM) Liver Hepatic injury LDH-5 (MMMM) Skeletal Skeletal muscle Muscle injury ▪ LDH-1 - most number of H subunit ▪ RI - renal infarction ▪ Hemolytic dis.- CK-MB normal, LDH-1 abnormal ▪ LDH-4 - liver specific, used for liver disorders ▪ Basis of nomenclature is electrophoretic migration • LDH1 - faster to migrate, closest to anode • LDH5 - slowest to migrate, farthest ▪ Relative concentration in normal serum • LDH-2 > LDH-1 > LDH-3 > LDH-4 > LDH-5 ▪ In AMI & intravascular hemolysis, LDH1 & LDH2 demonstrate flipped pattern (LDH1 > LDH2) ▪ Significance of Isoenzyme fraction o Isoenzyme activity measurement is more diagnostic that total enzyme activity due to increased sensitivity; more specific tissue is targeted for testing Correspondence between CPK & Isoenzyme Patterns ▪ • Sample 1: AMI • CK-BB - normal • CK-MB - abnormally high • CK-MM - abnormally low • LDH-1 - abnormally high • LDH-2 - LDH-5 - normal ▪ Sample 3: • CK-MM - normal Differentiation of Cardiac Markers CK-MB AST LDH Appearance 4-8 hours 6-8 hours 10-24 hours Peak 12-24 hours 24 hours 48-72 hours Stay Elevated 3 days 5 days 10 days o CK-MB - preferred for emergency AMI cases OTHER LIVER ENZYMES • Alkaline Phosphatase (ALP) o Aka alkaline orthophosporic monoester phosphohydrolase o Catalyze the hydrolysis of various phosphomonoesters at alkaline pH (9.0 - 10.0) o Liberate inorganic phosphate an organic phosphate ester with production of alcohol o Requires Mg2+ activator to increase binding an from 7 o o • Not specific, widely distributed, major tissue sources include liver, bones, placenta, intestine, kidneys ▪ Osteoblast - specific source in bone ▪ In healthy individuals, the predominant ALP is derived from liver & bones For evaluation of hepatobiliary and bone disorders. ▪ o • o o o Assay for Enzyme Activity ▪ Bowers and McComb • Based on molar absorptivity of p-Nitrophenol • Absorbance is measured at 405 nm o Colored reaction • pH 10.2 Other methods Methods (1-4) Bodansky, Shinowara, Jones, Reinhart (5) Bessy, Lowry & Brock (6) Bowers & McComb (7) King and Armstrong Substrate β-glycerophosphate End Product Inorganic PO4 + Glycerol pnitrophenyl phosphate Phenyl phosphate p-nitrophenol (yellow) ▪ ▪ phenol Reference Range ▪ 30 – 90 U/L (Adult) ▪ 70 – 220 U/L (0 – 3 months) ▪ 50 – 260 U/L (3 - 10 years) ▪ 60 – 295 U/L (10 - puberty) • ALP found in osteoblasts in bones • Osteoblasts - bone forming cell • During puberty, there is a surge in hormone release including growth hormone causing bone development that increases osteoblast activity leading to ↑ release of ALP in blood causing high blood ALP among growing ind. • Most specific pancreatic marker ▪ ALP Isoenzymes ▪ Differentiation by electrophoresis and other methods (chemical inhibition and heating) • In electrophoresis, isoenzymes are ▪ Major isoenzymes • Liver • Bone • Placental • Intestinal ▪ Liver ALP: fastest to migrate, most anodal • ↑ in liver disease • Two fractions: o Major liver - contribute to abundance of ALP in blood o Fast liver (α1 band) - responsible for fast migration ▪ Bone ALP: second fastest to migrate • Heat labile fraction (increased temperature (56C) decreases bone activity) • Derived from osteoblast • Elevation may be attributed to pathologic or physiologic condition ▪ ▪ Increase in bone disease (Paget’s), healing of bone fractures and physiologic bone growth o Paget’s disease - osteitis deformans; bone disorder; ↑ ALP; increased bone resorption • Increased during bone growth (puberty) Placental ALP: 3rd to migrate • Increased in pregnancy (16-20th wk gestation) • Most heat stable fraction - activity is measurable in high temperatures • Associated with malignancy/cancer (Carcinoplacental ALP) Intestinal ALP: slowest moving fraction, least anodic • Increased in blood groups B or O • Increased in fatty meal consumption (physiologic) and GIT disorders (pathologic) Carcinoplacental ALP • Variants of placental ALP associated with cancers/malignancies • Regan ALP: lung, breast, and gynecological cancers, bone ALP co-migrator, most heat stable ALP variant of placental ALP o Remains stable even after heating serum sample at 65C for 30 mins • Nagao ALP: adenocarcinoma of the pancreas and bile duct, pleural cancer • Inhibitors: phenylalanine, L-leucine o Phenylalanine can inhibit Regan ALP o Phenylalanine & L-leucine can inhibit the Nagao ALP Chemical inhibition • Placental & intestinal ALP are inhibited by phenylalanine (chemical inhibition) • 3M urea reagent - chemical inhibitor for bone ALP • Levamisole - inhibitor for bone & liver ALP • Enzyme activity = enzyme concentration • Addition of chemical inhibitor suppresses activity of other enzymes to measure specific isoenzymes • Chemical inhibitor - suppresses enzyme activity Differentiation by Heat Stability • Differ liver & bone ALP as cause of ALP increase • Total ALP elevations by liver & bone ALP is differentiated by heating serum at 56C for 10 mins • Liver ALP - ALP residual activity is ↓ to >20% • Bone ALP - ALP residual activity is ↓ to <20% • Order of ALP isoenzyme o Placenta → Intestinal → Liver → Bone • Liver ALP is present, 8 • Acid Phosphatase (ACP) o Aka acid orthophosporic monoester phosphohydrolase o Catalyze the hydrolysis of various phosphomonoesters at an acid pH o Liberate inorganic phosphate from an organic phosphate ester with production of alcohol o Non-specific, distributed in prostate gland (major source), RBCs, platelets, bones (osteoclasts) o For evaluation of metastatic carcinoma of prostate (tumor marker for prostate gland) ▪ When prostate-specific antigen (PSA) was discovered, it replaced ACP for prostate cancer marker due to increased specificity of PSA o Forensic investigation of rape ▪ ACP can be found in seminal fluid ▪ Vaginal washings - specimen for investigation • Tested for prostatic ACP activity ▪ Positive prostatic ACP = seminal fluid presence ▪ Presence of prostatic ACP only persist for 4 days o o To differentiate prostatic form from the non-specific form like RBC acid Phosphatase inhibitors are added ▪ L-tartrate ions – inhibits specific prostatic ACP ▪ Formaldehyde & Cupric ions – inhibit RBC ACP Assay for Enzyme Activity ▪ Reference Range: Prostatic ACP: 0 -3.5 ng/ml Methods (1) Quantitative end point o Substrate Thymolphthalein monophosphate α-naphthyl phosphate (2) Continuous monitoring Assay for Enzyme Activity Methods Substrate End Product Gutman And Phenyl PO4 Inorganic Gutman Phosphate Shinowara PNPP P-Nitrophenol Babson, Read Alpha Naphtyl Alpha Naphtol and Phillips PO4 Roy And Thymolphthalein Free Hillman MonoPO4 Thymolphthalein ▪ PNPP - para nitrophenylphosphate ▪ o AMS - nonspecific for acute pancreatitis; AMS may increase in other conditions Considered as smallest enzyme present in the plasma ▪ AMS is normally filtered by renal glomerulus ▪ AMS is normally found in urine ▪ Increased plasma AMS increases AMS in urine ▪ Urine AMS remains elevated for 7 days among patients with acute pancreatitis ▪ In acute pancreatitis, AMS - high in urine & plasma ▪ In renal disease, urine AMS decreases as most AMS are reabsorbed w/c increases plasma AMS o Amylase Isoenzymes ▪ Salivary AMS (S type) → ptyalin (fast moving) ▪ Pancreatic AMS (P type) → amylopsin (slow moving) o Amylase Methodologies/Assay for Enzyme Activity ▪ Amyloclastic • Measures the disappearance of starch substrate • Starch-Iodine Complex (Dark-blue) → decrease in color intensity • Only complex sugars react w/ iodine • AMS is inversely proportional to absorbance • If starch is not available, use glycogen o Glycogen + iodine → mahogany brown ▪ Saccharogenic • Measures the appearance of the product • Starch → reducing sugars • Product is directly proportional to the activity of amylase to the polysaccharide substrate • AMS is directly proportional to absorbance ▪ PANCREATIC ENZYMES • Amylase (AMS) o Aka alpha 1-4-glucan-4-glucohydrolase o A hydrolase that catalyzes the breakdown of starch and glycogen via α, 1-6 branching linkages o A digestive enzymes important for digestion & absorption of polysaccharides o Tissue sources: ▪ Major: pancreas, salivary glands (parotid gland) ▪ Minor: adipose tissues, small intestines, skeletal muscles, fallopian tubes o Increased in acute pancreatitis, renal failure and parotitis (inflammation of parotid gland) o Acute pancreatitis ▪ From alcoholism, flood intake ▪ Manifests during deep sleep through nightmarelike sounds (indication of extreme pain) • Pain due to ongoing pancreatitis ▪ AMS - fastest & first to elevate, 2-12 hrs rise after onset, peaks at 24 hrs, normalizes within 3-5 days ▪ Chromogenic • Measures the increasing color from production of product - chromogenic dye fragment • Insoluble starch-dye → soluble starch-dye fragments • AMS is directly proportional to absorbance Continuous monitoring - Coupling of several enzyme systems to monitor amylase activity • Increase in absorbance of reduced NAD at 340nm is measured • Secondary enzymes: α-glucosidase, hexokinase, G-6-PD • Oxidoreductase is always in final reaction conversion of coenzymes 9 • Lipase (LPS) o Aka triacylglycerol