Clinical Chemistry 2 Lecture Notes PDF

CLINICAL CHEMISTRY 2 LECTURE/TRINIDAD/LCB Section 1: INTRODUCTION TO ENZYMES ENZYMES SECONDARY STRUCTURE Theses are biological molecules (proteins) produced by living cel...

CLINICAL CHEMISTRY 2 LECTURE/TRINIDAD/LCB Section 1: INTRODUCTION TO ENZYMES ENZYMES SECONDARY STRUCTURE Theses are biological molecules (proteins) produced by living cells that hastens up chemical reactions in organic matter Conformation of the amphoteric - they’re able to react both as an acid or base segments of polypeptide Easily denatured with varying MW and mass chain If the MW is small, it is faster to denature Made up either helical or Synthesize in active state and operated in the presence of a beta pleated sheets cofactor maintained by hydrogen It helps in the proper digestion and metabolism bond These are found in all body tissues It has a twisting chain They appear in serum following cellular injury or structure (helical) They may come from degraded cell Changes of the concentration of enzymes may reflect on changes on health For example, large amounts of enzymes in serum, are clinically use as evidence of an organ damage TERTIARY STRUCTURE The catalyzed reactions are frequently specific and essential to physiologic functions: Arises from the interactions Hydration of C02 among side chains/groups of Nerve conduction the polypeptide chain Muscle contraction Structure are bent and folded Nutrient degradation and maintained by covalent Energy use disulfide bond TERMINOLOGIES a. Coenzymes - non-protein organic biochemical that takes part in the enzyme reaction Essential to the catalytic activity as a CO-SUBSTRATE Diffusible, heat stable, low molecular weight that when combined tightly to enzymes, the coenzyme will be called QUATERNARY STRUCTURE Prosthetic group E.g. NAD, Pyridoxal phosphate Separate bended & folded b. Activators - Inorganic ionic cofactor structures are put together to increase the catalytic activity of an enzyme when it binds to form a functional unit specific site There are more than one (1) Metabolic regulator of enzyme reaction polypeptide chain Usually metal ions (esp. divalent cations) E.g. Mg++, Na+, K+, Zn++ c. Holoenzyme - the combined enzyme & coenzyme d. Apoenzyme - enzyme with a cofactor e. Prosthetic group - A coenzyme that cannot be removed from its attachment to an enzyme using dialysis E.g. Pyridoxal phosphate in transaminase reaction f. Substrate - Substance acted upon by an enzyme & is converted into a new substance ENZYME COMPONENTS g. Product - Substance derived from a transformed substrate h. Active site – Site where substrate interacts with enzymes i. Allosteric site – Site other than the active site that may lead to either attachment of substrate to the enzyme’s active site or inhibition of attachment j. Isoenzymes – different form of an enzyme with different genetic origins but catalyze the same reaction k. Isoforms – Results when an enzyme is subject to different ACTIVE SITE post-transitional modification Often a water-free cavity, the substance on which the enzyme ENZYME STRUCTURE acts (the substrate) interacts with particular charged amino acid PRIMARY STRUCTURE residues. ALLOSTERIC SITE Refers to the sequence of amino acids joined by A cavity other than the active site peptide bonds to form a It may also bind regulator molecules polypeptide chain THEORIES RELATED TO E-S COMBINATION Each amino acid is joined by Lock and Key Theory a covalent peptide bond, First presented by Emil created in a condensation Fisher reaction The active site being Simplest enzyme structure complementary in shape & size to the substrate Induced Fit Model / Kochland’s Theory Also when performing enzymatic determination, it is needed The enzyme changes in that the substrate concentration is higher that is to promote shape during binding to saturation accommodate the substrate All reactions must be in the Zero-Order Kinetics that is to Considered the more correct permit the most accurate enzyme requirement. version Michaelis-Menten Curve FACTORS AFFECTING ENZYMATIC REACTIONS a. Time If the catalytic activity of an enzyme on a substrate is fast, this will mean a shorter reaction time thus liberating the enzyme to act again on the remaining substrate The rate of enzymatic reaction b. Molecular Compatibility The commonness between the enzyme and substrate Depends on the spade of either enzyme or substrate c. Space availability Km represents the substrate concentration in moles/L when the Number of substrate that can be reacted initial velocity is ½ Vmax If there is more substrate and there is least enzyme, the Show the relationship of the reaction velocity to the substrate enzymatic reaction will be longer concentration d. Specificity Equation: Capability of enzyme to recognize and bind only one or few molecules Absolute specificity - when an enzyme can act and catalyze one unique reaction Group specificity - when some enzymes act on different substrates belonging to the same group Not usually use because it is difficult to plot (hyperbolic curve) Stereoisomeric - an enzyme acts only on the specific Lineweaver-Burk Equation isomer Example: D and L isomers of glucose e. Substrate concentration Michaelis and Menten: they hypothesized the role of substrate concentration in the formation of the ES complex The rate of enzymatic reaction increases as the concentration of substrate increase Takes the reciprocal form of the Michaelis-Menten equation to produce a straight line equation The reciprocal is taken of both the substrate concentration and the velocity of an enzymatic reaction The maximum velocity is considered to be the plateau site because it means that it already reach the maximum concentration (it already formed a product) It is said to be at this level, the reaction is already f. Temperature saturated Optimum temperature - °T considered favorable for enzyme The curved we call it this reaction follows the First Order activity (30-37°C or 37 – 40°C) Kinetics Increasing the temperature usually increases the rate of FIRST ORDER KINETICS chemical reaction because there is an intermolecular The reaction rate is directly proportional to substrate collision that occurs in the reaction concentration Q10 value – reaction rate is doubled for every 10°C increase This happens during the first phase of the curve The rate of denaturation increases significantly when it ZERO-ORDER KINETICS reaches 40-50°C Second phase or the plateau level 50 – 60°C : enzyme undergoes inactivation and denaturation The reaction rate is independent of substrate g. pH - hydrogen ion concentration concentration Optimum pH - the point at w/c the reaction rate is greatest Take Note: By using an appropriate buffer solution When performing enzyme analysis in the laboratory, it is At pH 7.0 – 8.0, many enzymes show maximum activity therefore essential that the substrate be high enough to pH value are seen as low as 1.5 and as high as 10.5 promote saturation. Optimal pH may be different in forward and reverse reaction  Maintaining pH is important: it affects the three dimensional conformation