Clinical Chemistry Enzyme Handbook Review PDF

15 ENZYMOLOGY CHAPTER LEARNING OUTCOMES At the end of this chapter, the students should be able to: 1. explain the factors influencing enzymatic reactions;...

15 ENZYMOLOGY CHAPTER LEARNING OUTCOMES At the end of this chapter, the students should be able to: 1. explain the factors influencing enzymatic reactions; 2. outline the classification of enzymes and their catalytic actions; 3. distinguish the enzymatic theories, rate of reaction, kinetics, and units of measurement; 4. discuss the tissue sources of enzymes and correlate with their diagnostic significance; 5. design an algorithm for the clinical enzymes and disease correlations; and 6. discuss the samples and methods used in the detection and measurement of the clinically significant enzymes including assay interferences. Enzymes are proteins in nature produced by living cells that hasten chemical reactions in organic matter. They are large molecules that are normally confined within cells unless increased membrane permeability allows them to They frequently appear in the serum after cellular injury, degradation of cells or from storage areas. enter the blood. Enzymes are measured in terms of their activity and not in terms of their absolute values. Abnormal large amounts of enzymes in serum are used clinically as evidence of organ damage. Each enzyme catalyzes a single reaction or : limited number of chemical reactions and is specific for substrate that it converts to a defined product. The tissue and cellular locations of enzymes are crucial in the diagnosis and management of organ dysfunctions and diseases. Factors Affecting Enzymatic Reactions Enzyme Concentration The higher the enzyme concentration, the faster is the reaction because more enzyme is present to bind with the substrate. Substrate Concentration With the amount of enzyme exceeding the amount of substrate, the reaction rate steadily increases as more substrate is added. However, when substrate concentration reaches a maximal value, higher concentration of substrate no longer results in increased rate of reaction (saturation kinetics). Cofactors that must bind to particular enzymes before a reaction occurs. These are nonprotein entities a. Coenzyme It is an organic cofactor (second substrate). It is essential to achieve absolute enzymatic activity. Increasing its concentration will increase the velocity of an enzymatic reaction. 0 Examples: Nicotinamide adenine dinucleotide (NAD) Nicotinamide adenine dinucleotide phosphate (NADP)  196 CLINICAL CHEMIST REVIEW HANDBOOK IN b. Activators the enzyme for These are inorganic ions which alter the spatial configuration of proper substrac binding. Examples: Calcium, zinc, chloride, magnesium, and potassium C. Metalloenzymes These are inorganic ions attached to molecule. Examples: Catalase and cytochrome oxidase Inhibitors reaction. Enzymatic reactions may not progress if an inhibitor interferes with the a. Competitive Inhibitor It physically binds to the active site of an enzyme. Both the substrate and inhibitor compete for the same active site of the enzyme. With a substrate concentration significantly higher than the concentration of the inhibitor, the inhibition is reversible. Theeffect of the inhibitor can be counteracted by adding excess substrate to bind with the enzyme. Dilution of serum results in reduction in the concentration of this inhibitor, thus increasing the rate of reaction. It has the ability to alter the apparent Michaelis-Menten constant (Km). b. Non-Competitive Inhibitor C It does not compete with the substrate but look for areas other than the active site. The substrate and inhibitor (commonly metallic ion) may bind with an enzyme simultaneously. When the non-competitive inhibitor binds to the allosteric site, it alters the configuration of the active site and the tertiary structure of proteins. Because the inhibitor binds with the enzyme independently from the substrate, increasing the substrate ednuoins 5 concentration does not reverse the inhibition. The presence of the inhibitor when it is bound to the enzyme, slows the rate of the enzymatic reaction: while the substrate can still bind to the active site, the "fit has change" becomes imperfect. As a result of this change, the active site and substrate no longer share specificity, meaning the substrate cannot bind. C. Uncompetitive Inhibitor The inhibitor binds to the enzyme-substrate (ES) complex. The uncompetitive inhibitor can only bind to the enzyme only after the union of the enzyme and substrate. Increasing the substrate concentration results in more ES complexes to which the inhibitor binds and thereby increases the inhibition. Increasing substrate concentration results in increased inhibition. These are enzymes (polypeptide chains) having the same catalytic reactions but slightly different molecular structures - various forms occur because of differences intheaminoacid sequence of enzymes. The importance of the total enzyme activity is enhanced by fractionating the isoenzymes. Temperature Enzymes are active at 25° C, 30° C, or 37° C. The optimum temperature for enzymatic activity is at 37° C; however.enzymes such as creatine kinase may exhibit denaturation at this range. The rate of denaturation increases as the temperature increases, and it is usually significant at 40° C to 50°C. Increasing temperature usually increases the rate of a chemical reaction by the movement of molecules - the use of higher temperature provides catalyzed reaction rates andenhancing improves sensitivity of the assay. ENZYMOLOGY 197 Temperature Coefficient (Q10) means for every 10° C increase in temperature, there will be a twofold increase in enzyme activity. Temperature near 60° C to 65° C may result to inactivation of enzymes. Hydrogen Ion Concentration or ph Most physiologic reactions occur in the pH range of 7 to 8. Extreme pH level may denature an enzyme or influence its ionic state, resulting in structural change or change in the charge of amino acid residue in the active site. Storage Low temperatures (refrigeration/freezing) render enzymes reversibly inactive. Repeated freezing and thawing tends to denature proteins and should be avoided. Preservation for longer period of time (enzymes): -209 C Ideal storage temperature for substrate and coenzymes: 29 to 8° Room temperature is the ideal for storage of LDH (LD4 and LD5) enzyme. Hemolysis - mostly increases enzyme concentration Lactescense or milky specimen decreases enzyme concentration Enzyme Nomenclature To standardize enzyme nomenclature, the Enzyme Commission (EC) of the International Union of Biochemistry adapted a classification system in 1961, and revised the standards in 1972 and 1978. Each enzyme has two names: a practical or trivial name and a systematic name. The latter consists of a unique numeric code designation and the nature of the catalytic reaction (McPherson and Pincus, 2022). Enzymes are classified according to their biochemical functions, indicating substrate and class of reaction catalyzed, and are designated by individual identification numbers. First digit places the enzyme in its classifications (six classifications). Second and third digits represent the subclass to which the enzyme is assigned. Final and fourth number/s are serial numbers specific to each enzyme in a subclass. Acid phosphatase E.C. 3.1.3.2 8 0710 E.C. 6.4.1.1 Acetyl-CoA synthetase E.C. 3.1.3.1 Alkaline phosphatase E.C. 3.2.1.1 Amylase Alanine aminotransferase E.C. 2.6.1.2 E.C. 2.6.1.1 Aspartate aminotransferase E.C. 4.1.2.13 E.C. 3.4.15.1 Angiotensin-converting enzyme Creatine kinase E.C. 2.7.3.2. E.C. 2.3.2.2 Gamma-glutamyl transferase EC. 1.1.1.49 Glucose-6-phosphate dehydrogenase E.C. 3.1.1.3 Lipase E.C. 1.1.1.27 Lactic dehydrogenase E.C. 3.1.1.8 Pseudocholinesterase E.C. 3.1.1.7 True/Acetylcholinesterase E.C. 3.1.3.5 5' nucleotidase  198 REVIEW HANDBOOK IN CLINICAL CHEMIST Classification of Enzymes TABLE 42. Classification of Enzymes Example/s Class Function CO, LD, MD, ICD, G-6-PD Catalyze the removal or addition of electrons (redox