Use of Enzymes for Clinical Diagnosis

Clinical enzymology is branch off medical science deals with the usage of enzymes for diagnosis prognosis of various diseases. In general, each enzyme of clinical significance is found in many tissues of the body, and in healthy individuals, these enzyme exhibit very low levels in serum. In certain disease states or with cell injury, these intracellular enzymes are released into the blood and are indicative of the presence of a pathological condition. Quantification of enzyme levels in serum is useful in determining the presence of disease. Based on the individual’s physical symptoms, several enzymes may be chosen for analysis to determine if a pattern 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 can release enzymes into the circulation, these plasma enzyme levels can be used to develop a differential diagnosis of a patient with respect to specific organ disease and dysfunction(1).

Like others analyte use for clinical chemistry analysis, specific pre-analytical influences have to be taken into consideration. Analysis of enzyme measurement would involve the process from the start to the end that comprises the pre-analytical factors, analytical and post analytical factors. Pre- analytical issues in the enzyme measurement include the types of specimens, the specific anticoagulants and preservative in the tubes and the specimen collection procedure. Table 2.0 describe the type of enzymes, the specimen of choice and the pre-analytical factors that can affect the enzyme measurement.

Slight hemolysis can be accepted as there is no CK ain rbc, however severe or moderate hemolysis can cause enzymes and intermediates (adenylate kinase,ATP,glucose – 6-phosphatea) liberated from the erytrocytes and may affect the lag phase and the side reactions occurring in the assay system(3)

Lactate Dehydrogenase

LDH

Serum or heparanised plasma

Plasma containing anticoagulant especially oxalate, should not be used. Haemolysed specimen 150 times LDH in rbc than serum(3)

Alkaline Phosphatare

ALP

Serum or haparinized plasma

ALP-, 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 activity

EDTA concentration in the sample-reagent mixture, causing chelation of metallic cations, and this can affect the activity of the alkaline phosphatase

Gamma Glutamyl Transferase

GGT

Serum free from hemolysis preferred.

EDTA -plasma ( up to 1 mg/mL blood) can be used

Heparin produces turbidity in the reaction mixture; citrate, oxalate and fluoride depress activity by 10 – 15 %

The rate of disappearance of substrate or the rate of appearance of product had been utilized for enzyme measurement. Usually, measuring small increase in product it is much easier than to measure small decrease in a large amount of substrate. 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 indicator substance (1).

The rate of change in concentration of substrate or product is the principle of ‘kinetic’ method for most of the enzyme measurement. The accuracy of Kinetic 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 ΔP/ΔT, the change in amount of per unit time. The amount of enzyme in a sample is measured by the rate of reaction catalyzed by the enzyme. This rate is directly proportioned to the amount of enzyme and is expressed in enzyme unit, IU/L (4).

Substrate depletion phase is a period during an enzyme assay when the concentration of substrate is falling and the assay is not following zero-order kinetics(5). The amount of substrate must be present in sufficient quantity, so that the reaction rate is limited only by the amount of enzymes. In order to get optimal method of enzyme measurement, the substrate concentration is one of the important 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 activity
• High
• Moderate
• Low
• Substrate depletion
• Substrate depletion
• Lag phase
• Absorbance
• Time

Figure 1.0 – Enzyme activity can be calculated from a plot of absorbance versus time when monitoring an enzyme-catalysed 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 max); at this point, the rate of appearance of product (as measured from the slope of the line, ΔA/ΔT) 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 longer 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 Henry’s Clinical Diagnostic and Management by Laboratory Methods)

An organic component of enzymes is called coenzyme. Coenzymes participate in many of the enzyme analyses performed 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 example the use of pyridoxal phosphate for expression of enzyme activity for aspartate aminotransferase 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).

Enzyme

Co-enzyme

Clinical relevance

Creatinine kinase (CK)

Nicotinamide adenine dinucleotide

Elevations 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 cerebral vascular accidents.

CK-MB values greater than 6 % of total CK are suggestive 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,peak at 12-24 hours, and return to normal within 2-3 days(2).

Aspartate aminotransferase

pyridoxal phosphate

AST is used to evaluate hepatocellular disorders (up to 100 times upper 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, and 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 concurrent liver damage(2).

Lactate dehydrogenase (LD)

NAD

Elevated in cardiac disorders (AMI), hepatic 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).