of the enzyme h. Cofactors Non-protein entities that must bind to particular enzymes before a reaction occurs Example: a. Coenzymes an organic compound (second substrate) Essential to achieve absolute enzymatic activity example : NAD and NADP METHODS OF ENZYME ASSAY When bound tightly to the enzyme, the enzyme is FIXED TIME ASSAYS called a prosthetic group The reactants are combined b. Activators Reaction proceeds for a designated time & is stopped (by Inorganic cofactors, alters the spatial inactivating the enzyme) configuration of the enzyme for proper substrate Reaction is stopped by a weak acid, then measurement is made binding of the amount of reaction that has occurred Examples: Ca, Zn, Cl, Mg, K The reaction is assumed to be linear over the reaction time, the c. Metalloenzymes larger the reaction, the more enzyme is present Inorganic ion attached to a molecule CONTINUOUS MONITORING OR KINETIC ASSAY Example: catalase and cytochrome oxidase i. Enzyme concentration Multiple measurements are made at specific time intervals or by a continuous-recording spectrophotometer The higher the enzyme level, the faster the reaction will proceed Advantage: Linearity of the reaction is adequately verified because more enzyme is present to bind with substrate This is preferred because any deviation in linearity is readily j. Inhibitors observable enzymatic reactions may not progress if an inhibitor interferes UNIT OF MEASUREMENT with the reaction. IU - International Unit Competitive inhibition - inhibitors Binds to the active site, Proposed by the Commission on Enzymes (IUB) blocks access of the S to the E Used to standardize the system or reporting of quantitative Both the substrate and the inhibitor compete for the results same active site of the enzyme IU is the amount of enzyme that will catalyze the reaction of 1 The inhibition is reversible when the substrate µmol of substrate per minute under specified conditions of concentration is significantly higher than the temperature, pH, substrate and activators. concentration of inhibitor Expressed in terms of U/L or mU/L Katal Unit of enzyme activity w/c converts 1 mol of substrate per second Conforms w/ the Systemè International (SI) scheme of units Mole is the unit for substrate concentration while the unit of time is second Enzyme concentration is then expressed as katals per liter (kat/L) FACTOR THAT INFLUENCE RATE OF ENTRY Leakage of enzyme from cells Impaired energy production: promote deterioration of cell Non-competitive inhibition membrane It does not compete with the substrate but looks for Direct attack on the cell membranes (viruses or organic areas other than the active site chemicals) Because the inhibitor binds the enzyme independently Reduction in the supply of oxygenated blood perfusing any from the substrate, increasing substrate concentration tissue (e.g. MI) does not reverse the reaction. Altered enzyme production decrease The presence of the inhibitor when it is bound to the enzyme, slows the rate of the reaction Genetic deficiency of enzyme production Enzyme production is depressed as a result of disease CLEARANCE OF ENZYMES Urinary excretion NOT a major route for elimination Except: Amylase = ↑ blood levels (e.g. Acute pancreatitis) Enzyme inactivation in Plasma Inactivated enzymes are removed by the RES Half – life in plasma: 24 – 48 hours Uncompetitive Inhibition It occurs when an inhibitor binds to the ES complex to form an enzyme-substrate inhibitor complex that does not yield a product CLINICAL CHEMISTRY 2 LECTURE/TRINIDAD/LCB Section 2: ENZYME NOMENCLATURE AND CLASSIFICATION CLASSIFICATION OF ENZYMES - Based on the catalytic activity of an enzyme CLASS FUNCTION EXAMPLE 1. OXIDOREDUCTASES Catalyze the removal or addition of LDH, MDH, ICD, G-6-PD (DEHYDROGENASES/ OXIDASES electrons (redox reaction) A- + B ➝ A + B- Note: A is reductant and electron donor and B is oxidant and electron recipient For investigation of cardiac and liver disorders 2. TRANSFERASES Catalyze the transfer of a chemical group CK, AST, ALT other than hydrogen from one substrate another A + BX = AX + B Note: X is represents a functional group Gives information about liver damage 3. HYDROLASES - presence of H2O Catalyze hydrolysis or splitting of a bond by Three (3) Groups: the addition of water A-B + H2O ➝ A-OH + Esterases (ACP, ALP, Lipase) B-H Peptidase (Trypsin, Pepsin, Leucine, aminopeptidase) Glycosidase (AMS, galactosidases) 4. LYASES - w/o the presence of H2O Catalyze removal of groups from Glutamate decarboxylase, pyruvate, substrates without hydrolysis. The product decarboxylase, aldolase contains double bond A = B + C Assayed in disorders of skeletal muscles 5. ISOMERASES Responsible for the conversion of one Glucose phosphate isomerase, ribose isomer to another A > B phosphate isomerase All reactions are reversible 6. LIGASES Enzymes causing bond formation between Synthase two molecules to form a larger molecule A + B = AB ENZYME NOMENCLATURE The Enzyme Commission adopted a classification in 1961 AMS: Pancreas CK: Heart, Enzymes are classified according to the type of reaction they & Salivary Skeletal muscle catalyze Glands - & Brain - In naming an enzyme, the suffix “ase” is added to the name of the Lipase: ALP: Liver, substrate Pancreas Bone, Kidney & Systemic Name Pancreas Describes the nature of the reaction catalyzed ENZYME ORGAN ASSOCIATION Numerical code designation prefixed w/ letters E.C Example: E.C. 3.1.3.1 = ALP and E.C. 3.1.3.2 = ACP ORGAN/TISSUE ENZYMES 1st digit = denotes the class of the enzyme 2nd digit = subclass of the enzyme Prostrate ACP 3rd digit = sub sub-class 4th digit = specific serial number Brain CK-BB Pancreas Amylase and Lipase Bone ALP Skeletal muscle CK-MM, AST, LD & Aldolase Trivial Name Liver Hepatocellular disorder: AST, ALT, & LD5 Aka Non-specific, Practical name, Working name Biliary tract obstruction: ALP & GGT Examples: SGOT, SGPT TISSUE SPECIFICITY OF ENZYMES Heart CK-MB, AST & LD1>LD2 (Flipped) HIGH SPECIFICITY MODERATE LOW SPECIFICITY SPECIFICITY ACP: RBC & AST: Liver, LDH: All tissues Prostate Heart & Skeletal ALT: Liver - muscle - CLINICAL CHEMISTRY 2 LECTURE/TRINIDAD/LCB Section 3: ENZYMES OF CLINICAL IMPORTANCE AMINOTRANSFERASE Normally 1.0 but 2.0: associated with alcoholic hepatitis or hepatocellular Pyridoxal phosphate as obligate coenzyme - carcinoma Has two (2) isoenzyme fractions: Acute hepatocellular injury: AST>ALT initially: w/in 24-48 hrs, Cytoplasmic isoenzyme (more predominant) and ALT> AST Mitochondrial isoenzyme Higher AST activity in hepatocytes ASPARTATE AMINOTRANSFERASE (AST); E.C. 2.6.1.1 ALT has longer half-life in serum (16 & 24 hours, Formerly SGOT (serum glutamic-oxaloacetic transaminase) respectfully) Involved in the transfer of an amino group between aspartate and METHODS OF MEASUREMENT a-keto acids Continuous - Monitoring Method Reaction catalyzed: Transaminase reactions coupled to specific DH reactions Aspartate + a – ketoglutarate ⟷ Oxaloacetate + Glutamate Allows multiple measurements of the same sample TISSUE SOURCES The oxo-acids formed in the reaction