reaction) from CK, AST, ALT, OCT Transferases Catalyze the ransfer of a chemical group other than hydrogen one substrate to another Esterases: ACP, ALP, CHS, LPS Hydrolases Catalyze hydrolysis or splitting of a bond by the addition of water(hydrolytic reactions) Peptidases: Trypsin, Pepsin, LAP Glycosidase: AMS, Galactosidases glutamate decarboxylase, pyruvate Catalyze removal of groups from substrates without hydrolysis and the product contains double bonds decarboxylase, tryptophan decarboxylase and aldolase Catalyze the intramolecular arrangement of the substrate glucose phosphate Isomerase and ribose compound phosphate isomerase Catalyze the joining of two substrate molecules, coupled with Acetyl-CoA synthetase breaking of the pyrophosphate bonding ATP or similar compound CHS - Cholinesterase MD - Malate dehydrogenase ICD - Isocitrate dehydrogenase CO - Cytochrome oxidase LAP - Leucine aminopeptidase OCT - Ornithine carbamoyltransferase ACP - Acid phosphatase ALP - Alkaline phosphatase CK - Creatine kinase ALT Alanine aminotransferase LD - Lactate dehydrogenast ICD - Isocitrate dehydrogenase AMS - Amylase LPS - Lipase AST - Aspartate aminotransferase G-6-PD - Glucose-6-phosphate dehydrogenase General Properties of Enzymes Each enzyme contains: 1. Active site It is a water-free cavity where the substrate interacts with a particular charged molecule. It is an amino acid residue with a three-dimensional protein structure. 2. Allosteric site It is a cavity other than the active site. It may bind regulator molecules. It serves as an attachment site of a non-competitive inhibitor. Notes to Remember As protein, each enzyme is composed of (secondary structure), which then specific amino acid (primary structure), forming polypeptide chains The catalytic activity of an enzyme molecule depends generally on the integrity When all sites are filled, no of its structure. Terminologies hey et. When bound tightlyto the enzyme,the coenzyme Apoenzyme (enzyme (apoenzyme + prosthetic group = holoenzyme). Digestive enzymes in its inactive form originally secretedfrom the organ of production is called a proenzymeof Macroenzymes are enzymes-antibody complexes. ENZYMOLOGY 199 Enzyme Theory 1. Emil Fischer/Lock and Key Theory It is based on the premise that the shape of the key (substrate) must fit into the lock (enzyme). 2. Koshland/Induced Fit Theory It is based on the substrate binding to the active site of the enzyme. Enzyme Kinetics The Michaelis-Menten kinetics is a model for enzyme kinetics, where the enzymatic reaction is illustrated by the relationship between the rate of formation of a product (P) and concentration of the substrate (S). A chemical reaction may occur spontaneously if the free energy or available kinetic energy is higher for the substrate than the product. Enzymes catalyze physiologic reactions by lowering the activation energy level that the substrate must reach for the reaction to occur. An enzyme combining with only one substrate and catalyzing only one reaction is known as absolute specificity. Enzymes combining with all the substrates in a chemical group is called group specificity. Enzymes reacting with specific chemical bonds is known as bond specificity. Enzymatic Reaction ONE d 1. Zero-order reaction - The reaction rate depends only on enzyme concentration. 2. First-order reaction - The reaction rate is directly proportional to substrate concentration. A substrate concentration > 99 x Km is needed to achieve zero-order reaction. It is easier to measure small increases in product than to measure small decreases in a large amount of substrate. Substrate does not absorb light at a specific wavelength compared to a product. A pseudo-first-order reaction is a kind of "false second-order" reaction wherein one or the other reactant (like a substrate) is altered by increasing or decreasing its concentration. To measure the extent of enzymatic reactions, two general methods may be used 1. Fixed-time The reactants are combined; the reaction proceeds for a designated time; the reaction is stopped and measurement is made. 2. Continuous monitoring/kinetic assay - Multiple measurements of change in absorbance are made during the reaction; it is preferred than fixed-time; more accurate compared to fixed-time assay. Units for Expressing Enzymatic Activity per minute 1. International Unit (1U or U) - one micromole of substrate 2. Katal Unit (KU). one mole of substrate per second Enzymes are quantified based on their activity rather than absolute values. are activity units. The units used to report enzyme levels must consider change in pH, temperature, substrate, etc. The definition for activity unit Enzyme Activity Enzymes are measured in terms of: 1. Change in substrate concentration 2. Change in product concentration 3. Change in coenzyme concentration Enzyme Measurement Serum is the preferred specimen; for measurement of enzymes. some enzymes (AST and CK) are inhibited. Heparinized plasma is the alternate specimen; however, cause false decreased enzymatic activity. Citrated, EDTA, and fluoride plasma specimens may activity will be measured. The substrate (reagent) is specific to the enzyme whose diluted. if the sample will be The presence of inhibitors can be diminished cause interferences by dissolving enzymes and substrate. During measurement, proteases may  CLINICAL 200 REVIEW HANDBOOK IN CHEMIST. Causes of Elevated Plasma Enzyme Levels 1. Tissue necrosis and degeneration (death of enzyme-containing cells) 2. Impaired removal of enzyme from plasma 3. Normal cell turnover 4. Increased permeability of cell membrane 5. Increase in the production of enzymes by cells 6. Decreased clearance of enzymes from the circulation Notes to Remember reach equilibrium. An enzyme accelerates the rate of reaction, reducing the time required to A constant change in absorbance per unit time occurs only when the rate of the reaction is zero-order. An enzyme does not alter the free energy or direction of a reaction, but it alters the energy of activation by forminga metastable intermediate, the ES complex. Most enzymes are measured by monitoring the rate of absorbance change (kinetic assay) at 340 nm as NADHis reduced or consumed, and it allows direct reporting either by IU or KU. In first-order reaction, the enzymes are used as reagents to measure a specific analyte. Kinetic methods are more accurate and can easily identify any change in the reaction and samples requiring treatment (dilution). In non-kinetic assay, absorbance is made at 10-second intervals for 100 seconds. Endpoint measurement determines the concentration of substrate or product at a specific time after addition of the sample (bedside glucose testing using strips). Enzyme activity measurements may not be accurate if enzyme inhibitors are present- essential cofactors are not included in the assay-and when there is improper sample storage. The Lineweaver-Burk plot is an illustration of the reciprocal of the substrate concentration (x-axis, 1/S) and the reciprocal of the reaction velocity (y-axis, 1/V), in which both are components of the enzyme kinetics. MAJOR CLINICAL ENZYMES PHOSPHATASE Alkaline Phosphatase (ALP)/Alkaline Orthophosphoric Monoestr Phosphohydrolae It is a non-specific enzyme capable of reacting with many different substrates. It functions to liberate inorganic phosphate from an organic phosphate ester with the concomitant production of an alcohol. It is predominantly found in the cell membranes. Major tissue sources: liver, bone, placenta, and intestinal Reference range: 30-90 U/L Major Isoenzymes 1. Liver ALP 2. Bone ALP 3. Placental ALP 4. Intestinal ALP abundant plasma alkaline phosphatase (ALP) isoforms are The most coded by a single gene on chromosome 1, producing liver, bone, and kidney isoenzymes. Genes on chromosome 2 code for placental and intestinal ALPs. In healthy sera, ALP levels are derived from liver (hepatic sinusoid and canalicular surface) and bone (osteoblasts), hence, blood tests measure total ALP that comes from the hepatic tissues and bones. Bone isoenzyme increases due to osteoblastic activity and is normally elevated children during periods of growth and in adults older than age 50 years (geriatric). in During period of growth and muscle development, serum ALP and creatinine levels increase. In normal pregnancy, increased ALP activity can be detected between 16 and 20 weeks of pregnancy.  