acylhydrolase o Hydrolyzes the ester linkages of fats to produce alcohols and fatty acids o Hydrolysis of dietary triglycerides in the intestine to 2monoglyceride and fatty acids ▪ Involved in fat digestion & absorption ▪ Converts triglycerides into soluble monoglycerides to be absorbed in small intestine o Tissue source: pancreas o Early and specific marker for acute pancreatitis ▪ Most specific pancreatic marker due to pancreas as major tissue source ▪ For acute pancreatitis, LPS level rise within 6 hours after onset, peaks at 24 hours, and remains elevated for 7 days/1 week • AMY - first and fastest to elevate (2-12 hrs) • LPS - more specific • Acute pancreatitis can be fatal o Larger molecule and remains longer in circulation (7d) ▪ Size of LPS make it unfilterable through glomerulus which allow it to remain longer o Plasma levels are not affected by salivary gland disorders or renal disorders o Assay for Enzyme Activity ▪ Presence of hemoglobin in sample (plasma/serum) could affect lipase activity • Hgb can inhibit enzymatic activity of LPS to a substrate • Hemolysis can cause falsely decreased LPS o Outpatients should stay until centrifugation of sample to ensure immediate recollection in case of hemolysis Cherry Crandall Tietz Substrate 50% olive oil (triolein) Titrating agent 0.4N NaOH Indicator Phenolphthalein Thymolphthalein + veronal Endpoint Fatty acid (oleic acid) End color Pink blue ▪ Cherry Crandall - reference method for LPS • 50% oil = 50% water o Water promotes catalytic action • Triolein - more pure form of triglyceride • Titrating agent - allow fatty acid release • Higher LPS activity = Higher FA liberated ▪ Turbidimetric methods • Estimation of liberated fatty acids • • Turbidimetry - measurement of light absorbed by the suspension/solution o Insoluble molecules present in suspension would block the light o Bigger greater conc of insoluble molecules = higher absorbance o More turbid = more light blocked Substrate triglyceride - insoluble to water o Allows turbid solution o With lipase, it produces soluble products (fatty acids/monoglyceride) which decreases the turbidity o Decreased turbidity decreases abs • Activity of lipase to triacylglycerol is inversely proportional to the absorbance reading o High LPS = low turbidity = low abs MISCELLANEOUS ENZYMES • • • • • • 5’ Nucleotidase (5’N) o A phosphoric monoester hydrolase o Predominantly secreted in the liver ▪ Can be used as a liver enzyme o Marker for hepatobiliary disease and infiltrative lesions of the liver o Reference value: 0 - 1.6 units Cholinesterase/pseudocholinesterase o Index of parenchymal function o Secreted by the liver o Used to monitor the effect of muscle relaxants (succinylcholine) after surgery o Used as a marker for insecticide/pesticide poisoning ▪ Measures organophosphate ▪ Organophosphate - component of insecticide is that is poisonous that may cause adverse reaction and eventual death o Reference values: 0.5-1.3 units (plasma) Angiotensin-Converting Enzyme (ACE) o Aka peptidyldipeptidase A or kininase II o Responsible for converting Angiotensin I to Angiotensin II within the lungs ▪ Angiotensin I - inactive form ▪ Angiotensin II - active form that is responsible for the regulation of sodium by aldosterone, for the constriction of blood vessels, and water retention o Increased in: sarcoidosis, acute and chronic bronchitis, and leprosy o Tissue source: macrophage and epithelioid cells of the lungs o A possible indicator of neuronal disfunction (dementia, Alzheimer’s) Ceruloplasmin o Copper-carrying protein which acts as an enzyme o Act as an enzyme to catalyze o Marker for Wilson’s disease (hepatolenticular disease) Ornithine Carbamoyl Transferase (OCT) o Used for hepatobiliary diseases o Reference values: 8-20 mU/mL Glucose-6-Phosphtae Dehydrogenase (G-6-PD) o Functions to maintain NADPH in the reduced form in RBCs o Reduced NADP is important to stabilize RBC membrane for resistance of cell dame/destruction caused by harmful agents/drugs and protection against lysis o A newborn screening marker ▪ Newborn screening - screens/detects congenital disorders for early disease treatment/ management and prevention of disease triggers o Found in adrenal cortex, spleen, RBC, & lymph nodes o Deficiency can lead to drug-induced hemolytic anemia (primaquine, antimalarial drug) ▪ Premature RBC lysis occurs in the blood vessels o Increased levels: AMI, megaloblastic anemia o Specimen: RBC hemolysate, serum o Reference values: 10-15 U/g Hgb or 1200-2000 mU/mL packed RBC 10