UNIT: Enzymes II (Kinetic/Rate Reaction)

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1 UNIT: Enzymes II (Kinetic/Rate Reaction) 18enz2.wpd Task 1. Review of kinetic assays 2. Overview of selected medically significant enzymes 3. Isoenzymes 4. Creatine kinase procedure Objectives Upon completion of this exercise, the student will be able to: 1. Describe advantages of kinetic assay systems. 2. Identify and define the units that are used to report enzyme concentration 3. List substrates that can be used to measure phosphatase enzymes. 4. State the clinical significance of abnormal ALP, ACP, LD, and CK enzyme and isoenzyme concentrations. 5. Describe procedures used to identify isoenzymes. References Kaplan, Alex. Clinical Chemistry. Teitz, Norbert. Fundamentals of Clinical Chemistry. Sigma Diagnostics CK product insert. Procedure I Review of Kinetic Assays In the kinetic or continuous monitoring assay approach to enzyme measurement, enzyme concentration is determined through the observation of the enzyme's rate of activity over a short period of time. Three ways have been utilized to measure enzyme rate (decrease in substrate, increase in product, or a change in cofactor). Because the reaction time is usually short, there is little danger of enzyme inactivation. Furthermore, continuous monitoring permits multiple readings for the determination of the rate. A major advantage of this approach to enzyme measurement is that the depletion of the substrate is observable. (If a sample had an extremely high enzyme concentration, after a relatively short period of time the reaction rate would begin decreasing.) Continuous monitoring is used most commonly with those enzymes in which changes in NADH or NADPH are measured but can also be used for the determination of other enzyme activities (e.g., alkaline phosphatase) if a colored product is generated from a noncolored substrate. While in the past enzymes were reported in some unit determined by the person or company who developed the procedure, today it is more common to see enzyme results expressed in International Units per liter (IU/L). One International Unit is defined as the amount of enzyme that will convert one micromole of substrate per minute under the controlled conditions of an assay system. Companies that manufacture reagent kits state the conditions of the assay system that they have used to establish expected normal values. MLAB Clinical Chemistry Lab Manual I 157

2 In the future, enzyme results may be reported in Katal Units. Review lecture notes for additional information on Katal Units. Procedure II Enzyme Overview The following is a brief overview of selected medically significant enzymes. Phosphatases The group of enzymes known as phosphatases are found in a large number of body tissues. The phosphatases function in the tissue cells by facilitating the transfer of metabolites across cell membranes. As an enzyme in an assay system, the routinely measured phosphatases (acid and alkaline) will react on a number of phosphate substrates. The routinely measured phosphatases differ in their optimal ph preference. (Check lecture notes.) Methods used to measure phosphatase enzyme concentration include: 1. Bodansky method Serum is incubated with B-glycerophosphate for one (1) hour. 2. King-Armstrong method Serum is incubated with disodium phenyl phosphate for 30 minutes. 3. Bessey-Lowry-Brock method Serum is incubated with p-nitrophenol phosphate for one (1) hour. (The ACA uses this method for measurement of alkaline phosphatase. Results are reported as mmoles of p-nitrophenyl/l). 4. A method using thymolphthalein monophosphate as the substrate is used by the ACA in the measurement of acid phosphatase. Alkaline Phosphatase (ALP) Serum alkaline phosphatase enzyme is increased during times of increased bone activity and during a number of liver diseases. Isoenzyme fractionation should be done only on adults with increased alkaline phosphase concentration. Refer to lecture notes and textbook for information on the clinical significance of ALP. Acid Phosphatase (ACP) Many body tissues (spleen, kidney, liver, bone, blood platelets, etc.) contain ACP in low concentrations. Tissue of the prostate gland is a rich source of acid phosphatase. Acid phosphatase enzyme (ACP) acts optimally below ph 6.0. The ACP activity of normal serum is derived from some of the above tissues but primarily from blood platelets. Normal serum has a low activity of ACP but in metastasizing carcinoma of the prostate, its activity increases greatly and may rise to 3 to 15 x ULN. The carcinoma has to metastasize, i.e., to invade blood capillaries, lymph channels, and other tissues, before the elevation in the serum level of acid phosphatase occurs; a discrete prostatic cancer that has not penetrated beyond the capsule does not cause the rise in serum ACP. Because erythrocytes and blood platelets also contain an acid phosphatase, it is essential to distinguish between the ACP derived from these sources during the clotting of the blood specimen I 158 MLAB Clinical Chemistry Lab Manual