ENZYMOLOGY 201 The presence of intestinal ALP isoenzyme in serum depends on the blood group (secretor gene and H substance) of the individual; B or 0 blood group increases intestinal ALP after consumption of a fatty meal. ALP is also higher in individuals of groups B and 0 than in A of differences in and AB individuals because intestinal ALP levels. 16,19V1 Placental ALP is lower in pregnant women of blood groups A and AB. Carcinoplacental ALP 1. Regan ALP It is found in lung, breast, ovarian, and gynecological cancers. O It is the most heat-stable ALP (65' C for 30 minutes). It is inhibited by phenylalanine reagent, a bone ALP co-migrator. It is coded by the gene on chromosome 2. 2. Nagao ALP It is found in adenocarcinoma of the pancreas and bile duct, and in pleural cancer. It is a variant of Regan ALP. O It is inhibited by L-leucine and phenylalanine. Diagnostic Significance When total ALP levels are increased, it is the major liver fraction that is most frequently elevated, especially in obstructive jaundice. ALP is increased in obstructive jaundice due to greater rate of secretion. In biliary tract obstruction, serum ALP increases are primarily a result of increased synthesis of the enzyme by induced cholestasis. When there is obstruction in the flow of conjugated bilirubin into the canaliculus, it is accompanied by elevated plasma B2, ALP, and gamma-glutamyltransferase (GGT). For bone disorders, highest elevations occur in Paget's disease (osteitis deformans). In other bone disorders where there are active osteoblasts, such as in osteosarcoma, tumor metastatic to bone, and metabolic bone disease, serum ALP is also increased. Bone ALP isoform known as B1x is detected in the serum of dialysis patients. B1x isoform is used to study low bone mineral disease (BMD) in patients with chronic kidney disease. B1x isoform is also increased in the serum of individuals with BMD of the hip, which is composed mainly of trabecular bone. ALP is elevated in cases of abortion, and may be increased when there is difficulty during pregnancy and Serum birthing. Transient low serum ALP may occur after blood transfusion or cardiopulmonary bypass. Prolonged low levels of ALP occur in hypophosphatasia. ALP as a Tumor Marker Placental alkaline phosphatase (PLAP) is utilized tumor marker in serum and cerebrospinal fluid (CSF] for as a most germ cell tumors. CSF levels of PLAP are of diagnostic value in differentiating whether a tumor in the pineal cell tumor. body is a pinealoma or a germ Methods is the preferred sample. ALP is inactivated by EDTA, hence serum ALP requires magnesium ions for activation. Electrophoresis Liver and bone ALPs are the most anodal isoenzymes; intestinal ALP is the least anodal. Use of neuraminidase and wheat germ lectin improves the separation of bone and liver ALPs. gel and isoelectric focusing are capable of resolving High-resolution electrophoresis using polyacrylamide multiple bands of ALP.  202 REVIEW HANDBOOK IN CLINICAL CHEMISTA Heat Fractionation/Stability Test It is performed at 56°C for 10 to 15 minutes. Placental ALP is the most heat-stable; bone ALP is the most heat labile. Decreasing order of ALP heat stability: Placental, intestinal, liver, and bone Chemical Inhibition Test and levamisole solutions. This method uses different concentrations of phenylalanine, synthetic urea, ALP by 3M urea. Placental and intestinal ALPs are inhibited by phenylalanine reagent, and bone Levamisole reagent inhibits liver and bone ALP. Bowers-McComb (Szasz Modification) It is considered as the most specific method; IFCC-recommended method. It is currently the routine method for ALP. continuous-monitoring technique which requires a pH environment of 10.15 and should be It is a read at 405 nm. The use of hemolyzed specimens may cause a significant interference with this method. ALP p-nitrophenylphosphate p-nitrophenol + phosphate ion TABLE 43. Summary of ALP Methods Substrate End Product Method Bessy, Lowry, and Brock Para-nitro phenyl phosphate (PNPP) Para-nitrophenol or yellow nitrophenoxide ion Bowers and McComb Bodansky, Shinowara, Jones, and Reinhart Beta-glycerophosphate Inorganic phosphate + glycerol King-Armstrong Phenylphosphate Phenol Huggins-Talalay Phenolphthalein diphosphate Phenolphthalein red Moss Alpha-naphthol phosphate Alpha-naphthol Klein, Babson, and Reed Buffered phenolphthalein phosphate Free phenolphthalein Notes to Remember Zinc is a component of ALP, and magnesium is the enzyme activator. Ingestion of food leads to release of intestinal ALP into lymphatic fluid, and may transiently increase plasma levels of ALP. Hemolysis and diet (fatty meals) are sources of analytical errors and may cause elevated serum ALP. ALP is sensitive if stored at low temperature (4° C), leads to increased serum level. ALP is inhibited by phosphorus - the addition of 2-amino-2-methyl-1-propanol (AMP) buffer binds phosphorus under Bowers-McComb method. Decreased ALP is seen in zinc deficiency. ALP is also found in the leukocytes (Hazell and Ortiz, 1966). weak anion-exchange columns is able to establish six different isoforms of ALP in the sera of healthy individuals: bone/intestinal (B/I), bone isoforms (B1 and B2), and liver isoforms (L1, L2, and L3) (Haarhaus et al, 200%i Swolin-Eide et al., 2006, as cited in McPherson and Pincus, 2022). Increased ALP 1. Obstructive jaundice 5. Osteoblastic bone tumors (osteosarcoma) 2. Osteitis deformans 6. Sprue 3. Osteomalacia 7. Hyperparathyroidism 4. Rickets 8. Hepatitis and cirrhosis (slight increased) ENZYMOLOGY 203 Acid Phosphatase (ACP)/Acid Orthophosphoric Monoester Phosphohyrola It catalyzes the same reaction made byALP, except that it is active at pH 5.0. The major isoenzymes are coded for by different genes, with varied molecular weights, structures, and inhibition properties. ACP activity in the bones is associated with the osteoclasts. Normal men and women up to about age 55 have the same reference ranges of ACP. Tissue sources: Prostate (major source), RBCs, platelets, liver, and bone Prostatic and bone ACPs are the clinically significant isoenzymes. Reference range: Male = 2.5-11.7 U/L (Total ACP) 0-3.5 ng/mL (Prostatic ACP) Based on the differences at the structural level of the gene, ACP can be divided into 5 isoenzymes 1. Prostatic ACP: AcPP, human chromosomal location 3q21-q23 2. Erythrocytic ACP: AcP1, human chromosomal location 2p25 3. Lysosomal ACP: AcP2, human chromosomal location 11p12-p11 4. Testicular ACP: AcPT, human chromosomal location 19q13 5. Macrophagic ACP: AcP5, human chromosomal location 19p13.3-p13.1 Diagnostic Significance The major cause of elevated plasma ACP is prostatic disease. ACP is measured in the detection of prostatic adenocarcinoma. ACP assay aids in the detection of metastatic prostatic cancer, yet total ACP is not a sensitive marker, and prostate specific antigen (PSA) is a more useful screening and diagnostic tools. ACP IS also useful in forensic chemistry, in the investigation of rape cases vaginal washings are examined for seminal fluid-ACP activity, which can persist for up to 4 days. ACP activity > 50 IU/L indicates the presence of seminal fluid in the sample, in the case of forensic investigation. TRAP-5b is a marker for bone remodeling and for bone mineral disease (BMD) in patients with chronic kidney disease. TRAP-5b is associated with bone resorption while the bone ALP isoforms (B/I, B1, and B2) are related with bone deposition. TRAP-5b is considered to be a marker of the osteoclasts (Ishibashi, 2001). Increased ACP is observed in thrombocytopenia. ACP as a Tumor Marker Prostatic acid phosphatase (PAP) is used together with prostate specific antigen (PSA) monitor recurrence of to prostate cancer. PSA is more sensitive than PAP in detecting stages A and B prostatic