are measured indirectly by Widely distributed in human tissue enzymatic reduction to the corresponding hydroxyl acids Highest concentration (￪): cardiac tissue, liver & skeletal muscle The accompanying change in NADH concentration is being Smaller amounts (￬): kidney, pancreas & RBCs monitored spectrophotometrically DIAGNOSTIC SIGNIFICANCE Colorimetric Method Evaluation of hepatocellular disorders & skeletal muscle involvement Coupling w/ 2,4- DNPH (Reitman-Frankel) Liver disease-most important cause of elevated transaminase Still feasible activity in serum Phenylhydrazones of oxaloacetate & pyruvate are more In most liver disease, ALT is higher than AST (Except: chromogenic Alcoholic hepatitis, Hepatic Cirrhosis and liver neoplasia) Simple; limited but acceptable accuracy Mild degree of liver tissue injury: cytoplasmic isoenzyme is Derivative formed will give a strong blue color measured as 505 nm predominant ASSAY FOR AST ACTIVITY Severe tissue damage: release of mitochondrial isoenzyme shows marked increase in patients with extensive liver cell degeneration and damage It is also use for monitoring therapy with potentially hepatotoxic drugs AST ELEVATIONS Pulmonary embolism Following congestive heart failure Viral hepatitis: up to 100x ULN KARMEN METHOD Cirrhosis: ⁓ 4x ULN Coupled enzymatic reaction w/ MDH (indicator reaction) Skeletal muscle disorders: Muscular dystrophies & inflammatory Monitors change in absorbance at 340 nm (NADH to NAD) conditions ⁓ 4 – 8x ULN Optimal pH: 7.3 – 7.8 In AMI (Acute Myocardial Infarction): AST levels begin to rise w/in 6 – Sources of Errors: 8 hrs., peak at 24 hrs. & return to normal w/in 5 days Hemolysis: increase serum AST AST levels are NOT useful in the diagnosis of AMI AST activity in RBC is 15x higher than in serum REFERENCE VALUE: 5-37 U/L AST activity is stable in serum for 3 – 4 days at ref °T ALANINE AMINOTRANSFERASE (ALT); E.C. 2.6.1.2 Reference Range: 5 – 30 U/L (37°C) Formerly SGPT (serum glutamic pyruvate transaminase) Sample in AST should be hemolysis free because if the AST sample Catalyze the transfer of an amino group from alanine to is hemolyzed, it will increase the result 10x a-ketoglutarate with the formation of glutamate and pyruvate ASSAY FOR ALT ACTIVITY Reaction catalyzed: Pyruvate formed is converted to lactate by LDH Alanine + a-ketoglutarate ⟷ Pyruvate + glutamate LDH as the indicator enzyme TISSUE SOURCES NADH formed is oxidized to NAD Distributed in many tissues The accompanying change in NADH concentration is being Highly concentrations in the liver (more liver-specific enzyme of monitored spectrophotometrically the transferases) the disappearance of NADH is followed by measuring the decrease Low level in the heart and skeletal muscle in absorbance Isoenzyme: exclusively cytoplasmic form Change in absorbance is proportional to the micromoles of NADH RBC contains 5-8x as much ALT activity as does the serum oxidized that reflects the number of substrate transformed DIAGNOSTIC SIGNIFICANCE Evaluation of hepatic disorders (hepatocellular) Progressive inflammatory liver conditions : higher ALT elevations than AST Higher elevations are found in hepatocellular disorders than extrahepatic ot intrahepatic obstructive disorders ALT also monitor the course of liver-like hepatitis treatment and the effects of drug therapy More sensitive and specific for the screening of post-transfusion Sources of Errors: hepatitis or even occupational exposure compared to AST Relatively unaffected by hemolysis (ALT activity in RBCs is 7x higher De Ritis Ratio (AST:ALT ratio) than in normal serum) A diagnostic marker of alcoholic liver disease Stable for 3 – 4 days at 4°C Normal ratio between AST and ALT Reference Range: 6 – 37 U/L (37°C) Implication: most causes of liver cell injury, associated w/ greater ALT than AST, however, there are cases where in AST to ALT ratio is 2:1 or greater CLINICAL CHEMISTRY 2 LECTURE/TRINIDAD/LCB CREATINE KINASE CREATINE KINASE (CK) (E.C. 2.7.3.2) STRUCTURE Mw: 82,000 Da Dimer w/ 2 sub-units: B (brain) & M (muscle) Associated with ATP regeneration in contractile or transport systems Each sub-unit w/ a MW of ~ 40,000 ATP is utilized in energy consumption Three (3) isoenzymes: found in the cell cytosol Im muscle cells: involved in the storage of high energy creatine 1. CK-BB (brain type) CK1 phosphate Migrates fastest to the positively charge sides of Its dominant physiologic function is in muscle cells where it is electrophoresis (most anodic) involved in storage of high-energy creatine phosphate Locations: Brain (most abundant CK), prostate, gut, lung, (phosphocreatine - major phosphorylated compound in muscle) bladder, placenta, & thyroid This rxn is reversible: 2. CK-MB (hybrid type) CK2 Present in varying degrees in heart muscle (25 – 46% of CK activity) and minor degree in skeletal muscle 3. CK-MM (muscle type) CK3 4th most anodic Predominates in skeletal & cardiac muscle (normal serum: consist approx. 94-100% CK-MM) ATYPICAL FORMS OF CK When muscle contracts, ATP is consumed (to form ADP) & CK catalyzes CK-Mt: the rephosphorylation of ADP to form ATP, using Phosphocreatine 4th isoenzyme Found in different tissues or organs (heart, brain, skeletal muscles) Migrates cathodic of CK-MM Increased amounts of creatine kinase when they’re release into the Located b/w the inner & outer membranes of the mitochondria blood due to muscle damage Differs immunologically & in electrophoretic mobility When determining CK levels, it indicates that there is a muscle that is Constitutes up to 15% CK activity in the heart being destroyed by some abnormal process Its presence does not correlate with any specific disease however it Abnormal processes: muscle dystrophy or inflammation correlates with severe illness and cases of malignant tumor and cardiac abnormality MACROMOLECULAR FORM OF CK MACRO-CK TYPE 1 CK1 associated with IgG or CK3 w/ IgA Serum: 0.8-1.6% (not considered as clinically significant because it is found only in small amount) MACRO-CK TYPE 2 Oligomeric CK-Mt MITOCHONDRIAL (CK-Mi) This shows how CK is utilized Bound to the exterior surface of the inner mitochondrial membranes TISSUE SOURCE: of muscle, brain, and liver Greatest in skeletal muscle, brain tissue, & heart muscle Occurs in both dimeric state and oligomeric aggregates Smaller quantities in bladder, placenta, GIT, thyroid, uterus, kidney, MW: 350,000 Da lung, prostate spleen, and pancreas Incidence: 0.8-1.7% No activities in the liver and RBCs Clinical significance: malignant tumor & cardiac abnormalities Migrates fastest to the cathode DIAGNOSTIC SIGNIFICANCE: Pronounced Elevation (5 or more time normal) MIld or Moderate Elevations (2 to 4 times normal) Duchenne’s muscular dystrophy Severe exercise, trauma, surgical procedure Polymyositis Delirium tremens, alcoholic myopathy Dermatomyositis Pulmonary infarction Myocardial infarction hypothyroidism CK ISOENZYMES Tissue localization and sources of elevation ISOENZYME TISSUE CONDITION CK-MM Heart Myocardial infarction Skeletal Muscle Skeletal Muscle Disorder Muscular Dystrophy  Polymyositis Hypothyroidism