3 and that coming from the prostate. Two different techniques may be employed to assist in identifying the serum ACP derived from prostatic tissue. The first is to use a substrate that the prostatic ACP splits more readily than does the ACP from platelets and erythrocytes; sodium thymolphthlein monophosphate and -naphthylphosphate are such substrates. The second technique is to measure the ACP activity before and after adding tartrate to the mixture. Tartrate greatly inhibits the ACP from prostate, but is much less inhibitory for the ACP from erythrocytes or platelets. A combination of both techniques is considered to be the most satisfactory when employing -naphthylphosphate as the substrate. Although increased activity of the tartrate-inhibitable ACP is characteristically found in metastasizing carcinoma of the prostate, elevated activities of prostatic ACP may be found in Gaucher's disease or some bone diseases (Paget's disease) or female breast cancer that has metastasized to bone. Massage of the prostate increases ACP activity for 1 or 2 days. No physiologic significance is attached to a low serum ACP activity. Lactate Dehydrogenase (LD or LDH) LD is distributed widely in tissues and is present in high concentration in liver, cardiac muscle, kidney, skeletal muscle, erythrocytes, and other tissues. The measurement of the serum concentration of LD has proven to be useful in the diagnosis of myocardial infarction. The LD enzyme activity in serum does not rise as much as CK or AST after myocardial infarction, but it does remain elevated for a much longer period of time. This is quite important when the patient does not see a physician for 3 or 4 days following an infarct. In hepatocellular disease, the serum activity of LD rises, but the measurement of this enzyme is much less useful than that of AST or ALT because the test is less sensitive. The serum LD activity is increased in a wide variety of disorders because it is so widely distributed in tissues. The principle clinical uses of the LD test are the following: 1. In myocardial infarction, serum LD activity increases after myocardial infarction, but the rise occurs later than that for CK or AST and is of lesser intensity. Its great value in the diagnosis of myocardial infarction lies in the prolongation of its increased activity; it may remain elevated for 7 to 10 days, long after the CK and AST levels have returned to normal. The isoenzymes of LD also have an important role in the diagnosis of myocardial infarction. Refer to lecture notes and textbook for additional information. 2. Serum LD activity is increased in liver disease, but other enzymes are more sensitive and specific for liver disorders. The serum activity is also increased following muscle trauma, renal infarct, hemolytic diseases, and pernicious anemia. Hemolyzed blood specimens will have artificially elevated LD activities owing to LD enzymes coming from the ruptured red blood cells; the same is true if the serum is allowed to stand too long upon the clot. Refer to lecture notes (both liver lecture and enzyme lecture) and textbook for additional information. Creatine Kinase (CK or CPK) In the body CK is associated with the storage of phosphate (ATP) by catalyzing the following reaction: MLAB Clinical Chemistry Lab Manual I 159

4 This reaction allows the body to store the high energy phosphate in the form of creatine phosphate. Because the enzyme reaction is reversible the energy can quickly be made available to muscles. CK is present in high concentration in skeletal muscle, cardiac muscle, thyroid, prostate, and brain; it is present only in small amounts in liver, kidney, lung, and other tissues. An increase in serum CK activity is attributed primarily to damage to strained muscle (skeletal or cardiac) and in rare cases, to brain. Differentiation between these various diseases can frequently be made upon clinical grounds, but there are situations when this is not possible. Measurement of the CK-MB isoenzyme helps to solve the problem. Since CK is located primarily in skeletal muscle, myocardium, and brain, the serum activity increases after damage to these tissues and is not usually affected by the pathologic conditions in other organs. The following is a brief explanation of CK activity following tissue damage. 1. Damage to heart tissue. There is a sharp but transient rise in CK activity following myocardial infarction. The serum CK may be increased in some cases of coronary insufficiency without myocardial infarction. The simultaneous determination of the CK-MB isoenzyme and LD isoenzymes will help to make the diagnosis. 2. Damage to skeletal muscle. The serum CK activity may rise to high levels following injury to skeletal muscles. Some of the causes may be trauma, muscular dystrophy, massage of chest during a heart attack, an intramuscular injection, or even strenuous exercise. The serum activity parallels the amount of muscle tissue involved. In prolonged shock, the CK enzyme also leaves the ischemic muscle cells and appears in the serum. 3. Brain damage The CK levels in serum are increased in brain injury only when there is some damage to the blood brain barrier; the rise in the BB fraction. Damage to the blood brain barrier may be caused by trauma, infection, stroke, or severe oxygen deficiency. Procedure III Isoenzymes With the improved techniques for analyzing proteins, developed over the last twenty years, it has been demonstrated that a particular type of catalytic activity (enzymes) is frequently due to the existence of several distinct forms of an enzyme rather than to only one type of molecule. These enzyme variants may occur within a single individual, a single organ, or even within a single type of cell. The forms can be distinguished on the basis of differences in various physical properties, such as electrophoretic mobility or resistance to chemical or thermal inactivation. Although these differences may be significant, all forms of a particular enzyme retain the ability to catalyze its characteristic reaction. The multiple molecular forms of an enzyme are often described as isoenzymes (or isozymes). The existence of multiple forms of enzymes in tissues has important implications in the study of human disease. The presence of isoenzymes with distinctive properties in different organs helps I 160 MLAB Clinical Chemistry Lab Manual