cancer. After surgical treatment of prostate cancer, ACP levels fall faster than PSA, and plasma levels are expected to be undetectable following complete removal of tumor: Tartrate-resistant acid phosphatase (TRAP/TRACP) is present in some forms of cancer such as chronic leukemia, lymphomas, and hairy cell leukemia. TRACP-5a is found in Gaucher cells, or in the leukocytes of patients with hairy cell leukemia (Ishibashi, 2001). obstruction, acute urinary retention, extensive prostatic Other Causes of Increased Serum ACP: Urinary tract massage, prostatic inflammation, infarction/ischemia, and prostatic manipulations (needle biopsy and cystoscopy) TABLE 44. Summary of ACP Methods Substrate End Product Method Free thymolphthalein Roy and Hillman Thymolphthalein monophosphate Phenyl phosphate Inorganic phosphate Gutman and Gutman Para-nitrophenol PNPP (p-nitrophenyl phosphate) Alpha-naphthol Babson, Reed, and Phillips Alpha-naphthyl phosphate used in ACP. and substrates for he measurement of ALP activity are also NOTE: The methods  HANDBOOK IN CLINICAL CHEMISTRY 204 REVIEW Notes to Remember pH. Total ACP is measured by its ability to cleave groups at an acidic Serum sample must be free from hemolysis. total ACP. Icteric serum causes falsely decreased TRAP activity, but not for endpoint reaction. for quantitative Thymolphthalein monophosphate is the specific substrate and choice methods. a-naphthyl phosphate substrate is preferred for continuous monitoring 6.5. If not assayed immediately, serum should be frozen or acidified to a lower than pH With acidification, ACP is stable for 2 days at room temperature. Serum ACP decreases within to 2 hours if left room temperature. test for ACP if seminal fluid is present, provided a Fluid collected from the vagina on a cotton swab will give a positive stabilizing fluid with an acidic pH is used. Prostatic ACP is inhibited by 20-mM L-tartrate ions while 1-mM cupric sulfate and 2% formaldehyde ions inhibit red cell ACP. Increased ACP (with Metastatic Bone Involvement) 1. Prostatic carcinoma 2. Breast, lung, and thyroid carcinoma 3. Gaucher's disease 4. Niemann-Pick Disease TRANSFERASE/TRANSAMINASE Aspartate Aminotransferase (AST)/ Serum Glutamic Oxaloacetic Transaminase (SGOT) between aspartate and a-keto acids with the formation of It is involved in the transfer of an amino group oxaloacetate and glutamate. Isoenzyme Fraction: Cytoplasmic AST (ASTc) and mitochondrial AST (ASTm) Major tissue source: Cardiac tissue, liver, and skeletal muscle Other sources: Kidney, pancreas, and RBC ASTc is the abundant fraction in healthy serum. In case of tissue necrosis, ASTm is the predominant isoenzyme. ASTc and ASTm are true isoenzymes and are immunologically distinct (Botros and Sikaris, 2013). AST and LD have higher activity in the liver than ALT, but both are non-specific enzymes. AST and LD have equal concentrations in the hepatocytes. The kidney has a higher AST activity compared to ALT and LD. Reference range: 5-37 U/L Diagnostic Significance AST activity is vital in the evaluation of myocardial infarction, hepatocellular disorders, and skeletal muscle involvement. In acute myocardial infarction (AMI), AST levels begin to rise at 6 to normalize hours, peak at 24 hours, and within 5 days. It is released to a greater degree in chronic disorders of the liver with progressive damage. AST is used for monitoring therapy with potentially hepatotoxic drugs; a result more than 3x the upper borderof normal should signal cessation of therapy. AST is slight to moderately increased in muscular dystrophy or disorders minimal elevation or even normal. compared to ALT which has ENZYMOLOGY 205 Method Karmen Method It uses malate dehydrogenase (MD) and monitors the change in the absorbance at 340 nm continuously as is oxidized to NAD‡. Heparin may inhibit the activity of AST. AST activity is stable in serum for 3 to 4 days at refrigerated temperature. Hemolysis should be avoided because it increases AST 10x the upper reference limit (URL). Interferences: Hemolyzed and icteric samples (false increased) and presence of heavy metals (false decreased) AST Aspartate + a-ketoglutarate Oxaloacetate + Glutamate Oxaloacetate + NADH Malate + NAD* It catalyzes the transfer of an amino group from alanine to a-ketoglutarate with the formation of glutamate and Its highest concentration is in the liver, hence elevated plasma concentration denotes hepatic injury. It is almost present in the cytoplasm. It is more liver-tissue-specific compared to AST. Its enzymatic reaction is similar to AST. Major tissue source: Liver Other sources: kidney, pancreas, heart, skeletal muscles, lungs, and RBCs Reference range: 6-37 U/L Diagnostic Significance It is significant in the evaluation of hepatic disorders; has markedly increased concentration in acute inflammatory conditions involving the liver. Higher elevations are found in hepatocellular disease more than the extra-hepatic and intra-hepatic disorders. ALT is slightly increased in obstructive jaundice but markedly increased in necrotic jaundice. Though cardiac tissue has ALT activity, it is not included in the list of myocardial infarction (MI) markers since ALT has low activity in the heart and may not even exhibit plasma elevations in the presence of acute MI unless the hepatic is abnormally involved. It monitors the course of liver treatment (e.g., for hepatitis) and the effects of drug therapy. ALT levels are used to screen blood donors. ALT measurement is a more sensitive and specific screening test for post-transfusion hepatitis or occupational toxic exposure compared to AST. Method essential cofactor that should always be added in any Both ALT and AST require pyridoxal phosphate, an measurement. will result to diminished activity of both transferases. Absence of pyridoxal phosphate (vitamin B6) The use of icteric and lipemic samples may cause significant interference with this method. Use of hemolyzed sample cause false increase of AST activity while a slight elevation or none at all may be observed in ALT. effects of hemolysis on routine biochemistry parameters, ALT, GGT, In the study of Koseoglu et al (2011) on the inorganic phosphate, and cholesterol were not interfered up to severely hemolyzed levels with hemoglobin from 2.5 g/L to 4.5 g/L.  206 REVIEW HANDBOOK IN CLINICAL CHEMISTAN Coupled Enzymatic Reaction The reaction takes place at pH 7.5 and monitors the change in the absorbance at 340 nm continuously as NADH oxidized to NAD*, same with the AST reaction. Using hemolyzed sample may cause slight elevations in ALT activity. ALT Glutamate Alanine + a-ketoglutarate Pyruvate + NADH + H+ Lactate + NAD* TABLE 45. Comparison Between AST and ALT Using Reitman and Frankel Method ALT/SGPT AST/SGOT Substrate Aspartic Alpha-Ketoglutaric Acid Alanine Alpha-Ketoglutaric Acid End products Glutamic Acid + Oxaloacetic Acid Glutamic Acid + Pyruvic Acid Color developer 2,4 Dinitrophenyl hydrazone Color intensifier 0.4N NaOH Wavelength reading 505 nm (495 nm-535 nm) NOTE: Maximum incubation is up to 60 minutes room temperature and at 37° C. Notes to Remember Aminotransferases are present in human plasma, bile, CSF, and saliva. Aminotransferases require pyridoxal phosphate (vitamin B6) as coenzyme (prosthetic group). De Ritis described the AST/ALT ratio as being a useful indicator of the etiology of hepatitis such as acute viral hepatitis (Wroblewski, 1958, as cited in Botros and Sikaris, 2013). Erythrocytes contain three enzymes for synthesizing glutamate: alanine aminotransferae (ALT; EC 2.6.1.2), aspartate from a-ketoglutarate whereas GA synthesizes glutamate from glutamine (Ellinger et al., 2011). Serum ALT and AST Levels: Acute Hepatic injury versus Chronic Hepatic Injury In acute hepatitis, plasma AST, at first, will be higher than ALT due to increased activity of AST in the hepatocytes,; then, after 24 to 48 hours in the presence of continuous cellular damage, ALT will rise greater than AST because of its longer half-life. In chronic hepatocyte injury such as cirrhosis, initially ALT is higher than AST, however, as fibrosis