Malignant hyperthermia Physical activity, IM injection CK-MB Heart Myocardial infarction, Myocardial injury Skeletal Ischemia, Angina Inflammatory Heart disease, Cardiac surgery Duchenne type muscular dystrophy Polymyositis, Malignant hyperthermia Reye’s Syndrome Rocky Mountain Spotted Fever, Carbon Monoxide poisoning CK-BB Brain CNS shock Bladder Anoxic encephalopathy Lung Cerebrovascular accident, seizure Prostate Placental or uterine trauma Uterus Carcinoma Colon Reye’s syndrome Stomach Carbon monoxide poisoning Thyroid Malignant hyperthermia Acute and chronic renal failure DISEASES OF THE HEART Normal Serum CK activity is seen in Neurogenic muscle disease CK level: sensitive indicator of AMI (Total CK & CK-2) CK-MM major CK in serum of healthy people where Skeletal muscle CK-MB levels begin to rise w/in 4 – 8 hrs. Peak at 12 – 24 hrs. & contains almost exclusively of CK-MM and heart muscle activity is return to normal levels w/in 48 – 72 hrs. attributed to CK-MM Elevation of Total CK: Cardiac trauma ff. heart surgery (including Injury on both heart and skeletal will mean elevation of CK-MM transplantation) CK ACTIVITY IN MALIGNANCY CK-MB activity has been observed in other cardiac conditions, not CKBB is rarely seen in serum because of its molecular size entirely specific for AMI although specificity can increase if tested in Extensive damage to the brain may lead to the leakage of it in the conjunction with LDH serum The time course of CK is unique in AMI CK-BB is associated also with patients with carcinoma of various DISEASE OF THE CNS organs such as adenocarcinoma, lung tumors, tumors of the CKBB isoenzyme will be elevated in conditions such as cerebral prostate, kidney breasts and ovary the CKBB can be a useful tumor ischemia and cerebrovascular ischemia where blood flow to the brain marker is insufficient METHODS OF DETERMINATION Elevations are also noted during head injury and acute TANZER-GILBARG ASSAY cerebrovascular disease It is a forward reaction with opt. pH of 9.0, It is also observe that the activity may increase in conditions that Read at 340 nm in spectrophotometer affects children such as Reye’s syndrome where the liver and the PEP (phosphophenol pyruvate) brain is swollen as a side effect of viral infections The change in absorbance is proportional to the activity of CK DISEASE OF THE THYROID Reaction: 60% of hypothyroid subjects Major isoenzyme is CK-3 Hypothyroidism results in CK-MM elevations because of the involvement of muscle tissue (increased membrane permeability), the effect of thyroid hormone on enzyme activity, and, possibly, the slower clearance of CK as a result of slower metabolism. DISEASE OF THE SKELETAL MUSCLE All types of muscular dystrophy, esp. Duchenne type sex-linked progressive muscular dystrophy) which may show up to 50x the ULN  In addition, the atypical forms of CK-Mi and macro-CK are not inhibited by anti-M antibodies and also may cause erroneous results for MB activity. REFERENCE RANGE TOTAL CK: Male : 15 – 160 U/L (37°C) Female : 15 – 130 U/L (37°C) OLIVER-ROSALKI METHOD: CK – MB : Utilizes the reverse reaction < 6% Total CK Most common performed method because it is about 6 times faster NOTES TO REMEMBER: than the forward reaction (Tanzer-Gilbarg) Hemolysis may elevate CK activity Measured: The rate of NADPH formation is a measure of the CK RBCs do not contain CK activity This is due to the Adenylate kinase that is present in the Red NADPH : the NADPH will the be measured in a spectrophotometer at Blood cells. This enzyme is the result of CK activity (Adenylate 340 nm Kinase effect) HK (hexokinase) Serum should be stored in a dark place because CK is inactivated by Reaction: light Because of the effect of muscular activity and muscle mass on CK levels, it should be noted that people who are physically well trained tend to have elevated baseline levels Patients who are bedridden for prolonged periods may have decreased CK activity CK levels are higher in men Men have a bigger muscle mass than females SEPARATION TECHNIQUES ELECTROPHORESIS Reference method and the most useful method Bands are visualized by incubating the support with a concentrated CK assay using the reverse reaction NADPH formed is observed with the bluish-white fluorescence after excitation by ultraviolet light Allows visualization of AK (Adenylate kinase) ION EXCHANGE CHROMATOGRAPHY Ion Exchange Chromatography Potential for being more sensitive & precise than electrophoresis On unsatisfactory column: CK-MM may merge into CK-MB CK-BB may be eluted w/ CK-MB Macro-CK may elute w/ CK-MB IMMUNOASSAYS Measure the conc. of Enzyme protein rather than Enzyme activity Detects enzymatically inactive CK-2 IMMUNOINHIBITION an anti-CK-M subunit antiserum is used to inhibit both M subunits of CK-MM and the single M subunit Original CK-MB and allows determination of the enzyme activity of the B subunit of CK-MB and the B subunit of CK-BB The residual activity after inhibition is multiplied by 2 to account for MB activity (50% inhibited). The major disadvantage of this method is that it detects BB activity, which, although not normally detectable, will cause falsely elevated MB results when BB is present. CLINICAL CHEMISTRY 2 LECTURE/TRINIDAD/LCB LACTATE DEHYDROGENASE LDH STRUCTURE E.C. 1.1.1.27 MW: 128,000 Da Chemical name: Lactate Dehydrogenase 5 isoenzyme fractions: each w/ 4 sub-units Catalyzes oxidation of Lactate to Pyruvate Two (2) different polypeptide chain: NAD - a hydrogen transfer enzyme that uses the coenzyme H (heart) and M (muscle) NAD as a hydrogen acceptor TISSUE SOURCES Reaction catalyze: Widely distributed in the body Which is not used as a diagnostic markers for disorders Heart, RBCs, kidney (LD1 and LD2) Lungs, pancreas, spleen (LD 3) Skeletal muscles, liver, intestine (LD 4 and LD5) LDH ISOENZYMES ISOENZYMES DISTRIBUTION IN LOCALIZATION ASSOCIATED CONDITIONS % LDH1 (HHHH) 14-26 Heart and RBC Myocardial infarct and hemolytic anemia The highest levels of LDH is seen on " pernicious Anemia " LDH2 (HHHM) 29-39 (highest) Heart and RBC Megaloblastic anemia, acute renal infarct, and hemolyzed sample LDH3 (HHMM) 20-26 Lungs, lymphocytes, spleen, and pancreas P. embolism, lymphocytosis and acute pancreatitis LDH4 (HMMM) 8-16 Liver Hepatic injury LDH5 (MMMM) 6-16 (lowest) Skeletal muscle Skeletal muscle injury Note: Moderately increased in acute viral hepatitis and cirrhosis Markedly increased in hepatic carcinoma and toxic hepatitis LDH6 (Alcohol Migrates cathodic LDH5 is elevated concurrently with the appearance Patients w/ arteriosclerotic CV failure dehydrogenase) to LDH5 of LDH6, this may be caused by hepatic congestion which signifies grave prognosis & due to cardiovascular disease.Therefore it is impending death suggested that LDH6 may reflect liver injury secondary to severe circulatory insufficiency Elevated in drug hepatotoxicity and obstructive jaundice LABORATORY DETERMINATION: Wacker Method ASSAY FOR LDH ACTIVITY Forward rxn Either a forward or reverse reaction has been used in clinical assays Use a pH of 8.8 (alkaline) Lactate is a more specific substrate