Enzymes PDF

Enzymes are specific biologic proteins that catalyze biochemical reactions without altering the equilibrium point of the reaction or being consumed or changed in composition. The other substances in the reaction are converted to products. The catalyzed reactions are frequently specific and essential...

Enzymes are specific biologic proteins that catalyze biochemical reactions without altering the equilibrium point of the reaction or being consumed or changed in composition. The other substances in the reaction are converted to products. The catalyzed reactions are frequently specific and essential to physiologic functions, such as the hydration of carbon dioxide, nerve conduction, muscle contraction, nutrient degradation, and energy use. Found in all body tissue, enzymes frequently appear in the serum following cellular injury or, sometimes, in smaller amounts, from degraded cells. Certain enzymes, such as those that facilitate coagulation, are specific to plasma and, therefore, are present in significant concentrations in plasma. Plasma or serum enzyme levels are often useful in the diagnosis of particular diseases or physiologic abnormalities. This chapter discusses the general properties and principles of enzymes, aspects relating to the clinical diagnostic significance of specific physiologic enzymes, and assay methods for those enzymes. GENERAL PROPERTIES AND DEFINITIONS Enzymes catalyze many specific physiologic reactions. These reactions are facilitated by the enzyme structure and several other factors. As a protein, each enzyme contains a specific amino acid sequence (primary structure), with the resultant polypeptide chains twisting (secondary structure), which then folds (tertiary structure) and results in structural cavities. If an enzyme contains more than one polypeptide unit, the quaternary structure refers to the spatial relationships between the subunits. Each enzyme contains an active site, often a water-free cavity, where the substance on which the enzyme acts (the substrate) interacts with particular charged amino acid residues. An allosteric site—a cavity other than the active site—may bind regulator molecules and, thereby, be significant to the basic enzyme structure. Even though a particular enzyme maintains the same catalytic function throughout the body, that enzyme may exist in different forms within the same individual. The different forms may be differentiated from each other based on certain physical properties, such as electrophoretic mobility, solubility, or resistance to inactivation. The term isoenzyme is generally used when discussing such enzymes; however, the International Union of Biochemistry (IUB) suggests restricting this term to multiple forms of genetic origin. An isoform results when an enzyme is subject to posttranslational modifications. Isoenzymes and isoforms contribute to heterogeneity in properties and function of enzymes. In addition to the basic enzyme structure, a nonprotein molecule, called a cofactor, may be necessary for enzyme activity. Inorganic cofactors, such as chloride or magnesium ions, are called activators. A coenzyme is an organic cofactor, such as nicotinamide adenine dinucleotide (NAD). When bound tightly to the enzyme, the coenzyme is called a prosthetic group. The enzyme portion (apoenzyme), with its respective coenzyme, forms a complete and active system, a holoenzyme. Some enzymes, mostly digestive enzymes, are originally secreted from the organ of production in a structurally inactive form, called a proenzyme or zymogen. Other enzymes later alter the structure of the proenzyme to make active sites available by hydrolyzing specific amino acid residues. This mechanism prevents digestive enzymes from digesting their place of synthesis. ENZYME CLASSIFICATION AND NOMENCLATURE To standardize enzyme nomenclature, the Enzyme Commission (EC) of the IUB adopted a classification system in 1961; the standards were revised in 1972 and 1978. The IUB system assigns a systematic name to each enzyme, defining the substrate acted on, the reaction catalyzed, and, possibly, the name of any coenzyme involved in the reaction. Because many systematic names are lengthy, a more usable, trivial, recommended name is also assigned by the IUB system.1 In addition to naming enzymes, the IUB system identifies each enzyme by an EC numerical code containing four digits separated by decimal points. The first digit places the enzyme in one of the following six classes: 1. 2. 3. 4. 5. 