Use of Enzymes for Clinical Diagnosis

Use of Enzymes for Clinical DiagnosisClinical enzymology is kickoff off medical science deals with the usage of enzymes for diagnosis prognosis of divers(a) diseases. In general, each enzyme of clinical signifi lavatoryce is found in galore(postnominal) tissues of the body, and in healthy individuals, these enzyme exhibit very low levels in blood serum. In certain disease states or with cell injury, these intracellular enzymes are released into the agate line and are indicative of the presence of a pathological condition. Quantification of enzyme levels in serum is habitful in determining the presence of disease. Based on the individuals strong-arm symptoms, several enzymes may be chosen for analysis to determine if a dominion develops that aids in identifying the tissue source of the enzyme elevation in the serum(2). The understanding of enzyme kinetics allows for laboratory measurement of plasma levels. Damaged or dying cells within organ rear release enzymes into the ci rculation, these plasma enzyme levels can be utilise to develop a differential diagnosis of a patient with respect to specific organ disease and dysfunction(1).Like others analyte engagement for clinical chemistry analysis, specific pre-analytical influences catch to be taken into consideration. outline of enzyme measurement would involve the process from the start to the end that comprises the pre-analytical factors, analytical and extend analytical factors. Pre- analytical issues in the enzyme measurement include the types of precedents, the specific anticoagulants and protective in the tubes and the specimen collection procedure. Table 2.0 discover the type of enzymes, the specimen of choice and the pre-analytical factors that can affect the enzyme measurement.Slight hematolysis can be accepted as there is no CK ain rbc, however strong or mode step hemolysis can make believe enzymes and intermediates (adenylate kinase,ATP,glucose 6-phosphatea) liberated from the erytr ocytes and may affect the lag phase and the side replys betidering in the assay system(3) go down on DehydrogenaseLDHSerum or heparanised plasmaPlasma containing anticoagulant especially oxalate, should not be utilize. Haemolysed specimen 150 times LDH in rbc than serum(3) basic PhosphatareALPSerum or haparinized plasmaALP-, free hemolysis. Complexing anticoagulants such as citrate,oxalate, and edta must be avoided.Storage and doing test later than 4 hours can cause loss of legal actionEDTA concentration in the warning-reagent mixture, causing chelation of metallic cations, and this can affect the application of the alkaline phosphataseGamma Glutamyl TransferaseGGTSerum free from hemolysis preferred.EDTA -plasma ( up to 1 mg/mL blood) can be utilizeHeparin produces turbidity in the reaction mixture citrate, oxalate and fluoride depress exercise by 10 15 %The rate of disappearance of substratum or the rate of appearance of overlap had been utilized for enzyme measurement. Usually, measuring small increase in product it is much easier than to measure small decrease in a large measuring rod of substratum. In some enzyme measurements, neither the product not the substrate of a chemical reaction can be measured conveniently. In such cases the enzymatic reaction can be coupled to another reaction that uses the product of the enzyme catalyzed reaction to produce an index substance (1).The rate of flip-flop in concentration of substrate or product is the principle of kinetic method for most of the enzyme measurement. The accuracy of energizing makes it easier to detect changes in reaction conditions and samples requiring dilution. In a kinetic reaction, the rate of reaction can be expressed as P/T, the change in amount of per unit time. The amount of enzyme in a sample is measured by the rate of reactioncatalyzed by the enzyme. This rate is without delay proportioned to the amount of enzyme and is expressed in enzyme unit, IU/L (4).Substrate depletion phase is a gunpoint during an enzyme assay when the concentration of substrate is falling and the assay is not following zero- bless kinetics(5). The amount of substrate must be present in sufficient quantity, so that the reaction rate is restoreed only by the amount of enzymes. In order to get optimal method of enzyme measurement, the substrate concentration is one of the primary(prenominal) parameters. It is essential for the concentration of the substrate(s) is saturating during the measured period of the reaction(6). At saturating substrate concentrations, the reaction velocity is pseudo zero order with respect to the substrate and the velocity is proportional only to the enzyme concentration. Figure 1.0 describe the importance of substrate depletion in enzyme measurement.Enzyme activityHighModerate brokenSubstrate depletionSubstrate depletionLag phaseAbsorbanceTimeFigure 1.0 Enzyme activity can be calculated from a plot of absorbance versus time when observe an enzyme-cat alysed reaction. When reagents and serum are mixed, there may initially be a period of a time when mixing and any preliminary reactions occur this is termed the lag period. Following this phase, the reaction will proceed at zero-order kinetics (V pocket) at this point, the rate of appearance of product (as measured from the slope of the line, A/T) is directly proportional to the enzyme activity present. As the reaction proceeds and substrate is depleted, the rate of reaction will fall below V max and the plot is no eight-day linear. At this point, the reaction is no longer zero order with respect to substrate concentration rate of reaction is now dependent on both amount of substrate (which is declining) and amount of enzyme present, making it difficult to calculate amount of enzyme present. (Adapted from Henrys Clinical Diagnostic and focusing by Laboratory Methods)An organic component of enzymes is called coenzyme. Coenzymes collapseicipate in many of the enzyme analyses perfo rmed in the clinical laboratory. As the coenzyme make up a part of the active site, the role of this coenzyme in enzymatic transamination is crucial as an archetype the use of pyridoxal phosphate for expression of enzyme activity for aspartate aminopherase and alanine aminotransferase measurement(7). Table 3.0 describe the enzyme, the coenzyme and the clinical relevance of the enzyme measurement for laboratory diagnosis.In conclusion, the type of assay method, sample preparation, age and storage conditions are the variables that have to be taken into consideration in the determination of enzyme activity. Other important variables in determining enzyme activity include temperature, pH, concentration of substrate, concentration of cofactors of the assay, use of other enzyme reactions as indicators, and whether the forward or backward reaction is used to measure the enzyme. All of these variables can lead to significant differences in enzyme activity between methods (1).EnzymeCo-enzy meClinical relevanceCreatinine kinase (CK)Nicotinamide adenine dinucleotideElevations of total CK in serum are associated with cardiac disorders, such as AMI, and skeletal muscle disorder, such as muscular dystrophy. Occasionally,elevations are due to central nervous system, including seizures and rational vascular accidents.CK-MB values greater than 6 % of total CK are connotative of AMI. When AMI is suspected, troponin is assayed in conjunction with CK-MB, and sometimes myoglobin is assayed. Following AMI, Ck-MB levels rise within 4-6 hrs, prime of life at 12-24 hours, and return to commonplace within 2-3 days(2).Aspartate aminotransferasepyridoxal phosphateAST is used to evaluate hepatocellular disorders (up to 100 times speeding reference limit in infectious mononucleosis, and up to 4 times upper reference limit in cirrhosis), skeletal muscle disorders ( up to 8 times upper reference limit) and acute pancreatitis(8).In AMI, AST rises within 6-8 hours, peaks at 18-24 hours, a nd return to normal within 4-5 days. AST is not used to diagnose AMI, but awareness of the AST pattern may be useful when ruling out other disorders, including simultaneous liver damage(2).Lactate dehydrogenase (LD)NADElevated in cardiac disorders (AMI), liverwort diseases (viral hepatitis,cirrhosis,infectious mononucleosis),skeletal muscle diseases, haemolytic and haematological disorders (acute lymphoblastic leukemia)In AMI, LD levels rise within 8-12 hours, peak at 24-48 hours, and return to normal in 7 10 days. Although LD and LD isoenzymes are not used to diagnose AMI, knowledge of their pattern may be useful when assessing concurrent liver damage(2).