compared to pyruvate Most commonly used method; produces NADH; not affected by SOURCES OF ERRORS product inhibition Hemolysis LD1 prefer this method/rxn LDH in RBCs is 100 – 150x than that in serum; any degree is Rxn catalyzed: unacceptable LDH is unstable in serum regardless of the temperature at which it is stored. If sample cant be processed immediately, store at 25oC within 24 hrs Since LDH 5 is the most labile isoenzyme, loss of activity occurs at Wrobleuski La Due / Wroblewski and LaDue 4oC Reverse rxn Most stable is LD1 Uses a lower pH of 7.2 (still alkaline) Reference range: 100-225 U/L About 2x faster than the forward reaction and is preferred method for Notes: dry slide technology In the sera of healthy individuals, the major isoenzyme fraction is - No wet reagents involved LDH2 followed by LDH1, LDH3, LDH4 and LDH5. Smaller specimen volume requirement LDH1 migrates fastest towards the anode followed in sequence by Cheaper in cost the other fractions with LDH5 migrating slowest LD5 prefer to be tested in this method/rxn LDH1 & LDH2 are present to the same extent in tissues however, Rxn catalyzed: cardiac tissue and RBC contains a higher conc. Of LDH1 and for this reason, in conditions involving cardiac necrosis (AMI) and intravascular hemolysis, LDH1 will increase to a point at w/c it is  presented in greater conc. Than LDH2 (LDH flipped Pattern) LDH1>LDH2 LD2 is always higher than LD1 LD2, LD3, & LD4 are considered as cancer markers. Predominantly with LD3 LDH6 is responsible for the metabolic conversion of methanol and ethylene glycol to toxic compounds LDH2 is the major isoenzyme found in the sera of healthy individuals LD can use other substrate in addition to lactate such as α-hydroxybutyrate LD activity in pleural fluids is useful for differentiating transudate from exudates Total LD increases temporarily after blood transfusion but returns to baseline within 24 hours. CLINICAL CHEMISTRY 2 LECTURE/TRINIDAD/LCB ACID PHOSPHATASE (ACP) ACP TISSUE SOURCES E.C. 3.1.3.2 Prostate ⬆source of ACP ACP belongs to the same group of phosphatase enzymes as ALP Bone A hydrolase Liver Major difference between ACP and ALP is the pH where ACP activity Spleen takes place at a pH of 5.0 Kidney Erythrocytes Platelets ISOENZYMES Band 1 Prostate ACP (inhibited by tartrate) Band 2 and 4 Granulocytes Band 3 - major form in plasma Platelets, RBCs, and Monocytes Band 5 Bone - osteoclasts (resistant to tartrate inhibition) ACP ELEVATION Medico-legal: PROSTATIC ISOENZYME Proved to be useful in forensic chemistry and suspected rape Prostatic Cancer: cases Particularly Metastatic Carcinoma of the prostate Vaginal washings are examined for seminal fluid where ACP ACP is inferior to PSA (Prostatic Specific Antigen) activity can be observed from the first 12 hours up to 4 days. ACP is an insensitive marker for prostatic cancer only when Elevations of ACP activity is presumptive evidence of rape in tumor has metastasize such cases Because PSA is more useful screening and diagnostic tool BONE ISOENZYME for prostate cancer Elevations of the bone isoenzyme is seen in active osteoclast-mediated bone resorption, Note: Gaucher’s cells and; After surgical treatment for prostate cancer. the levels of ACP falls Autosomal recessive inherited disorder of metabolism , where a faster than PSA. type of fat called glucose cerebroside , cannot be equally Plasma levels are undetectable following complete removal of degraded tumor Hairy cell leukemia Prostatic hyperplasia & prostatic infarction Elevations is also found in urinary tract obstruction, carcinoid tumors of rectum & prostatic massage, prostatic manipulations METHODS OF MEASURING ACP ACTIVITY METHODS SUBSTRATE END-PRODUCTS Gutman & Gutman Phenylphosphate Inorganic PO4 Shinowara PNPP (p-Nitrophenyl Phosphate) p-nitrophenol Babson, Read & Alpha-naphthyl PO4 Alpha-naphthol Phillips (the preferred substrate for continuous methods for the detection of ACP) Roy and Hillman Thymolphthalein monophosphate Free thymolphthalein (substrate of choice for quantitative end point reaction) Note: Assays for total ACP use the same techniques as in ALP assay but are performed in acidic pH Reaction products are colorless in reaction pH, and alkali solution is used to stop reaction and transform products into chromogens which is measured spectrophotometrically Chemical Inhibition Reaction is measured before addition of Tartrate (Total ACP) After addition: Residual activity Total ACP – Residual activity = Prostatic ACP Prostatic ACP Thymolphthalein monophosphate – preferred substrate for quantitative endpoint reactions (Modified by Roy) α-naphthyl phosphate – continuous monitoring methods (Hillman method) NOTES TO REMEMBER: Serum must be free from hemolysis If hemolysis happened, ACP in the RBCs increases Serum ACP decreases within 1-2 hours if left at room temperature If not assayed immediately; seum should be frozen or acidified to pH lower than 6.5 CLINICAL CHEMISTRY 2 LECTURE/TRINIDAD/LCB ALKALINE PHOSPHATASE (ALP) ALP Intestinal isoenzyme E.C. 3.1.3.1 CLINICAL SIGNIFICANCE Belongs to a group of enzymes that catalyzes the hydrolysis of ALP elevations (isoenzymes) is found in the following conditions: various phosphomonoesters in alkaline pH Liver: obstructive hepatic disorder Opt. pH for the reaction is 9 to 10 (alkaline) Bone: Paget’s disease (Osteitis deformans) and increased Considered a non-specific enzyme, because it ables to react with osteoblastic activity many different substrate Intestinal: DM, renal failure and cirrhosis Activator: Magnesium Placental: seen in pregnant women It liberates inorganic phosphate from an organic phosphate ester w/ Note: for ALP, it focuses in hepatobiliary and bone disorders the concomitant production of an alcohol ALP isoenzymes in cancer Rxn catalyzed: These are fractions associated with neoplasm They are referred to as carcinoplacental alkaline phosphatase because of their similarities to the placental Isoenzyme Regan isoenzyme - characterized as an example of an ectopic production of an enzyme by malignant tissue Heat stable Detected in lung, ovarian, color, and gynecologic cancer MAJOR ISOENZYMES Nagao isoenzyme - found in adenocarcinoma of the Liver isoenzyme pancreas and bile duct Came from the sinusoidal and canalicular membranes of the Variant of Regan isoenzyme liver Inhibited by L-leucine and phenylalanine Bone isoenzyme Kasahara isoenzyme - found in hepatoma Placental isoenzyme DIAGNOSTIC SIGNIFICANCE Pronounced Elevation Moderate Elevation Slight Elevation Biliary obstructions Granulomatous or infiltrative disease of the Viral Hepatitis Bile duct obstruction liver Cirrhosis Osteitis deformans (Paget’s disease) Infectious mononucleosis Healing fracture Hyperparathyroidism Metastatic tumor in bones MEASUREMENT OF ALP ACTIVITY BOWERS AND McCOMB METHOD (SZASZ MODIFICATION) Uses p-NPP substrate A colorless compound is hydrolyzed p-nitrophenol (yellow) and subsequent liberation of phosphate ions Kinetic or continuous monitoring method method (pH 10.2) which is based on the molar absorptivity of para-nitrophenol Chelator falsely