progresses, ALT activity declines and AST gradually increases, so by the time full blown cirrhosis is present, AST is often higher (slight increased) than ALT. acute fulminant hepatic failure, an massive destruction of liver tissue In uncommon but highly fatal condition, results in complete liver failure (McPherson and Pincus, 2022). Though the exact cause of acute fulminant hepatitis is still unknown, conditions such as hepatitis B-delta (HBDV) superinfection, cirrhosis, Reye syndrome, high acetaminophen intake, alcoholism, and Budd- Chiari syndrome may contribute to the aminotransferases especially AST. development of the disease with extremely elevated activity of the In end-stage cirrhosis, the serum levels of both ALT and AST generally are not elevated and may result of massive tissue destruction. be low as the Acute liver failure by definition is also known as acute fulminant hepatic failure. Increased Transferase (ALT and AST) Toxic hepatitis 7. Hepatic cancer Myocardial Infarction* 2. Acute 8. Reye's syndrome 3. Wolff-Parkinson White Syndrome* 9. Viral hepatitis 4. Trichinosis* 10. Muscular Dystrophy* 5. Chronic alcoholism 11. Acute pancreatitis* 6. Dermatomyositis* *most elevations are seen in AST ENZYMOLOGY 207 The highest elevations of transferases, that is, combined serum levels, are hepatitis (A, B, and C) is seen in acute hepatitis. Viral levels of ALT and AST. one of the most common causes of acute liver injury resulting in increased plasma High serum levels of ALT and AST occur most commonly in acute hepatitis and/or mechanical damage to the liver (McPherson and Pincus, 2022). Severe viral or toxic hepatitis may produce elevations of transferase up to 20x the normal limits. Moderate elevation of transferases is observed in chronic hepatitis, hepatic cancer, and infectious mononucleosis, while minimalincrease in hepatic cirrhosis and obstructive jaundice may be reflected in the patient clinical data. The De Ritis ratio (ALT:AST) in acute hepatitis is > 1.0, whereas in alcohol-induced hepatic injury, the AST:ALT ratio is 3:1. High AST/ALT ratios suggest advanced alcoholic liver disease (Nyblom et al., 2004, as cited in McPherson and Pincus, 2022). The De Ritis ratio therefore reflects the time course of acute viral hepatitis and is generally a vital clue to the patient's prognosis (Botros and Sikaris, 2013). Deficiency in vitamin B6 seen in alcoholism results to decreased serum AST and ALT. In severe hepatocellular disease such as cirrhosis, most patients have low serum level or at times normal activity of ALT and AST. 10762 TABLE 46. Summary of Liver Function Tests for Common Disorders Disorders B2 Albumin ALT AST ALP Biliary obstruction N H N N N H Cirrhosis H N/*L N/*L N/ Sl. Inc Hepatitis H H N H H H *end-stage cirrhosis AMYLASE (AMS)/ ALPHA-1-4 GLUCAN-4-GLUCOHYDROLASE It catalyzes the breakdown of starch and glycogen. It is an important enzyme in the physiologic digestion of starch. It is a calcium-containing enzyme. It is the smallest enzyme in size (with a MW of 50,000 to 55,000 daltons), thus, it is filtered by the renal glomeruli and normally appears in the urine. It is the earliest pancreatic marker. The major isoenzymes are both present in normal, healthy sera. Major isoenzymes: S-type (ptyalin) and P-type (amylopsin) and S3 Isoforms of salivary amylase: S1, S2, Isoforms of pancreatic amylase: P1, P2, and P3 (predominant isoform in acute pancreatitis) glands Major sources: Acinar cells of the pancreas and the salivary muscles, and adipose tissue Other sources: Fallopian tubes, small intestine, skeletal Reference value: 60-180 *SU/dL 95-290 U/L IU) Conversion factor: 1.85 (Somogyi units to "SU, Somogyi unit Diagnostic Significance 12 hours after onset, peak at 24 hours, and normalize within 3 to to In acute pancreatitis, AMS levels rise 2 days. Increased plasma AMS is accompanied by increased urinary excretion. AMS in urine remains elevated for up to 7 days.  REVIEW HANDBOOK IN CLINICAL CHEMIS 208 also release AMS into the circulation, Causing Salivary gland inflammation (parotitis) due to mumps can elevated serum AMS. accompanied by decreased AMS is In renal failure in the absence of acute pancreatitis, increased plasma AMS. relapsing pancreatitis. Elevated renal clearance can be used as marker for acute and less specific than lipase Because amylase can also be produced by the salivary glands, amylase is as a marker in pancreatitis (McPherson and Pincus, 2022). Macroamylasemia It is characterized by elevated macroamylase in serum. frequent in men and usually disCovered more It not a disease but rather an acquired benign condition, 1989, as cited in McPherson and incidentally in the fifth through seventh decades (Remaley and Wilding, Pincus 2022). (either IgG or IgA); this enzyme-protein Macroamylase is a amylase attached to protein combination of an it remains in the blood circulation. complex cannot be excreted in urine due to its "macro" size, hence, serum lipase is normal Clinical findings: Persistent increased serum amylase without symptoms; Methods inactivation. Samples with high activity of AMS should be diluted with NaCl to prevent Elevated triglyceride may inhibit serum AMS activity. The administration of morphine and other opiates for pain relief before blood sampling will lead to Falsely elevated serum AMS levels. Samples with citrate, oxalate, and heparin should be avoided as they may inhibits AMS. Amylase is stable in serum and urine specimens for 7 days at room temperature. Specimen precaution: Avoid contamination with saliva (false increase of serum AMS) Substrate: Starch Saccharogenic It is the classic reference method expressed in Somogyi unit (SU). It measures the amount of reducing sugars produced by the hydrolysis of starch by the usual glucose methods. It measures amylase activity by following the decreases in substrate concentration (degradation of starch). Chromogenic It determines amylase activity by the increase in color intensity of the soluble dye-substrate solution produced in the reaction. Coupled-enzyme It quantifies amylase activity by a continuous-monitoring technique. AMS Maltose Glucosidase Maltrotriose + Maltose 5-glucose 5-glucose + 5 ATP 5-glucose-6-phosphate + 5 ADP G-6-PD 5-glucose-6-phosphate + NAD 5,6-phosphogluconolactone + 5 NADH Notes to Remember Generally, enzyme molecules are too large to pass through the excretion not a major route for elimination except amylase. healthy glomerulus of the kidneys, which makes Many endogenous inhibitors of AMS such as wheat germ are present in serum. ENZYMOLOGY 209 If threefold amylase increase with glycol precipitation. normal 24-hours urine amylase is observed, repeat serum AMS after polyethylene P3, the predominant amylopsin, is detected in renal failure. Electrophoresis: Isoamylases migrate to the beta and gamma region, and the most observed fractions are P2, S1, and S2. There is no uniform expression of AMS activity although Somogyi units are frequently used. Normal amylase/creatinine ratio: 1%-4% (0.01-0.04) Amylase:Creatinine (AC) ratio: > 4% ( up to 15%) Urine amylase Serum creatinine Formula for A/C ratio: x 100 Serum amylase Urine creatinine Increased Serum Amylase 1. Acute pancreatitis 2. Ectopic pregnancy 3. Peptic ulcers 4. Alcoholism 5. Mumps It is an enzyme that hydrolyzes the ester linkages of fats to produce alcohol and fatty acid. It catalyzes partial hydrolysis of dietary TAG in the intestine to the 2-monoglyceride intermediate, with the production of long chain fatty acids. It is the most specific pancreatic marker. Its primary and major source is the pancreas and is not affected by renal disorders. Plasma concentrations are normal in the disorders involving the salivary gland no lipase activity is yet to appear in the salivary gland. It is not present in normal urine. Pancreatic LPS (predominant in serum), intestinal LPS, lipoprotein LPS, and gastric LPS Isoforms of LPS: L1, L2 (sensitive and specific marker), and L3 Major tissue source: Pancreas Other sources: Stomach, liver, intestine, adipose tissues, breast milk, and WBCs Reference range: 0-1.0 U/mL Diagnostic Significance hours (average, 6 hours) after onset of attack, peak at 24 hours, In acute pancreatitis, LPS serum levels rise 4 to 8-14 days. remains elevated for 7 days, and normalize in and weeks. Serum LPS may be elevated for weeks may indicate the presence of pancreatic Persistent and prolonged elevations of serum lipase more than 2 cyst. In chronic pancreatitis, acinar cell degradation occurs, resulting in loss of amylase and lipase production. renal disorders, lipase is found in the urine. In patients with failure of renal tubular reabsorption caused by Urine lipase activity in the absence of pancreatic disease is inversely related to creatinine clearance (McPherson and Pincus, 2022). Methods In the traditional method, olive oil is a substrate because other esterases can hydrolyze TAG and synthetic Addition of colipase (protein secreted by the pancreas) and bile salts will make assay more sensitive and specific for the detection of acute pancreatitis. Calcium is an important activator for lipase.  