6. Oxidoreductases. Catalyze an oxidation–reduction reaction between two substrates Transferases. Catalyze the transfer of a group other than hydrogen from one substrate to another Hydrolases. Catalyze hydrolysis of various bonds Lyases. Catalyze removal of groups from substrates without hydrolysis; the product contains double bonds Isomerases. Catalyze the interconversion of geometric, optical, or positional isomers Ligases. Catalyze the joining of two substrate molecules, coupled with breaking of the pyrophosphate bond in adenosine triphosphate (ATP) or a similar compound The second and third digits of the EC code number represent the subclass and sub-subclass of the enzyme, respectively, divisions that are made according to criteria specific to the enzymes in the class. The final number is the serial number specific to each enzyme in a sub-subclass. Table 13.1 provides the EC code numbers, as well as the systematic and recommended names, for enzymes frequently measured in the clinical laboratory. TABLE 13.1 Classification of Frequently Quantitated Enzymes Adapted from Competence Assurance, ASMT. Enzymology, An Educational Program. Bethesda, MD: RMI Corporation; 1980. Table 13.1 also lists common and standard abbreviations for commonly analyzed enzymes. Without IUB recommendation, capital letters have been used as a convenience to identify enzymes. The common abbreviations, sometimes developed from previously accepted names for the enzymes, were used until the standard abbreviations listed in the table were developed. 2,3 These standard abbreviations are used in the United States and are used later in this chapter to indicate specific enzymes. ENZYME KINETICS Catalytic Mechanism of Enzymes There are some chemical reactions that may occur at a slow rate if there is not enough kinetic energy to drive the reaction to the formation of products (uncatalyzed reaction). Other chemical reactions may occur spontaneously if the free energy or available kinetic energy is higher for the reactants than for the products. The reaction then proceeds toward the lower energy if a sufficient number of the reactant molecules possess enough excess energy to break their chemical bonds and collide to form new bonds. The excess energy, called activation energy, is the energy required to raise all molecules in 1 mole of a compound at a certain temperature to the transition state at the peak of the energy barrier. At the transition state, each molecule is equally likely to either participate in product formation or remain an unreacted molecule. Reactants possessing enough energy to overcome the energy barrier participate in product formation. One way to provide more energy for a reaction is to increase the temperature, which will increase intermolecular collisions; however, this does not normally occur physiologically. Enzymes catalyze physiologic reactions by lowering the activation energy level that the reactants (substrates) must reach for the reaction to occur (Fig. 13.1). The reaction may then occur more readily to a state of equilibrium in which there is no net forward or reverse reaction, even though the equilibrium constant of the reaction is not altered. The extent to which the reaction progresses depends on the number of substrate molecules that pass the energy barrier. FIGURE 13.1 Energy versus progression of reaction, indicating the energy barrier that the substrate must surpass to react with and without enzyme catalysis. The enzyme considerably reduces the free energy needed to activate the reaction. The general relationship among enzyme, substrate, and product may be represented as follows: (Eq. 13-1) where E is enzyme, S is substrate, ES is enzyme–substrate complex, and P is product. The ES complex is a physical binding of a substrate to the active site of an enzyme. The structural arrangement of amino acid residues within the enzyme makes the three-dimensional active site available. At times, the binding of ligand drives a rearrangement to make the active site. The transition state for the ES complex has a lower energy of activation than the transition state of S alone, so that the reaction proceeds after the complex is formed. An actual reaction may involve several substrates and products. Different enzymes are specific to substrates in different extents or respects. Certain enzymes exhibit absolute specificity, meaning that the enzyme combines with only one substrate and catalyzes only the one corresponding reaction. Other enzymes are group specific because they combine with all substrates containing a particular chemical group, such as a phosphate ester. Still other enzymes are specific to chemical bonds and thereby exhibit bond specificity. Stereoisomeric specificity refers to enzymes that predominantly combine with only one optical isomer of a certain compound. In addition, an enzyme may bind more than one molecule of substrate, and this may occur in a cooperative fashion. Binding of one substrate molecule, therefore, may facilitate binding of additional substrate molecules. Factors That Influence Enzymatic Reactions Substrate Concentration The rate at which an enzymatic reaction proceeds and whether the forward or reverse reaction occurs depend on several reaction conditions. One major influence on enzymatic reactions is substrate concentration. In 1913, Michaelis and Menten hypothesized the role of substrate concentration in formation of the enzyme–substrate (ES) complex. According to their hypothesis, represented in Figure 13.2, the substrate readily binds to free enzyme at a low substrate concentration. The reaction rate steadily increases when more substrate is added, with the amount of enzyme exceeding the amount of substrate. When this occurs, the reaction is following first-order kinetics because the reaction rate is directly proportional to substrate concentration. Eventually, when the substrate concentration is high enough to saturate all available enzyme, the reaction velocity has reached its maximum. When product is formed, the resultant free enzyme immediately combines with excess free substrate. The reaction is in zero-order kinetics, and the reaction rate depends only on enzyme concentration. FIGURE 13.2 Michaelis-Menten curve of velocity versus substrate concentration for enzymatic reaction. Km is the substrate concentration at which the reaction velocity is half of the maximum level. The Michaelis-Menten constant (Km), derived from the theory of Michaelis and Menten, is a constant for a specific enzyme and substrate under defined reaction conditions and is an expression of the relationship between the velocity of an enzymatic reaction and substrate concentration. The assumptions are made that equilibrium among E, S, ES, and P is established rapidly and that the E + P → ES reaction is negligible. The rate-limiting step is the formation of product and enzyme from the ES complex. Then, maximum velocity is fixed, and the reaction rate is a function of only the enzyme concentration. As designated in Figure 13.2, Km is specifically the substrate concentration at which the enzyme yields half the possible maximum velocity. Therefore, Km indicates the amount of substrate needed for a particular enzymatic reaction. The Michaelis-Menten hypothesis of the relationship between reaction velocity and substrate concentration can be represented mathematically as follows: (Eq. 13-2) where V is measured velocity of reaction, Vmax is maximum velocity, [S] is substrate concentration, and Km is Michaelis-Menten constant of enzyme for specific substrate. Theoretically, Vmax and then Km could be determined from the plot in Figure 13.2. However, Vmax is difficult to determine from the hyperbolic plot and often not actually achieved in enzymatic reactions because enzymes may not function optimally in the presence of excessive substrate. A more accurate and convenient determination of Vmax and Km may be made through a Lineweaver- Burk plot, a double-reciprocal plot of the Michaelis-Menten constant, which yields a straight line (Fig. 13.3). The reciprocal is taken of both the substrate concentration and the velocity of an enzymatic reaction. The equation becomes FIGURE 13.3 Lineweaver-Burk transformation of Michaelis-Menten curve. Vmax is the reciprocal of the x-intercept of the straight line. Km is the negative reciprocal of the x-intercept of the same line. (Eq. 13-3) Enzyme Concentration Because enzymes catalyze physiologic reactions, the enzyme concentration affects the rate of the catalyzed reaction. As long as the substrate concentration exceeds the enzyme concentration, the velocity of the reaction is proportional to the enzyme concentration. The higher the enzyme level, the faster the reaction will proceed because more enzyme is present to bind with the substrate. pH Enzymes are proteins that carry net molecular charges. Changes in pH may denature an enzyme or influence its ionic state, resulting in structural changes or a change in the charge on an amino acid residue in the active site. Hence, each enzyme operates within a specific pH range and maximally at a specific pH. Most physiologic enzymatic reactions occur in the pH range of 7.0 to 8.0, but some enzymes are active in wider pH ranges than others. In the laboratory, the pH for a reaction is carefully controlled at the optimal pH by means of appropriate buffer solutions. Temperature Increasing temperature usually increases the rate of a chemical reaction by increasing the movement of molecules, the rate at which intermolecular collisions occur, and the energy available for the reaction. This is the case with enzymatic reactions until the temperature is high enough to denature the protein composition of the enzyme. For each 10 degree increase in temperature, the rate of the reaction will approximately double until, of course, the protein is denatured. Each enzyme functions optimally at a particular temperature, which is influenced by other reaction variables, especially the total time for the reaction. The optimal temperature is usually close to that of the physiologic environment of the enzyme; however, some denaturation may occur at the human physiologic temperature of 37°C. The rate of denaturation increases as the temperature increases and is usually significant at 40°C to 50°C. Because low temperatures render enzymes reversibly inactive, many serum or plasma specimens for enzyme measurement are refrigerated