lower activity Chelators are chemical compounds that react to metal ions to form a stable water complex Relatively stable at 4oC up to a week Most specific method recommended by IFCC (International Federation of Clinical Chemistry and Laboratory Medicine) Increase in Abs is directly proportional in ALP activity Read at 500 nm spectrophotometrically SEPARATION OF ISOENZYMES Rxn catalyzed: INHIBITION METHOD Phenylalanine - reduces activity of intestinal & placental isoenzymes Detects both liver and bone isoenzyme but cannot differentiate those isoenzymes Levamisole - inhibits bone & liver isoenzymes Detects placental and intestinal isoenzymes but cannot differentiate but cannot differentiate those isoenzymes 3M urea - inhibits bone ALP HEAT FRACTIONATION / STABILITY TEST Measurement before and after heating It shows stability of isoenzyme at 56oC  Placental isoenzyme is the most heal stable followed by ELECTROPHORESIS intestinal, liver then bone isoenzyme is the most heat labile Disadvantage: there is still overlapping of the liver and bone fraction isoenzymes. However this is considered as the most useful single Placental isoenzymes resist denaturation at 65oC for 30 minutes technique for ALP isoenzyme which makes it the most heat stable isoenzyme. Liver and bone ALPs are the most anodal isoenzymes; intestinal ALP Imprecise method is the least anodal Affected by different factors upon heat activation: Use of neuraminidase and wheat germ lectin improves separation of Temperature bone and liver ALP Time 2 fractions of liver isoenzymes: Analytical methods sensitive enough to detect small 1. Major liver band - increased in hepatobiliary conditions amounts of residual ALP activity 2. Fast liver (𝛂-1) - increased in metastatic carcinoma of the liver Performed at 56oC for 10-15 minutes Makes use of agarose gel, polyacrylamide gel and cellulose acetate Decreasing order of ALP heat stability: Placental, Intestinal, Liver, Liver fraction migrates the fastest, followed by bone, placental then and Bone the intestinal is the slowest isoenzyme. Rxn utilized: USE OF ENZYMES OR LECTINS Because of the similarity between bone and liver forms, enzymes or lectins may be used to further separate the two SUMMARY OF ALP METHODS METHODS SUBSTRATE END PRODUCTS Bodansky Shinowara Beta-glycerol phosphate Inorganic PO4 + glycol Jones Reinhart King and Armstrong Phenylphosphate Phenol Bessy, Lowry, and Brock P-nitrophenyl phosphate (PNPP) P-nitrophenol or yellow nitrophenoxide ion Bowers and McComb Huggins and Talalay Phenolphthalein phosphate Phenolphthalein red Moss Alpha naphthol PO4 Alpha-naphthol Klein, Babson and Reed Buffered Phenolphthalein PO4 Free phenolphthalein SOURCES OF ERROR: Hemolysis - ALP is approx 6x more concentrated in erythrocyte than in serum Activity in serum increases approx 3-10% on standing at 25oC or 4oC for several hours Diet may induce elevations in ALP activity in blood group B and O individuals who are secretors Patients under this blood groups have higher ALP levels because they have a secretor gene and H substance present in their intestines ALP here is about intestinal isoenzyme Values may be 25% higher following ingestion of a high fat meal NOTES TO REMEMBER: Ingestion of food leads to release of intestinal ALP into lymphatic fluid, and may transiently increase plasma levels of ALP Specifically to blood groups B and O ZInc is a component of ALP If there is a decrease of result in ALP, that indicates of decrease in zinc Placental ALP - useful tumor marker in serum and CSF for most germ cell tumors CLINICAL CHEMISTRY 2 LECTURE/TRINIDAD/LCB GAMMA-GLUTAMYL TRANSFERASE (GGT) GGT Smoking – moderate = 10% E.C. 2.3.2.1 smoking increase the risk of liver cancer and liver Catalyzes the transfer of γ-glutamyl residue from cirrhosis, promotes production of cytokines and γ-glutamyl peptides to amino acids, other peptides, or chemicals that cause inflammation to the liver H2O GGT Decrease: In short, catalyzes the hydrolytic cleavage of Pregnancy: 1st trimester = dec. 25% peptides to form amino acids or smaller peptides Oral contraceptives: dec. 20% In most biological systems, glutathione serves as Estrogens and oral contraceptives are both the gamma glutamyl donor associated with several liver related Rxn catalyzed: complications including intrahepatic cholestasis, sinusoidal dilatation, hepatic adenomas, hepatocellular carcinoma FUNCTION: ASSAY FOR ENZYME ACTIVITY NO SPECIFIC physiologic function has not yet γ-glutamyl -p-nitroanilide been established. Most widely used substrate in GGT analysis It is for protein and peptide synthesis Nitroanilide is chromogenic product w/ strong abs Regulation of tissue glutathione levels, and; at 405-420 nm Transport of amino acids across cell membranes. Sample: TISSUE SOURCES: Stable for 1 week at 4oC Found primarily in tissue of the kidney, brain, No interference of hemolysis prostate, pancreas and liver GGT enzyme lack erythrocyte GGT assay is confined mainly to evaluation of Plasma can also be used (heparin) liver and biliary system disorders Common anticoagulant in CC is heparin They are cell membrane bound with increased Citrate, Oxalate, and Fluoride depresses GGT secretory and absorptive properties activity by 10 to 15% Half-life: 7-10 days but may persist up to 28 days in Reference values are lower in females cases of alcoholic liver disease. because of suppression of enzyme activity DIAGNOSTIC SIGNIFICANCE: resulting from estrogenic or progestational Hepatobiliary disorders and Biliary tract obstruction hormones In the liver, GGT is located in the canaliculi of the Normal Values: hepatic cell and the epithelial cell lining of the Male: 6-45 U/L biliary ducts. Because of its location, it is elevated Female: 3-30 U/L in virtually all hepatobiliary disorders Patients receiving warfarin, phenobarbital, and phenytoin (increased and 4x ULN) In the parenchyma, GGT exists to a large extent in the smooth endothelial reticulum. Therefore, it is subject to Hepatic microsomal induction Alcoholism (2-3x ULN) (level return to normal within 2-3 weeks after to cessation) Chronic alcoholism GGT is the most sensitive marker of acute alcoholic hepatitis GGT assay is useful for monitoring the effects of alcohol abstention Source of elevation on: Acute pancreatitis: pancreas DM pancreas Myocardial Infarction: unknown Useful in differentiating the source of an elevated ALP GGT levels are normal in skeletal muscle disorders and during pregnancy CLINICAL CHEMISTRY 2 LECTURE/TRINIDAD/LCB AMYLASE AMS Considered to be the smallest enzyme E.C. 3.2.1.1 Can pass through glomerular filter Chemical Name: α-1,4-glucan-4-glucanohydrolase This is the only enzyme that could tested in urine Hydrolase Opt. pH: 6.9-7.0 in serum Catalyzes the breakdown of starch and glycogen TISSUE SOURCES: Starch is made up of: Pancreas - greatest concentration Amylose - long unbranched chain of glucose linked by a,1-4 Salivary glands - also great in concentration glycosidic bonds (glucose molecule) Skeletal muscles Amylopectin - branched chain polysaccharide with a,1-6 Small intestines linkage (polysaccharide) Fallopian tube How does the digestion of starch start? Digestion of starches begins in the mouth with the hydrolytic action of It is an earliest pancreatic marker salivary AMY. Salivary AMY activity, however, is of short duration a-AMS attacks only the a,1-4 glycosidic bonds to produce the because, on swallowing, it is inactivated by the acidity of the gastric degradation products: Glucose, Maltose and an intermediate chain contents. Pancreatic AMY then performs the major digestive action of (Dextrin) starches once the polysaccharides reach the intestine MW: 50,000 to 55,000 daltons Important enzyme in the digestion of starches Activator: Ca and Cl ions ISOENZYMES OF AMYLASE S-type Isoamylase (Ptyalin) P-type Isoamylase (Amylopsin) Its activity starts in the salivary gland where it initiates the Synthesized by the pancreatic acinar cells and secreted hydrolysis of starch while food is in the mouth and into the intestinal tract via the pancreatic duct system ACTIVITY esophagus Its action is favored by the mildly alkaline condition of the Its action is terminated by the acidity of the stomach duodenum inhibited by monoclonal antibodies and protein isolated inhibited by monoclonal antibodies but not inhibited by INHIBITION from wheat protein isolated from wheat NOTES: MACROAMYLASES The isoenzymes of salivary origin migrates most quickly (S1,S2,S3) , Abnormal amylase (usually the S-type) in combination with P type are slower (P1,P2,P3) Immunoglobulins (IgA or IgG) or other high MW proteins The most commonly observed fractions: P2, S1 and S2 Macroamylasemia - a condition that results when the amylase In acute pancreatitis: P3 predominates molecule will combine immunoglobulins to form a complex that S type represent 2/3 of AMS activity of normal serum is too large to be filtered by the glomerular filter P type predominates in the normal urine Amylase: decrease in the urine while it increases in the serum The primary reason for an elevated amylase is Acute Pancreatitis Not found in urine and concentrated in serum/plasma No clinical significance, it should be differentiated from other causes of hyperamylasemia DIAGNOSTIC SIGNIFICANCE The primary reason for an elevated amylase is Acute pancreatitis Note: The degree of elevation of AMS is helpful in differentiating diagnosis of acute pancreatitis HENRY TIETZ AMS ACTIVITY 2-12 hours 5-8 hours Rise upon onset of symptoms 48 hours 12-72 hours Peak activity (fourfold to sixfold) 3rd-5th day 3rd-4th day Returns to normal Other conditions: Mumps, Perforated peptic ulcer, Appendicitis, Ruptured ectopic pregnancy, Dissecting aortic aneurysm, and Biliary tract disease METHODS OF AMS DETERMINATION SUMMARY: CONTINUOUS MONITORING METHOD more controlled and consistent hydrolysis conditions makes use of coupling of several enzyme systems to monitor amylase activity HISTORICAL METHODS AMYLOCLASTIC METHOD SOURCES OF ERROR Little loss of activity occurs at room temperature for 1 week or at 4°C Iodometric method for 2 months. AMY is allowed to act on a starch substrate to which iodine has been Plasma triglycerides suppress or inhibit serum AMY activity = attached. As AMY hydrolyzes the starch molecule into smaller units, NORMAL IN ACUTE PANCREATITIS WITH HYPERLIPIDEMIA. the iodine is released and a decrease occurs in the initial dark-blue Morphine and other opiates will lead to falsely elevated serum AMY color intensity of the starch–iodine complex. levels. The decrease in color is proportional to the AMY concentration The drugs presumably cause constriction of the sphincter of the SACCHAROGENIC METHOD pancreatic ducts Salivary amylase is inhibited by wheat germ lectin The S and P type can be estimated by measuring the total amylase in the presence and absence of lectin IMPORTANT POINTS: Normal serum contains both salivary and pancreatic AMS Normal amylase / creatinine ratio = 1.0% – 4.0% (0.01 – 0.04) A:C ratio (Acute Pancreatitis) = >4.0% (up to 15%) An elevated amylase-creatinine clearance ratio has been established as being highly specific for the diagnosis of acute pancreatitis By Nelson Soogyi and modified by Henry & Chiamon This uses a starch substrate that is hydrolyzed by the action of AMY to its constituent carbohydrate molecules that have reducing properties. The amount of reducing sugars is then measured where the concentration is proportional to AMY activity Reference method for determining AMY activity, is reported in Somogyi units Somogyi units are an expression of the number of milligrams of glucose released in 30 minutes at 37°C under specific assay conditions. CONVERSION FACTOR: Somogyi and IU: 1.85 CHROMOGENIC METHOD Klein, Foreman, & Searcy This uses a starch substrate to which a chromogenic dye has been attached, forming an insoluble dye–substrate complex. The increase in color intensity of the soluble dye–substrate solution is proportional to AMY activity. TURBIDIMETRY AND NEPHELOMETRY Peralta & Reinhart Measures the change in turbidity of starch solution over a short reaction period CLINICAL CHEMISTRY 2 LECTURE/TRINIDAD/LCB LIPASE LPS CHERRY CRANDALL METHOD E.C. 3.1.1.3 Chemical Name: Triacylglycerol Acylhydrolase Hydrolyzes the ester linkages of fats to produce alcohols and fatty acids. LPS catalyzes the partial hydrolysis of dietary triglycerides in the intestine to the 2-monoglyceride intermediate, with the production of long-chain fatty acids. Rxn. catalyzed PRINCIPLE: Hydrolysis of olive oil after incubation for 24 hours at 37oC and titration of fatty acids using NaOH SUBSTRATE: 50% Olive oil END PRODUCT: fatty acid Triolein is one substrate now used as a more pure form of triglyceride. Note: complicated by the lack of stable and uniform substrates This reaction is accelerated by the presence of colipase and bile salt TURBIDIMETRY METHOD Importance: Responsible for triglyceride metabolism Simpler and more rapid than titrimetric assays Small molecule and filtered in the glomeruli but not normally seen in Emulsion of fats produces milky appearance urine bec. It is totally reabsorbed by the renal tubules As the fats are hydrolyzed by LPS, the particles disperse, and the Acts only on emulsified substrate rate of clearing can be measured as an estimation of LPS activity Most specific pancreatic mark COLORIMETRIC METHOD TISSUE SOURCES: Based on coupled reactions with enzymes such as peroxidase or Pancreas (primary) glycerol kinase GIT OTHERS: Leukocytes Titrimetric Methods: fatty acid is titrated w/ alkali solution Adipose cells Spectrophotometric Method: Myrtle and Zell method: fatty acids Colostrum are extracted using Petroleum ether DIAGNOSTIC SIGNIFICANCE Fluorometric Method: Fatty acid + chemical reagent: Fluorescein For Acute Pancreatitis: (4-methyl bellifuzone) HENRY TIETZ LIPASE ACTIVITY For Short-Chain TG Substrate: ○ Advantages: Analytical, greater solubility in aqueous medium 4-8 hours 4-8 hours Rises after onset of ○ Disadvantage: non physiologic substrate symptoms Erlanson & Bergstrom – Tributyrin Radio-immunoassay : makes use of 1251-labeled lipase Latex Agglutination : antibodies to pancreatic lipase are bound