CLINICAL 210 REVIEW HANDBOOK IN CHEMIST The enzymatic reaction is at optimum pH 8.8. (McPherson and Pincus, Icterus, lipemia, and hemolysis do not interfere with turbidimetric lipase assays 2022) Cherry-Crandall (reference method) of fatty acids using NaOH. It involves hydrolysis of olive oll after incubation for 24 hours at 37'C and titration Substrate: 50% olive oil End product: Fatty acid Reference range: 0-1.5 U/mL (0-417 U/L) Conversion factor:(278, U/mLtoU/L) aimine LPS Triglyceride + 2 H20 2-monoglyceride + 2 fatty acids (Olive oil) Tietz and Fiereck Substrate: Olive oil (Triglyceride) pH of the buffer: 8.0 Incubation: 3 hours End product: Fatty acid Peroxidase Coupling It is currently the most commonly used method. Substrate: Triolein (pure form of TAG) End product: Fatty acid LACTATE DEHYDROGENASE (LD/LDH) It catalyzes the interconversion of lactic and pyruvic acids. It is a zinc-containing enzyme that is part of the glycolytic pathway and Krebs cycle. It is a hydrogen-transfer enzyme that uses the coenzyme nicotinamide dinucleotide (NAD*). It is a tetrameric molecule containing four subunits of two possible forms (H and M). It is present in almost all cells in the body. In plasma, the majority of LD activity originates from the breakdown of erythrocytes and platelets, with varying contributions from other organ sources. The heart and red blood cells are the predominant sources of LD. Tissue sources: Heart, erythrocytes, kidneys lungs, pancreas, spleen (LD-3); skeletal muscles. liver, intestine (LD-4 and LD-5) Reference range: 100-225 U/L (Forward reaction) 80-280 U/L (Reverse reaction) TABLE 47. Differential Characteristics of LD Isoenzyme Tissue Source (HM subunits) Isoenzyme Activity as : Percentage of Normal Total LD LD-1 (HHHH) Heart, erythrocytes, and renal cortex 17-27% LD-2 (HHHM) 27-37% LD-3 (HHMM) Lungs, renal cortex, pancreas, spleen, platelets, and adrenal gland 18-25% LD-4 (HMMM) Skeletal muscles, liver, intestine, and skin 3-8% 0-5% LD Tissue Sources LD-1 is relatively abundant in cardiac mnuscles, whereas LiD-s is more abundant in sheletal muscle. LD-1 is not found in the skeletal muscles and liver. ENZYMOLOGY 211 LD-2 is the major isoenzyme in the sera of healthy persons. LD-2 serum activity is greater than RBCs and cardiac tissues contain high levels of LD-1 and LD-2. LD-3 and LD-4 are also present in minimal activity in the heart and L-4 is the most abundant isoenzyme in the skeletal muscle. LD-5 is most abundant isoenzyme in the liver but almost undetectable in the heart, RBCs, and renal cortex. LD will be clinically significant if separated into isoenzyme fractions. Diagnostic Significance Highest LD serum levels are seen in In acute myocardial infarction, LD levels begin to rise within 12 to 24 hours, peak levels within 48 to 72 hours, and remains elevated for 10 to 14 days. LD-1 > LD-2, also known as the "flipped pattern," is seen in myocardial infarction and hemolytic anemia. LD activity in pleural fluids is useful for differentiating transudates (low LD) from exudates (high LD). Pheochromocytoma tissue as well as normal human adrenal tissue contained LDH, maximally type 3; the amount of LDH in tumors far exceeded that in normal adrenal glands, suggesting that the tumor tissue is the source of the excessive serum LDH in these patients (O'Connor and Gochman, 1983). Viral hepatitis and cirrhosis would give LD slightly increased values (2-3x URL) - this elevation is temporary, usually the plasma level will return to normal after onset of symptoms.I She BundsO-baweldowW ebontam Hepatic carcinoma and toxic hepatitis will have tenfold increase. LD-5 is moderately increased in acute viral hepatitis and cirrhosis and markedly increased in hepatic cancer (CA) and toxic hepatitis. LD activity may be considered as a marker in occupational hazards such as in silica-induced toxicity. In the study of Aggarwal (2014), the LDH activity in blood plasma samples of silica-exposed agate workers was found to increase about 25x, while the activity in the blood cells was reduced to 10% in control subjects; while the ratio of LDH activity (blood plasma/cells) was found to be 6.6 in the silica-exposed agate workers, it was 0.02 in control (non-exposed) subjects. Therefore, the total LDH activity and the LDH ratio (plasma/cells), along with occupational exposure history, are markers for silica exposure-induced toxicity in agate workers. An elevated total LD is a non-specific result because of its presence from several tissues. TIE LL YON LD as a Tumor Marker LD-2, LD-3, and LD-4 are cancer markers (predominantly LD-3) for acute leukemia, germ cell tumors, and breast and lung cancers. Serum LD is increased in metastatic CA due to its multiple organ sources. prognostic CA marker as it may determine response therapy. to Serum LD level is for prostate cancer, elevated LD-5 levels have been found to be In patients who have undergone radiotherapy rates and relapse following radiotherapy (Koukourakis et al., strongly indicative of high tumor proliferation 2014, as cited in McPherson and Pincus, 2022). Other LD Isoforms V LD-6 represents the alcohol dehydrogenase enzyme; the sixth band in electrophoresis; responsible for to toxic compounds; present in patients with the metabolic conversion of methanol and ethylene glycol arteriosclerotic failure; may be elevated in drug hepatoxicity and obstructive jaundice. in semen; not found yet in human of four C subunits is detected in spermatozoa and serum or even LD composed in individuals with seminoma. Methods Lactate is a more specific substrate compared to reaction. reverse LD-1 prefers the forward reaction, whereas LD-5 prefers the 6 days on storage at 4° C. LD is stable at room temperature for 48 hours and at least Direct Immunoassay It is currently the most widely used method in the clinical laboratory.  