or frozen to prevent activity loss until analysis. Storage procedures may vary from enzyme to enzyme because of individual stability characteristics. Repeated freezing and thawing, however, tends to denature protein and should be avoided. Because of their temperature sensitivity, enzymes should be analyzed under strictly controlled temperature conditions. Incubation temperatures should be accurate within ±0.1°C. Laboratories usually attempt to establish an analysis temperature for routine enzyme measurement of 25°C, 30°C, or 37°C. Attempts to establish a universal temperature for enzyme analysis have been unsuccessful and, therefore, reference ranges for enzyme levels may vary significantly among laboratories. In the United States, however, 37°C is most commonly used. Cofactors Cofactors are nonprotein entities that must bind to particular enzymes before a reaction occurs. Common activators (inorganic cofactors) are metallic (Ca2+, Fe2+, Mg2+, Mn2+, Zn2+, and K+) and nonmetallic (Br− and Cl−). The activator may be essential for the reaction or may only enhance the reaction rate in proportion with concentration to the point at which the excess activator begins to inhibit the reaction. Activators function by alternating the spatial configuration of the enzyme for proper substrate binding, linking substrate to the enzyme or coenzyme, or undergoing oxidation or reduction. Some common coenzymes (organic cofactors) are nucleotide phosphates and vitamins. Coenzymes serve as second substrates for enzymatic reactions. When bound tightly to the enzyme, coenzymes are called prosthetic groups. For example, NAD as a cofactor may be reduced to nicotinamide adenine dinucleotide phosphate (NADP) in a reaction in which the primary substrate is oxidized. Increasing coenzyme concentration will increase the velocity of an enzymatic reaction in a manner synonymous with increasing substrate concentration. When quantitating an enzyme that requires a particular cofactor, that cofactor should always be provided in excess so that the extent of the reaction does not depend on the concentration of the cofactor. Inhibitors Enzymatic reactions may not progress normally if a particular substance, an inhibitor, interferes with the reaction. Competitive inhibitors physically bind to the active site of an enzyme and compete with the substrate for the active site. With a substrate concentration significantly higher than the concentration of the inhibitor, the inhibition is reversible because the substrate is more likely than the inhibitor to bind the active site and the enzyme has not been destroyed. A noncompetitive inhibitor binds an enzyme at a place other than the active site and may be reversible in the respect that some naturally present metabolic substances combine reversibly with certain enzymes. Noncompetitive inhibition also may be irreversible if the inhibitor destroys part of the enzyme involved in catalytic activity. Because the inhibitor binds the enzyme independently from the substrate, increasing substrate concentration does not reverse the inhibition. Uncompetitive inhibition is another kind of inhibition in which the inhibitor binds to the ES complex—increasing substrate concentration results in more ES complexes to which the inhibitor binds and, thereby, increases the inhibition. The enzyme–substrate–inhibitor complex does not yield product. Lastly, a mixed inhibitor has the ability to bind to either the E or ES complex at a different site from the substrate active site. Each of the four types of inhibition are unique with respect to effects on the Vmax and Km of enzymatic reactions (Fig. 13.4). In competitive inhibition, the effect of the inhibitor can be counteracted by adding excess substrate to bind the enzyme. The amount of the inhibitor is then negligible by comparison, and the reaction will proceed at a slower rate but to the same maximum velocity as an uninhibited reaction. The Km is a constant for each enzyme and cannot be altered. However, because the amount of substrate needed to achieve a particular velocity is higher in the presence of a competing inhibitor, the Km appears to increase when exhibiting the effect of the inhibitor. FIGURE 13.4 Normal Lineweaver-Burk plot (solid line) compared with each type of enzyme inhibition (dotted line). (A) Competitive inhibition Vmax unaltered; Km appears increased. (B) Noncompetitive inhibition Vmax decreased; Km unchanged. (C) Uncompetitive inhibition Vmax decreased; Km appears decreased. The substrate and inhibitor, commonly a metallic ion, may bind an enzyme simultaneously in noncompetitive inhibition. The inhibitor may inactivate either an ES complex or just the enzyme by causing structural changes in the enzyme. Even if the inhibitor binds reversibly and does not inactivate the enzyme, the presence of the inhibitor when it is bound to the enzyme slows the rate of the reaction. Thus, for noncompetitive inhibition, the maximum reaction velocity cannot be