5 in understanding organ-specific patterns of metabolism (whereas genetically determined variations in enzyme structure between individuals account for such characteristics as differences in sensitivity to drugs and hereditary metabolism disorders). Multimolecular forms (isoenzymes) have been noted in many human enzymes. Those that have diagnostic implications are isoenzyme fractions of ALP, ACP, LDH, and CK. Isoenzymes of amylase exist but the diagnostic value of their identification is still being questioned. Alkaline Phosphatase (ALP) Isoenzymes 1. Electrophoresis produces 1-4 bands (fast liver, liver, bone, and intestine) 2. Heat-inactivation. Serum heated to 56 C for 15 minutes will lose any ALP activity due to bone isoenzyme. 3. Chemical inhibition. High concentrations of urea readily inhibits bone isoenzyme, while liver has intermediate resistance, and placental is most resistant. 4. Immunochemical techniques. Monospecific antisera to placental and intestinal alkaline phosphatase provide the best measurements of these isoenzymes. Acid Phosphatase (ACP) Isoenzymes Acid phosphatase is found in a variety of tissues (RBCs, liver, spleen) but the object of diagnostic assays is almost always to determine the prostatic fraction. 1. Substrate preference. Selection of a substrate preferred by the prostatic ACP isoenzyme.! thymolphthalein monophosphate! naphthyl phosphate (preferred for continuous monitoring procedures. 2. Chemical inhibition. Prostatic ACP is inhibited by tartrate. Refer to lecture notes and textbook for additional information. Lactate Dehydrogenase (LD or LDH) Isoenzymes Isoenzymes of LDH differ from each other in the primary sequence of the constituent polypeptide chains. The LDH enzyme molecule consists of 4 polypeptide subunits, of which there are 2 types: H and M chain. Thus, there are 5 possible combinations of the H+M chains. LD1 (HHHH) LD2 (MHHH) LD3 (MMHH) LD4 (MMMH) LD5 (MMMM) 1. Electrophoretic separation. Since the H and M subunits have different net charges, each of the individual LD isoenzymes have different net charges. This difference in charge can be used to separate the isoenzymes by electrophoresis. 2. Ion-exchange chromatography can also be used to separate LD isoenzymes. Creatine Kinase (CK or CPK) Isoenzymes There are three isoenzymes of CK separated by electrophoresis: CK-BB (CK 1), CK-MB (CK 2) and CK-MM (CK ). The MM isoenzyme is found primarily in skeletal and cardiac muscles but low 3 MLAB Clinical Chemistry Lab Manual I 161