REVIEW HANDBOOK IN CLINICAL CHEMISTO 212 Wacker Method (Forward/Direct Reaction) it is not It is preferred over the reverse method because it produces a positive rate (NADH) and affected by product inhibition. The reaction is at pH 8.8 using 340 nm. Lactate + NAD Wroblewski-Ladue (Reverse/Indirect Reaction) It is about 2x faster as the forward reaction. It is used in dry slide technology. The completion of the reaction is at pH 7.2. Advantage: Small volume of the sample MENOM Disadvantages: ALT and pyruvate dehydrogenase react with the pyruvate substrate, early loss of linearity, and influenced by LD inhibitors. Lactate NAD Pyruvate + NADH Other methods: Wroblewski-Cabaud and Berger-Broida™019/5 Notes to Remember Hemolyzed samples should be totally avoided due to very high levels of LD in the red blood cells. Serum LD is higher than the plasma LD due to release of the enzyme from platelets. LD activity in serum or plasma is temperature-dependent (mostly LD-1 and LD-5), and it varies when LD is suspended at room temperature or in cold storage. Decreased activity is observed when samples are frozen (LD-5 is cold-labile), therefore samples should be processed within 24 hours after collection and stored at 25° C. Frozen serum is unacceptable for measurement of total LD and its isoenzymes, nevertheless the LD activity of serum samples frozen -20° C has been found to be stable for one month (Chua, 2018, as cited in McPherson and Pincus, 2022). LD can use other substrates in addition to lactate and pyruvate, such as a-hydroxybutyrate. a-hydroxybutyrate dehydrogenase (a-HBD) represents the LD-1 activity. a-hydroxybutyrate activity is elevated in conditions in which both LD-1 and LD-2 are increased. There is transient elevation of total LD after blood transfusion but plasma level returns to baseline within 24 hours. Increased LD 1. Anemias (pernicious, hemolytic, and megaloblastic) 2. Myocardial infarction 3. Leukemia 4. Renal infarction 5. Hepatitis and hepatic cancer Muscular dystrophy 7. Delirium tremens 8. Malignancy 9. Pneumocystis jerovecii pneumonia CREATINE KINASE (CK)/ATP-CREATINE-N-PHOSPHOTR ARCELENIL It catalyzes the transfer of a phosphate group between creatine phosphate and adenosine diphosphate. It is involved in the storage of high-energy creatine phosphate in the muscles It is a dimeric molecule with small molecular size, composed called of a pair of two different monomers M and B. ENZYMOLOGY 213 It is found in small CK from brain tissue amounts throughout the body, but high concentrations are in the muscles and brain, although never crosses the blood-brain barrier to reach normal plasma. Major tissue sources: Brain, smooth and skeletal muscles, and cardiac muscles Reference range: Male = 15-160 U/L Female = 15-130 U/L CK-MB = < 6% of total CK Isoenzymes: CK-BB (brain type), CK-MB (hybrid type), and CK-MM (muscle type) CK-1 is the most anodal and labile isoenzyme; CK-3 is the least anodal. CK-BB is the dominant isoenzyme of CK found in brain, intestine, and smooth muscle. Serum of adults rarely contains CK-BB of brain origin due to its high molecular size; it may be normally present in neonatal sera. Cardiac tissues contain significant amounts of CK-MB (20%) myocardium is the only tissue from which CK-MB enters the serum in significant quantities. CK-MB in serum of healthy person is < 5 g/L. CK-MB is utilized as a serodiagnostic test for myocardial infarction. CK-MMis both abundantly present in the cardiac and skeletal muscles. ith In the sera of healthy persons, CK-MM is the major isoenzyme (94-100%). Physically well-trained individuals tend to have elevated baseline levels of total CK. Direct muscle trauma, as seen in contact sports, surgery, strenuous exercise, and intramuscular injection, are common causes of mild elevations of serum CK (up to about five to six times reference limits). Intramuscular injections are known to increase CK (< 5x URL). Bedridden patients may have decreased CK activity. Causes of chronic muscle injury:IV drug abuse, medications such as statin, Duchenne muscular dystrophy, inflammatory disorders (polymyositis and dermatomyositis), hypothyroidism, and alcohol abuse 1. Intramitochondrial CK It is also known as the MtCK or non-muscle/ubiquitous MtCK (uMtCK). lizang wond ot babamon alaidf Elevated serum MtCK is seen in cirrhosis or hepatitis. It may be a reliable independent predictor of development of hepatocellular carcinoma. 2. Sarcomeric Muscle CK It supports the myofibrillar structure and contractility (Li et al., 2015). Diagnostic Significance Highest elevation of total CK is seen in Duchenne's muscular dystrophy (50x URL). CK-BB is increased in cerebrovascular injury. CK-MB is found mainly in myocardial tissue - it is used as a serodiagnostic test for AMI. Demonstration of elevated levels of CK-MB, ≥ 6% of the total CK, is considered the most specific indicator of myocardial damage, particularly acute myocardial infarction (AMI). to rise within 4 to 8 hours, peak at 12 to 24 hours, and normalize within Following AMI, the CK-MB levels begin 48 to 72 hours. majority of CK-MM elevations. Injury to both cardiac and skeletal muscle accounts for the muscle from crush injury, convulsions, tetany, surgical Total CK is markedly elevated after trauma to skeletal incision, or intramuscular injections. for neuroleptic malignant syndrome. Only CK is located in the smooth muscles; not ALT, AST, and LD. cardiac troponins in the assessment of AMI. CK-MB is not elevated in angina, and it has been replaced by CK-MB though increased in AMI within 4 hours of chest pain, had low sensitivity and specificity as compared to GPBB and myoglobin (Singh et al., 2018).  214 REVIEW HANDBOOK IN CLINICAL CHEMISTRy CHEMIC Other biomarkers (e.g., CK MB) are less sensitive and less specific in determining myocardial injury (Coodmaner al., 2006, as cited in Thygesen et al., 2018). acute AMI, it has been diagnosis of To increase both the sensitivity and specificity of CK-MB in the 2022). found necessary to perform serial determinations of MB fraction (McPherson and Pincus, Methods Tanzer-Gilvarg Assay (Forward/Direct method) The reaction takes place at pH 9.0 and wavelength of 340nm. Creatine + ATP Creatine PO4 + Phosphoenolpyruvate Pyruvate + NADH Lactate + NAD Oliver-Rosalki (Reverse/Indirect method) It is the commonly used method due to faster reaction at pH 6.8 and wavelength of 340 nm. Creatine PO4 + ADP Creatine + ATP ATP + glucose ADP + Glucose-6-PO G-6-PD Glucose-6-PO + NADP 6-phosphogluconate + NADPH CK Relative Index (CKI) It is an expression of the percentage of the total CK that is attributed to CK-MB. This is computed to know possible release of CK-MB from non-cardiac tissues when total CK is very high. CK - MB 1g/L or IU/L x 100 Total CK IU/L Notes to Remember Liver cells and RBC do not contain CK, and any disorder affecting those cells will not significantly alter the plasma concentration of CK. assay, particularly with hemolysis of > 320 mg/L. Adenosine monophosphate (AMP) is added to the reverse hemolysis since AK hydrolyzes ADP. method to inhibit AK which may be present in the serum from N-acetylcysteine is added to CK reagent to activate the enzyme (aside from Mg+2) oxidized sulfhydryl groups. and partially reverse the inhibition of Imidazole serves as a buffer; urate and cystine are potent CK inhibitors. Cleland's reagent and glutathione partially restore lost activity of CK. CK is light- and pH-sensitive; it is also lost with excessive storage. Sodium azide when added to reagents inhibit bacter 1 growth and increase both the sensitivity and the interfering enzymes (peroxidase and catalase). To specificity of CK-MB in the diagnosis of acute AMI, it has been found necessaly to perform serial determinations of MB fraction (at 3- to 4-hour intervals over a 12- show a progressive rise that reaches a peak, followed by a fall to low levels. to 16-hour period) that CK mass unit assay is more sensitive than electrophoresis but electrophoresis, is still the reference method for CK ENZYMOLOGY 215 Purpose of EDTA and Sulfhydryl in CK assay The inclusion of EDTA in the creatine kinase reagent recommended by the Scandinavian Committee on Enzymes was shown to increase reagent stability from less than 24 hours to days. Part of this effect can be explained by the fact that EDTA delays the formation of inhibitory products formed when N-acetyl cysteine is oxidized. The addition of EDTA to the reagent also results in increased measured CK activity. This effect is more pronounced for CK-BB than for CK- MM. Calcium and ferric ions are shown to inhibit the enzyme and the chelation of these ions can partly explain the observed increase of CK activity (Gerhardt et al., 1978) Addition of sulfhydryl groups to the reaction mixture, to stabilize serum creatine kinase (creatine phosphokinase, CPK), results in apparent increases in the activity of this enzyme in many sera. n addition, in sera from patients just after myocardial infarction, assays for sulfhydryl-activated CPK have a different clinical pattern than do those for CPK assayed without sulfhydryl activators: activities are greater and remain abnormally high longer in assays in which the enzyme is sulfhydryl activated (Dalal, 1972). Increased Total CK 1. Duchenne's muscular dystrophy 