achieved. Increasing substrate levels has no influence on the binding of a noncompetitive inhibitor, so the Km is unchanged. Because uncompetitive inhibition requires the formation of an ES complex, increasing substrate concentration increases inhibition. Therefore, maximum velocity equal to that of an uninhibited reaction cannot be achieved, and the Km appears to be decreased. In addition, mixed inhibitors have the potential to interfere with substrate binding and enzyme catalysis. Consequently, the apparent Vmax may decrease, and the apparent Km may decrease or increase. Measurement of Enzyme Activity Because enzymes are usually present in very small quantities in biologic fluids and often difficult to isolate from similar compounds, a convenient method of enzyme quantitation is measurement of catalytic activity. Activity is then related to concentration. Common methods might photometrically measure an increase in product concentration, a decrease in substrate concentration, a decrease in coenzyme concentration, or an increase in the concentration of an altered coenzyme. If the amount of substrate and any coenzyme is in excess in an enzymatic reaction, the amount of substrate or coenzyme used, or product or altered coenzyme formed, will depend only on the amount of enzyme present to catalyze the reaction. Enzyme concentrations, therefore, are always performed in zero-order kinetics, with the substrate in sufficient excess to ensure that no more than 20% of the available substrate is converted to product. Any coenzymes also must be in excess. NADH is a coenzyme frequently measured in the laboratory. NADH absorbs light at 340 nm, whereas NAD does not, and a change in absorbance at 340 nm is easily measured. In specific laboratory methodologies, substances other than substrate or coenzyme are necessary and must be present in excess. NAD or NADH is often convenient as a reagent for a coupled enzyme assay when neither NAD nor NADH is a coenzyme for the reaction. In other coupled enzyme assays, more than one enzyme is added in excess as a reagent and multiple reactions are catalyzed. After the enzyme under analysis catalyzes its specific reaction, a product of that reaction becomes the substrate on which an intermediate auxiliary enzyme acts. A product of the intermediate reaction becomes the substrate for the final reaction, which is catalyzed by an indicator enzyme and commonly involves the conversion of NAD to NADH or vice versa. When performing an enzyme quantitation in zero-order kinetics, inhibitors must not be present and other variables that may influence the rate of the reaction must be carefully controlled. A constant pH should be maintained by means of an appropriate buffer solution. The temperature should be constant within ±0.1°C throughout the assay at a temperature at which the enzyme is active (usually, 25°C, 30°C, or 37°C). During the progress of the reaction, the period for the analysis also must be carefully selected. When the enzyme is initially introduced to the reactants and the excess substrate is steadily combining with available enzyme, the reaction rate rises. After the enzyme is saturated, the rates of product formation, release of enzyme, and recombination with more substrate proceed linearly. After a time, usually 6 to 8 minutes after reaction initiation, the reaction rate decreases as the substrate is depleted, the reverse reaction is occurring appreciably, and the product begins to inhibit the reaction. Hence, enzyme quantitations must be performed during the linear phase of the reaction. One of two general methods may be used to measure the extent of an enzymatic reaction: (1) fixed-time and (2) continuous-monitoring or kinetic assay. In the fixed-time method, the reactants are combined, the reaction proceeds for a designated time, the reaction is stopped (usually by inactivating the enzyme with a weak acid), and a measurement is made of the amount of reaction that has occurred. The reaction is assumed to be linear over the reaction time; the larger the reaction, the more enzyme is present. In continuous-monitoring or kinetic assays, multiple measurements, usually of absorbance change, are made during the reaction, either at specific time intervals (usually every 30 or 60 seconds) or continuously by a continuous- recording spectrophotometer. These assays are advantageous over fixed-time methods because the linearity of the reaction may be more adequately verified. If absorbance is measured at intervals, several data points are necessary to increase the accuracy of linearity assessment. Continuous measurements are preferred because any deviation from linearity is readily observable. The most common cause of deviation from linearity occurs when the enzyme is so elevated that all substrate is used early in the reaction time. For the remainder of the reaction, the rate change is minimal, with the implication that the coenzyme concentration is very low. With continuous