6 activity exists in lung and kidney. Cardiac muscle cells contain a mixture of the CK-MM and CK-MB isoenzymes; the major portion is MM but the MB content is considerable and may comprise from 15 to 20% of the total CK activity. By contrast, skeletal muscle CK consists of approximately 99% MM fraction and only about 1% of MB. The BB isoenzyme is present in brain tissue, gastrointestinal and genitourinary tracts (colon, prostate, uterus), with lower activity in thyroid and lung. The predominant CK isoenzyme in the serum of normal individuals is the MM fraction, which comprises 94 to 98% of the total. The MB isoenzyme may be present up to 6% of the total but is usually only 2 to 4% (1 to 4U). In normal serum, the BB isoenzyme is undetectable by electrophoretic methods but it may increase appreciably in women immediately postpartum, in patients with cardiovascular accidents (stroke), acute renal disease, adenocarcinomas of the prostate or other tissues, severe hypoxia (oxygen lack), and brain injury that damages the blood brain barrier. The most important diagnostic use for CK isoenzymes is for the diagnosis of myocardial infarction (MI). Following a moderate to severe MI, the MB isoenzyme rises rapidly, reaches a maximum within 24 hours, and then falls rapidly. Its relative increase in serum is greater than for total CK but it returns to normal values a little earlier than the latter. After a small MI, the MB isoenzyme may become elevated even though the total CK remains within normal limits. Myocardial Infarction A myocardial infarct is a necrotic area in the heart caused by a deficient blood flow to the area as the result of a clot in a coronary vessel and/or narrowing of the vessel lumen by atheromatous plaques. When the cardiac cells in the necrotic area die, their intracellular enzymes diffuse out of the cell into tissue fluid and end up in plasma. Since it is not always possible to make a definitive diagnosis of myocardial infarction by an electrocardiogram, appropriate enzyme tests are extremely helpful for this purpose. The enzyme tests that have proven to be most helpful in the diagnosis of myocardial infarction are: creatine kinase (CK), aspartate aminotransferase (AST or SGOT), lactate dehydrogenase (LD or LDH), isoenzyme CK-MB, and isoenzymes of LD (flipped pattern). Some of the enzyme activities increase early after an infarct (CK and CK-MB), some appear a little later (AST), and some increase even later and remain elevated for prolonged periods (LD, LD 1, and LD 2). Each enzyme has its own particular time course when the serum activity of the enzyme is plotted against time after the myocardial infarct. Since the laboratory has no control over when the patient may elect to see the physician or when the enzyme tests are ordered, it is necessary to have some tests available that can help to diagnose a myocardial infarction in a time period that may vary from 4 hours to 10 days. Procedure IV Creatine Kinase Background and Principle of Test Creatine phosphokinase (CPK) catalyzes the reversible phosphorylation of ADP by phosphocreatine to form ATP and free creatine. Various methods for CPK determination have I 162 MLAB Clinical Chemistry Lab Manual

7 been proposed in which the reaction rate is followed by measuring the formation of either of the end products. In 1955, Oliver described a procedure based on Kornberg's assay for ATP. The ATP generated by the CPK-catalyzed reaction is utilized in a hexokinase/glucose-6-phosphate dehydrogenase coupled enzyme system which ultimately yields an amount of reduced NADP (NADPH) proportional to the CPK activity. NADPH formation is followed spectrophotometrically at 340 nm. Upon addition of sample to the test system, an equilibration interval of several minutes is required to permit the reaction kinetics to become linear (zero-order). An initial reading is then taken, followed by a second reading 5 minutes later. The change in absorbance at 340 nm ( A 340) during the 5-minute period is used to calculate the CPK activity. The described procedure involves the following reactions: Step 1: Step 2: Step 3: When NADP is reduced to NADPH the A 340 sharply increases and is proportional to the CPK activity. Supplies and Equipment 1. A narrow-bandwidth spectrophotometer capable of transmitting & detecting light at 340 nm. 2. Conventional or automatic pipets 3.0 ml (volumetric) 50 ul 2.0 ml (serologic) 3. Thermometer Specimen Collection and Storage Plasma collected in heparin or EDTA, as well as serum may be used. Since red cells are practically devoid of CPK, slight hemolysis does not affect serum CPK levels. Preliminary tests indicate that serum containing hemoglobin concentrations up to 200 mg/dl do not alter results. Serum may be refrigerated (2-6 C) for 5 days with no appreciable change in CPK levels. Serum stored at room temperature will slowly begin losing CPK activity (<10% loss in 24 hour). No loss of CPK activity in serum frozen up to 2 months. Procedure (Single Assay Vial) 1. The temperature of the reaction mixture should be maintained at 25 C or some other constant temperature. Refer to Temperature Correction Factors (TCF) table in product insert if procedure is conducted at any other temperature. 2. If using semimicro cuvets which accommodate 1.5 ml of TEST mixture, it is possible to use one CPK Single Assay Vial to perform two assays as follows: MLAB Clinical Chemistry Lab Manual I 163