to 4-8 hours 24 hours Peak activity latex particles Coupled enzymatic method 8-14 days 7-14 days Activity decreases SOURCES OF ERROR LPS is stable in serum. NOTE: Negligible loss in activity at room temperature for 1 week or for 3 Diagnostic marker for AP weeks at 4°C The increase in serum LPS is not necessarily proportional to the Hemolysis cause falsely low values/decreased LPS severity of the attack Because hemoglobin inhibits the activity of serum LPS In Acute Pancreatitis, LPS persist up to 8 days, AMS persist 2-3 days Both level rise quickly Note: elevations do not correlate with severity of Disease. Useful in differentiating serum AMY elevation as a result of pancreatic versus salivary involvement. Elevations: Duodenal ulcers Perforated Peptic ulcer Intestinal obstruction Acute cholecystitis Mesenteric vascular obstruction LIPASE ISOENZYME L1 & L2 Pancreatic isoenzymes or isoforms of lipase L2: Most clinically specific and sensitive lipase isoenzyme METHOD OF DETERMINATION: Specimen: serum (stable at RT), pleural fluid, ascitic fluid Inhibitors: heavy metals, quinine & some esterase inhibitors Not inhibited by fluoride or arsenilate Bacterium increases concentration of LPS Some important lipase-producing bacterial genera include Bacillus, Pseudomonas and Burkholderia CLINICAL CHEMISTRY 2 LECTURE/TRINIDAD/LCB TRYPSIN TRY After an attack of acute pancreatitis, serum TRY-1 EC 3.4.21.4 rises in parallel with serum AMY activity to peak It is a Pancreas-specific serine protease values ranging from 2 to 400 times the upper Solely produced by pancreatic acinar cells reference limit. The acinar cells of the human pancreas High in neonates synthesize two different trypsin (1 and 2) in the Cases in cystic fibrosis - a common inherited form of the inactive proenzymes (or zymogens), genetic disease in pediatric patients, newborns trypsinogen-1 and -2 has severe pancreatic insufficiency, accumulation Hydrolyzes the peptide bonds formed by the carboxyl of thick mucus secretion in the pancreatic ducts groups of lysine or arginine with other amino acids that inhibit secretion of pancreatic digestive Activator: Enterokinase enzymes Inactivated in plasma by: α-1-antitrypsin & TRYPSIN 2 α-2-macroglobulin Anionic trypsin TWO (2) MAJOR TRYPSINS Increases more in acute pancreatitis (10- fold TRY-1 is also described as cationic and TRY-2 as greater) anionic because of their differing electrophoretic Larger amounts are excreted in the urine mobility. DIAGNOSTIC SIGNIFICANCE TRYPSIN 1 For differentiating the cause of acute pancreatitis Cationic trypsin Trypsinogen-2 & Trypsinogen-2-AAT Major form found in the serum (Trypsin-2-Alpha-1-Antitrypsin): more elevated in Related to the severity of acute pancreatitis alcohol-associated pancreatitis Elevated in chronic renal failure as amylase and Trypsin 1, Amylase & Lipase: more elevated in lipase biliary pancreatitis MISCELLANEOUS ENZYMES ANGIOTENSIN-CONVERTING ENZYME (ACE) DISEASE CORRELATION E.C. 3.4.15.1 Diagnosis and monitoring Sarcoidosis (decrease in Also known as peptidyl dipeptidase A/ Kininase II ACE) Considered as a hydrolase Sarcoidosis: This is an inflammatory disease It converts angiotensin I to angiotensin II within the that affects multiple organs in the body, mostly lungs the lungs and the lymph glands. In people with Takes part in the RAAS - System that control sarcoidosis, abnormal masses or nodules (called blood pressure by regulating the volume of fluid in granulomas) consisting of inflamed tissues form the body by regulating aldosterone (sodium in certain organs of the body are found balance) Elevations are more likely in pulmonary involvement if there is perceived volume depletion or low Example: Acute and Chronic Bronchitis sodium filtered, Renin is produced TRUE CHOLINESTERASE juxtaglomerular apparatus (proteolytic enzyme), E.C. 3.1.1.7 initiate cleavage of angiotensinogen Cleaves AcCholine (neurotransmitter) into acetic acid Converts Angiotensin I to the vasoconstrictor and choline angiotensin II Major sources: CNS, RBCs, Lung and Spleen It increased blood pressure and stimulate Measurement of activity: it uses acetylcholine aldosterone release Cholinesterase activity is inhibited Possible indicator of Neuronal dysfunction DIAGNOSTIC SIGNIFICANCE Example: Alzheimer’s disease Diagnosis of organophosphate insecticide poisoning Inactivation of bradykinin, encephalin, tachykinin (cholinesterase activity is inhibited) PSEUDOCHOLINESTERASE OR ACYLCHOLINE TISSUE SOURCES ACYL HYDROLASE (PChE) Most active in the lungs and epithelioid cells E.C. 3.1.1.8 Lungs Secreted by the liver Testes Cleaves succinylcholine (muscle relaxant used during Macrophages and; surgery) Epithelioid cells Tissue sources: myocardium & pancreas Acts as an “antixenobiotic enzyme”  because it catalyzes the removal of benzyl group A marker of hepatobiliary disease and infiltrative from cocaine lesions of the liver It is used to monitor the effect of muscle relaxant Measurement: Measurement of activity: Chelating agents interfere Pseudocholinesterase uses butyrylthiocholine Chelating Agents: these are chemical Released thiocholine reacts with Ellman’s reagent compounds that bind to different metal ions Product: measured photometrically In medicine: used to remove toxic metals from DIAGNOSTIC SIGNIFICANCE the body Diagnosis of organophosphate insecticide poisoning The assay uses large amounts of other non The Cholinesterase activity is inhibited = ⬇levels nucleoside substrates * Liver function test: Pseudocholinesterase production Non-nucleoside substrates: substrates that Decrease activity in malnutrition binds directly to reverse transcriptase Diagnosis of genetic variants DIAGNOSTIC SIGNIFICANCE: Example: Prolonged Apnea after using Increased in Acute hepatitis, Ovarian carcinoma, succinylcholine during anesthesia & Rheumatoid Arthritis Involved in the metabolism of Anticholinergic drugs Together with other enzymes such as ALP, LDH it Anticholinergic drugs: used to treat a wide variety can also serve as a tumor marker of conditions with the activation of the parasym- pathetic nervous system and also for the involve- ement in the central nervous system. Decreased in: acute hepatitis, cirrhosis, carcinoma metastatic to liver and malnutrition Method used: Ellman technique and potentiometric LEUCINE AMINOPEPTIDASE (LAP) E.C. 3.4.11.1 Hydrolyzes amino acids from the N-terminal end of the peptides They are widely distributed or ubiquitous in nature Measurement used: Starch Gel Electrophoresis LAP ISOENZYMES: 1. LIVER ISOENZYME Major isoenzyme Has similar activity to GGT and ALP It is canalicular membrane-bound DIAGNOSTIC SIGNIFICANCE Increased in obstructive liver disease (NOT in bone disease) * More sensitive than ALP & 5’-nucleotidase Less sensitive and specific than GGT Elevated in systemic lupus erythematosus Autoimmune disease with butterfly rash Present in the breast, endometrial and ovarian carcinomas, germ cell tumors of the ovary and testis 2. PLACENTAL ISOENZYME Important in hydrolysis of oxytocin and angiotensin II Oxytocin: hormone that acts as a chemical mess