monitoring, the laboratorian may observe a sudden decrease in the reaction rate (deviation from zero-order kinetics) of a particular determination and may repeat the determination using less patient sample. The decrease in the amount of patient sample operates as a dilution, and the answer obtained may be multiplied by the dilution factor to obtain the final answer. The sample itself is not diluted so that the diluent cannot interfere with the reaction. (Sample dilution with saline may be necessary to minimize negative effects in analysis caused by hemolysis or lipemia.) Enzyme activity measurements may not be accurate if storage conditions compromise integrity of the protein, if enzyme inhibitors are present, or if necessary cofactors are not present. Calculation of Enzyme Activity When enzymes are quantified relative to their activity rather than a direct measurement of concentration, the units used to report enzyme levels are activity units. The definition for the activity unit must consider variables that may alter results (e.g., pH, temperature, substrate). Historically, specific method developers frequently established their own units for reporting results and often named the units after themselves (i.e., Bodansky and King units). To standardize the system of reporting quantitative results, the EC defined the international unit (IU) as the amount of enzyme that will catalyze the reaction of 1 μmol of substrate per minute under specified conditions of temperature, pH, substrates, and activators. Since specified conditions may vary among laboratories, reference values are still often laboratory specific. Enzyme concentration is usually expressed in units per liter (IU/L). The unit of enzyme activity recognized by the International System of Units (Systè me International d'Unité s [SI]) is the katal (mol/s). The mole is the unit for substrate concentration, and the unit of time is the second. Enzyme concentration is then expressed as katals per liter (kat/L) (1.0 IU = 17 nkat). When enzymes are quantitated by measuring the increase or decrease of NADH at 340 nm, the molar absorptivity (6.22 × 103 mol/L) of NADH is used to calculate enzyme activity. Measurement of Enzyme Mass Immunoassay methodologies that quantify enzyme concentration by mass are also available and are routinely used for quantification of some enzymes, such as creatine kinase (CK)-MB. Immunoassays may overestimate active enzyme as a result of possible cross-reactivity with inactive enzymes, such as zymogens, inactive isoenzymes, macroenzymes, or partially digested enzyme. The relationship between enzyme activity and enzyme quantity is generally linear but should be determined for each enzyme. Enzymes may also be determined and quantified by electrophoretic techniques, which provide resolution of isoenzymes and isoforms. Ensuring the accuracy of enzyme measurements has long been a concern of laboratorians. The Clinical Laboratory Improvement Amendment of 1988 (CLIA '88) has established guidelines for quality control and proficiency testing for all laboratories. Problems with quality control materials for enzyme testing have been a significant issue. Differences between clinical specimens and control sera include species of origin of the enzyme, integrity of the molecular species, isoenzyme forms, matrix of the solution, addition of preservatives, and lyophilization processes. Many studies have been conducted to ensure accurate enzyme measurements and good quality control materials.4 Enzymes as Reagents Enzymes may be used as reagents to measure many nonenzymatic constituents in serum. For example, glucose, cholesterol, and uric acid are frequently quantified by means of enzymatic reactions, which measure the concentration of the analyte due to the specificity of the enzyme. Enzymes are also used as reagents for methods to quantify analytes that are substrates for the corresponding enzyme. One example, lactate dehydrogenase (LDH), may be a reagent when lactate or pyruvate concentrations are evaluated. For such methods, the enzyme is added in excess in a quantity sufficient to provide a complete reaction in a short period. Immobilized enzymes are chemically bonded to adsorbents, such as agarose or certain types of cellulose, by azide groups, diazo, and triazine. The enzymes act as recoverable reagents. When substrate is passed through the preparation, the product is retrieved and analyzed, and the enzyme is present and free to react with more substrate. Immobilized enzymes are convenient for batch analyses and are more stable than enzymes in a solution. Enzymes are also commonly used as reagents in competitive and noncompetitive immunoassays, such as those used to measure human immunodeficiency virus (HIV) antibodies, therapeutic drugs, and cancer antigens. Commonly used enzymes include horseradish peroxidase, alkaline phosphatase (ALP), glucose-6-phosphate dehydrogenase, and β-galactosidase. The enzyme in these assays functions as an