8 a. Reconstitute 1 vial with 3.0 ml water, cap and invert several times to dissolve contents. b. Pipet 1.5 ml of the solution into a small test tube. c. Pipet 0.05 ml of serum and mix. d. Pipet 0.05 ml of a second sample to the remaining solution in the vial. Mix and transfer to another small test tube. 3. Parafilm and invert several times to mix. DO NOT SHAKE! 4. Wait approximately 5 minutes to allow reaction kinetics to become linear (zero order). 5. Read and record absorbance at 340 nm using water as reference. This is INITIAL A. 6. Exactly five minutes later, again read and record absorbance. This is FINAL A. CALCULATIONS Final A - Initial A = A/5 minutes A/5 minutes x Vial Factor F x TCF = CPK Sigma units/ml Vial Factor F appears either on vial label or on box containing vials. TCF = temperature correction factor. Temperature correction factor at 25 C = 1. If procedure is carried out at any other temperature, consult product insert for appropriate TCF. Notes 1. To express activity in terms of International Units (U), which are equal to micromoles of substrate converted per minute under the conditions of this procedure, use the following equation: Where: 3.1 = volume (ml) of reaction mixture 1000 = conversion of micromolar units/ml to micromolar units/l TCF = (Temperature Correction Factor) 1.0 at 25 C 5 = conversion of A per 5 min to A per min 6.22 = millimicromolar absorptivity for NADPH at 340 nm 0.1 = sample volume (ml) At 25 C the above formula reduces to: International Units/L = A per 5 min x 1000 To correct International Units/L at 25 C to International Units/L at 30 C, multiply by To correct International Units/L at 25 C to International Units/L at 37 C, multiply by If the A for 5 minutes is greater than 0.35, repeat determination using 0.05 ml serum in Step 1 and multiply result by 2. I 164 MLAB Clinical Chemistry Lab Manual

9 3. If sample has a high CPK value or if procedure is performed at a temperature as high as 37 C, it is suggested that 0.05 ml of sample be used and result multiplied by 2. Expected Values Normal Serum CPK Activity Subjects Serum Units/mL International Units/L (25 C) Males Females Performance Characteristics Linearity of reaction rate has been observed with a A per 5 minutes as high as Reproducibility studies revealed a coefficient of variation of 3% obtained for 11 replicate assays of a commercial serum enzyme control, having an average value of 36 Sigma Units/mL. Name Date Enzyme being tested Reaction temperature Kinetic Enzyme Report Form Spectrophotometer Used Vial Factor (F) Wavelength Initial A Final A A Concentration Sigma Units/ml Concentration U/L Control 1 Control 2 Patient 1 Patient 2 MLAB Clinical Chemistry Lab Manual I 165

10 Example Calculations Quality Control Your Results Controls range of expected results. In control? Yes / No Level 1 ID Level 2ID Accepting Patient Results? Reason I 166 MLAB Clinical Chemistry Lab Manual

11 Name Date Enzyme being tested Reaction temperature Kinetic Enzyme Report Form Spectrophotometer Used Vial Factor (F) Wavelength Initial A Final A A Concentration Sigma Units/mL Concentration U/L Control 1 Control 2 Patient 1 Patient 2 Example Calculations Quality Control Your Results Controls range of expected results. In control? Yes / No Level 1 ID Level 2ID Accepting Patient Results? Reason MLAB Clinical Chemistry Lab Manual I 167

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13 Name Study Questions Date Instructions: Legibly write your answers in the space provided. Unless otherwise indicated, each question is worth one point. 1. What is the major advantage of the continuous monitoring approach to enzyme measurement? substrate depletion observable 2. Define International Units (U/L). The amount of enzyme that will convert one micromole of substrate per minute under the controlled conditions of an assay system 3. Define Katal Units. expression of enzymes activity as moles/second Kat/L = moles/second/liter 1 IU = 60 ukatal 4. List the substrates used for the following methods to determine phosphatase enzyme concentration: (3 pts) Bodansky method King-Armstrong method Bessey-Lowry-Brock method glycerophosphate disodium phenylphosphate p-nitrophenolphosphate 5. Under what normal condition(s) would an increased ALP be expected? bone growth in children (possibly following bone fracture in an adult) 6. What abnormal conditions would be detected by increased ALP? metastasizing carcinoma of the prostate 7. What abnormal conditions would be detected by an increased ACP? bone disease; icteric liver disease MLAB Clinical Chemistry Lab Manual I 169

14 8. List at least three (3) ways that can be used to identify isoenzyme fractions. (3 pts)! electrophoretic mobility! resistance to chemical inactivation! resistance to thermal inactivation! ion exchange! immunological 9. Which ALP isoenzyme fraction is heat labile? bone 10. What is the purpose of tartrate in the procedure to identify prostatic ACP? inhibits ACP prostatic 11. What two subunits make up the various LD isoenzymes? H & M 12. What CK isoenzyme fraction provides diagnostic evidence of myocardial infarction? CK-MB CK Why aren't the normal values (in IU/L) in this CK procedure the same as those quoted in the lecture? reaction rates are at different temperature I 170 MLAB Clinical Chemistry Lab Manual