6. Rhabdomyolysis 2. Myocardial infarction 7. Carbon monoxide poisoning 3. Hypothyroidism 8. Rocky Mountain Spotted Fever 4. Pulmonary infarction 9. Cerebral vascular accident (occasional) 5. Reye's syndrome ALDOLASE (ALDO)/FRUCTOSE 1,6-DIPHOSPHATE ALDOLASE It is a glycolytic enzyme that splits fructose-1,6-diphosphate into two triose phosphate molecules in the metabolism of glucose. It is included in the panel of markers for skeletal muscle injury. Increased: Skeletal muscle disease, leukemia, hemolytic anemia, and hepatic cancer odgonom- Major Isoenzymes and Tissue Sources Aldolase A: Skeletal muscle Aldolase B: Liver, kidney, and WBC Aldolase C: Brain Tissue 8.1L8.0.8:2009 eantoisentlodbobused Gamma-Glutamyl Transferase (GGT): E.C. 2.3.2.2 It catalyzes the transfer of glutamyl groups between peptides or amino acids through linkage at a gamma- carboxyl group. It is located in the canaliculi of the hepatic cells and particularly in the epithelial cells lining the biliary duct; also in the kidney, prostate, and pancreas. the hepatic surface while the ALP is on the canalicular canalicular system in the liver, it is located on In the surface. It affects the cell membrane and microsomal fractions. It is elevated among individuals undergoing warfarin, phenobarbital, and phenytoin therapies. GGT due to impaired pancreas. Patients with diabetes mellitus (DM) may have increase Increased: Obstructive jaundice, alcoholism, and DM Tests for obstructive jaundice: ALP, GGT, 5' NT, and LAP Substrate: Gamma-glutamyl-p-nitroanilide Methods: Szass, Rosalki, and Tarrow; Orlowski Other methods: Electrochemical and Pincus, 2022) Reference range: 5-40 U/L (McPherson 8-61 U/L (Mayo Clinic, n.d.)  IN CLINICAL 216 REVIEW HANDBOOK CHEMIST Diagnostic Significance It is useful in differentiating the source of an increased ALP level. remains normal even In which during Serum levels of GGT differ from those of ALP during pregnancy Cholestasis in pregnancy (McPherson and Pincus, 2022). It is elevated in all hepatobiliary disorders such as biliary tract obstructions. It is the most sensitive marker of acute alcoholic hepatitis. It is a sensitive indicator of alcoholism loceult alcoholism); however, it is often elevated in alcoholics even without liver disease. It is useful in monitoring the effects of abstention from alcohol. It is found to be elevated in Individuals taking high dosage of anti-inflammatory and anti-seizure drugs such as acetaminophen, carbamazepine, and phenytoin. It is also increased in pancreatitis and prostatic disorders. 5' Nucleotidase (5'-NT): E.C. 3.1.3.5 It is a phosphoric monoester hydrolase; predominantly secreted from the liver. It is a differential test for serum ALP to detect the source of the elevated ALP, if liver or bone. It is a marker for hepatobiliary disease and infiltrative lesions of the liver. It is routinely increased in cholestatic disorder. It has slightly increased concentration during pregnancy. elevation in 5'-NT in plasma Acute hepatitis causes an increase in 5'-NT synthesis by the liver and a slight (Fukano et al., 1990, as cited in McPherson and Pincus, 2022). Measurement pitfalls: Some enzymes like ALP may react with the 5-'NT substrate and affect the detection limit of 5-'NT. Substrate: 5'-monophosphate (5'-IMP) Common method: Quantitative Enzymatic Other methods: Dixon and Purdon, Campbell, Belfield and Goldberg Reference range: 0-1.6 U (McPherson and Pincus, 2022) 1-15 U/L (Mayo Clinic, n.d.) Pseudocholinesterase (PChE): E.C. 3.1.1.8 It is also known as the butyrylcholinesterase (BuChe) or acetylcholine acylhydrolase. It is secreted by the liver, hence, measurement of this enzyme reflects synthetic function rather than hepatocyte injury; a secondary liver function test. Itis a marker of insecticide/pesticide poisoning (organophosphate poisoning). It also determines high exposure to nerve poisons such as sarin (methyl isopropyl fluorophosphate). It is used to monitor the effect of musclerelaxants (succinylcholine) after surgery; pre-operative screening for patients with a history or family history of prolonged paralysis and apnea after the use of the muscle relaxant, succinylcholine, for anesthesia (Mayo Clinic, n.d.). It catalyzes the removal of benzyl group from cocaine by acting as an "antixenobiotic enzyme." It is involved in the metabolism of anticholinergic drugs. It is readily available in plasma compared to acetylcholinesterase. Tissue source: Liver, heart, pancreas, and CNS (white matter of the brain) Serum level in poisoning: Low PCHe Decreased: Malnutrition and liver disease (acute hepatitis, cirrhosis. and carcinoma metastatic to the liver) Substrate: Butyrylcholine Methods: Ellman technique and potentiometric Reference range: 0.5-1.3 pH units (plasma) ENZYMOLOGY 217 TABLE 48. Differential Characteristics Between Acetylcholinesterase and Butyrycholinester Indicators Acetylcholinesterase (AChE) Butyrylcholinesterase (BuChE) Enzyme Commission E.C. 3.1.1.7 E.C. 3.1.1.8 Number Description True cholinesterase Pseudocholinesterase Terminates neuronal transmission and signaling Function Metabolism of drugs (muscle relaxant, between synapses to prevent AChE dispersal and anesthetics, illicit drugs, etc.) activation of nearby receptors Tissue Sources RBCs, lungs, spleen, skeletal muscle, and CNS (gray Liver, heart, pancreas, and CNS (white matter matter of the brain) of the brain) Diagnostic Significance Detects chronic exposure Detects acute exposure (Organophosphate AChE is inhibited by organophosphate, an important poisoning) component of pesticides and nerve agents (Trang and 10 000 Khandhar, 2022). Sample EDTA whole blood Serum Reference Range *0.65-1.3 pH units *0.5-1.3 pH units ** 31.2-61.3 U/g of hemoglobin **Males: 320-12,920 U/L (Quantitative Amniotic AChE is a test for neural tube *Females: 0-15 years: 5,320-12,920 U/L defects.) 16-39 years: 4,260-11,250 U/L 40-41 years: 5,320-12,920 U/L or =42 years: 5,320-12,920 U/L *McPherson and Pincus, 2022 **Mayo Clinic (https://endocrinology.testcatalog.org/show/ACHS) Angiotensin-Converting Enzyme (ACE): E.C. 3.4.15.1 It is also known as peptidyl-dipeptidase A or kininase II. It is an aspartic acid protease. It is a hydrolase enzyme that requires zinc for activation. It has two forms encoded by one and the same gene. It is found primarily in the vascular endothelium of the lungs and kidneys (Fountain and Lappin, 2021). It is a component of the renin-angiotensin system. I beablbong al 11 It converts the inactive angiotensin to its active form, the angiotensin (I, within the lungs. It is diagnostic test for sarcoidosis. It is a possible indicator of neuronal dysfunction (Alzheimer's disease using CSF sample). It is a critical target for inhibitory drugs designed to lower blood pressure. It normally has low concentration in plasma, since it is mostly cell-bound (endothelial cell membranes). ACE2 is the cellular receptor of SARS and SARS-CoV-2. for serum or other body fluid assay for ACE2 assays (McPherson There appears to be little clinical use at present and Pincus, 2022). Forms of ACE: somatic (sACE-present in many tissues) and testes (tACE) Main function: To cleave histidine-leucine sequence from a decapeptide called angiotensin 1 Major tissue sources: Lungs and Kidneys Other sources: Heart, muscles, neurons, macrophages, and epitheloid cells Diagnostic significance: Diagnosis and monitoring of sarcoidosis Increased: Sarcoidosis, multiple sclerosis, pneumonia, acute and chronic bronchitis, leprosy, HIV infection, and Addison's disease Sample: Serum Method: Spectrophotometric assay  218 REVIEW HANDBOOK IN CLINICAL CHEMIST Glucose-6-Phosphate Dehydrogenase (G-6-PD): E.C. 1.1.1.49 It functions to maintain NADPH in the reduced form in the erythrocytes. It is a newborn screening marker. Deficiency of this enzyme can lead to drug-induced hemolytic anemia after taking primaquine, an antimal drug. When exposed to an oxidant drug (e.g., antimalarial drug), affected individuals experience : hemolytic episode and the severity of the hemolysis is directly related to the drug concentration. in the same