indicator that reflects either the presence or absence of the analyte. ENZYMES OF CLINICAL SIGNIFICANCE Table 13.2 lists the commonly analyzed enzymes, including their systematic names and clinical significance. Each enzyme is discussed in this chapter with respect to tissue source, diagnostic significance, assay method, source of error, and reference range. TABLE 13.2 Major Enzymes of Clinical Significance CASE STUDY 13.1 A 57-year-old moderately overweight Caucasian male visits his family physician with a symptom of “indigestion” of 5 days' duration. He has also had bouts of sweating, malaise, and headache. His blood pressure is 140/105 mm Hg; his family history includes a father with diabetes who died at age 62 of AMI secondary to diabetes mellitus. An electrocardiogram revealed changes from one performed 6 months earlier. The results of the patient's blood work are as follows: Questions 1. Can a diagnosis of AMI be ruled out in this patient? 2. What further cardiac markers should be run on this patient? 3. Should this patient be admitted to the hospital? Creatine Kinase CK is an enzyme with a molecular weight of approximately 82,000 that is generally associated with ATP regeneration in contractile or transport systems. Its predominant physiologic function occurs in muscle cells, where it is involved in the storage of high-energy creatine phosphate. Every contraction cycle of muscle results in creatine phosphate use, with the production of ATP. This results in relatively constant levels of muscle ATP. The reversible reaction catalyzed by CK is shown in Equation 13-4. Tissue Source (Eq. 13-4) CK is widely distributed in tissue, with highest activities found in skeletal muscle, heart muscle, and brain tissue. CK is present in much smaller quantities in other tissue sources, including the bladder, placenta, gastrointestinal tract, thyroid, uterus, kidney, lung, prostate, spleen, liver, and pancreas. Diagnostic Significance Due to the high concentrations of CK in muscle tissue, CK levels are frequently elevated in disorders of cardiac and skeletal muscle (myocardial infarction [MI], rhabdomyolysis, and muscular dystrophy). The CK level is considered a sensitive indicator of acute myocardial infarction (AMI) and muscular dystrophy, particularly the Duchenne type. Extreme elevations of CK occur in Duchenne-type muscular dystrophy, with values reaching 50 to 100 times the upper limit of normal (ULN). Although total CK levels are sensitive indicators of these disorders, they are not entirely specific indicators inasmuch as CK elevation is found in various other abnormal cardiac and skeletal muscle conditions. Levels of CK also vary with muscle mass and, therefore, may depend on gender, race, degree of physical conditioning, and age. Elevated CK levels are also occasionally seen in central nervous system disorders such as cerebrovascular accident, seizures, nerve degeneration, and central nervous system shock. Damage to the blood–brain barrier must occur to allow enzyme release to the peripheral circulation. Other pathophysiologic conditions in which elevated CK levels occur are hypothyroidism, malignant hyperpyrexia, and Reye's syndrome. Table 13.3 lists the major disorders associated with abnormal CK levels. Serum CK levels and CK/progesterone ratio have been useful in the diagnosis of ectopic pregnancies.5 Total serum CK levels have also been used as an early diagnostic tool to identify patients with Vibrio vulnificus infections.6 TABLE 13.3 Creatine Kinase Isoenzymes—Tissue Localization and Sources of Elevation Because enzyme elevation is found in numerous disorders, the separation of total CK into its various isoenzyme fractions is considered a more specific indicator of various disorders than total levels. Typically, the clinical relevance of CK activity depends more on isoenzyme fractionation than on total levels. CK occurs as a dimer consisting of two subunits that can be separated readily into three distinct molecular forms. The three isoenzymes have been designated as CK-BB (brain type), CK-MB (hybrid type), and CK-MM (muscle type). On electrophoretic separation, CK-BB will migrate fastest toward the anode and is therefore called CK-1. CK-BB is followed by CK-MB (CK-2) and, finally, by CK-MM (CK-3), exhibiting the slowest mobility (Fig. 13.5). Table 13.3 indicates the tissue localization of the isoenzymes and the major conditions associated with elevated levels. Separation of CK isoforms may also by visualized by high-voltage electrophoretic separation. Isoforms occur following cleavage of the carboxyl-terminal amino acid from the M subunit by serum carboxypeptidase N. Three isoforms have been described for CK-MM and two isoforms for CK-MB; the clinical significance is not well established. FIGURE 13.5 Electrophoretic migration pattern of normal and atypical CK isoenzymes. The major isoenzyme in the sera of healthy people is the MM form. Values for the MB isoenzyme range from undetectable to trace (