Serum Enzyme Assay

Metabolites of carbon tetrachloride may also give a positive result with tiiis test, but carbon tetrachloride is only partially metabolised to trichloromethyl compounds and the test may fail to detect this agent. If carbon tetrachloride poisoning is suspected, evidence of hepato-toxicity should be sought by carrying out appropriate serum-enzyme assays. 

Following massive crush injury, myoglobin released from damaged muscle fibers colors the urine dark red. Myoglobin can be detected in plasma following a myocardial infarction, but assay of serum enzymes (see Chapter 7) provides a more sensitive index of myocardial injury. 

The sensitivity of enzyme assays can also be exploited to detect proteins that lack catalytic activity. Enzyme-linked immunoassays (ELlSAs) use antibodies covalently finked to a reporter enzyme such as alkafine phosphatase or horseradish peroxidase, enzymes whose products are readily detected. When serum or other samples to be tested are placed in a plastic microtiter plate, the proteins adhere to the plastic surface and are immobilized. Any remaining absorbing areas of the well are then blocked by adding a nonantigenic protein such as bovine serum albumin. A solution of antibody covalently linked to a reporter enzyme is then added. The antibodies adhere to the immobilized antigen and these are themselves immobilized. Excess free antibody molecules are then removed by washing. The presence and quantity of bound antibody are then determined by adding the substrate for the reporter enzyme. 

Russell, C. D. Cotlove, E. Serum glutamic-oxaloacetic transaminase Evaluation of a coupled-reaction enzyme assay by means of kinetic theory. Clin. Chem. (1971), 17, 1114-1122. 

An enzymic assay involving the reaction of neomycin with aminoglycoside 4 adenyltransferase has been described l. a linear response for neomycin was observed over the range 2.5 to 20yg/ml serum. 

Swine serum Direct assay Direct competitive enzyme-linked 13 55... 

Cell Culture. KB cells were maintained in a humidified atmosphere of 5% carbon dioxide - 95% air at 37°C in the presence of modified Eagle s medium containing calf serum (10%), penicillin (100 jug/ml) and streptomycin (100 units/ml). Cells were routinely subcultured with 0.25% trypsin and stocks were discarded after twenty passages. All drugs were administered with fresh media 24 hours after subculture in the following concentrations TPA, 1.6 uM RA, 1.6 juM butyric acid, 2mM. Drug treatments were for 20-24 hours. Cells were harvested for enzyme assays with phosphate-buffered saline containing 0.05% EDTA and stored at -20°C in 0.32 M sucrose. 

Kidney Currently, there are no kidney-specific leakage enzymes assayed in serum. Renal function is evaluated by assaying serum levels of nitrogenous wastes—blood urea nitrogen (BUN) and creatinine—and monitoring protein levels in the urine (indicator of glomerular damage). 

Serum enzyme levels have clinical diagnostic significance the presence of enzymes in the serum can be indicative of tissue and cellular damage. Commonly assayed biomarker enzymes include ... 

The enzyme assay was prepared by mixing 500 yL of 0.100 M phosphate buffer (pH 7.20), 50 yL of ethylhomocholine, 25 yL of 500 mM acetylcholine, and 25 fiL of serum diluted 1 4 with isotonic saline. Stock solutions of the substrate and internal standard were prepared in 10 mM acetate buffer (pH 4.5). The assay proceeded for 10 minutes ambient temperature (22°C) before being stopped by the addition of 25 yL of 2.50 M perchloric acid. This amount of perchloric acid did not stop the reaction instantaneously, but a correction can be made by running a zero-time control. The samples were filtered through 0.45 /urn filters before injection of 20 yL aliquots into the HPLC. 

El55 Gerard, S. and Khayam-Bashi, H. (1984). Negative interference with the Ektachem (Kodak) enzymic assay for creatinine by high serum glucose. Clin. Chem. 30, 1884. 

The pronounced effects of pH on enzyme reactions emphasize the need to control this variable by means of adequate buffer solutions. Enzyme assays should be carried out at the pH of optimal activity, because the pH-activity curve has its minimum slope near this pH, and a small variation in pH will cause a minimal change in enzyme activity. The buffer system must be capable of counteracting the effect of adding the specimen (e.g, serum itself is a powerful buffer) to the assay system, and the effects of acids or bases formed during the reaction (e.g., formation of fatty acids by the action of lipase). Because buffers have their maximimi buffering capacity close to their pK values, whenever possible a buffer system should be chosen with a pK value within IpH unit of the desired pH of the assay (see Chapter 1). Interaction between buffer ions and other components of the assay system (e.g., activating metal ions) may eliminate certain buffers from consideration. 

The application of quality control procedures to ensure that satisfactory analytical performance of enzyme assays is maintained on a day-to-day basis is complicated by the tendency of enzyme preparations to undergo denaturation with loss of activity. This maltes it difficult to distinguish between poor analytical performance and denaturation as possible causes of a low result obtained for a control sample introduced into a batch of analyses. Assured stability within a defined usable time span is therefore the prime requirement for enzyme control materials, as it is for enzyme calibrators. However, specifications for the two types of materials can differ in other respects. Because the function of a calibrator is to provide a stated activity under defined assay conditions, it is not necessary for it to show sensitivity to changes in the assay system identical to those of the samples under test therefore within certain Umits, enzymes from various sources can be considered in the search for stability. However, it is the function of a control to reveal small variations in reaction conditions, so it must mimic the samples being analyzed. The preparation of enzymes from human sources is not by itself a guarantee of an effective control. For example, human placental ALP is very stable, but it differs significantly in kinetic properties from the liver and bone enzymes that contribute most of the ALP activity of human serum samples it is therefore not an ideal enzyme for use in control material for the determination of ALP. 

Fossati P, Prencipe L, Berti G. Use of 3,5-dichloro-2 hydroxybenzenesulfonic acid/4-amino-phenazone chromogenic system in direct enzymic assay of uric acid in serum and urine. Clin Chem 1980 26 227-31. 

Ethanol, when given orally to mice at doses of 0.125-2.0 g/kg, potentiated both the lethality and behavioral effects (inverted screen test) of inhaled 1,1,1-trichloroethane at concentrations ranging from = 200 to 10,000 ppm (Woolverton and Balster 1981). In another study, a 3-day pretreatment of mice with ethanol enhanced 1,1,1-trichloroethane-induced liver toxicity, as indicated by an assay of liver function (bromosulfophthale in retention in plasma), but not an assay of liver damage (SGPT levels) (Klaassen and Plaa 1966). Other studies, using only serum enzyme levels to assay liver... 

Enzyme assays employed in the diagnosis of diseases are one of the most frequently used clinical laboratory procedures. The most commonly used body fluid for this purpose is serum, the fluid that appears after the blood has clotted. The liquid portion of unclotted blood is called plasma. Serum is used for many enzyme assays because the preparation of plasma requires addition of anticoagulants (e.g., chelating agents) that interfere with some assays. Enzymes in circulating plasma are either plasma-specific or nonplasma-specific. Plasma-specific enzymes are normally present in plasma, perform their primary function in blood, and have levels of activity that are usually higher in plasma than in tissue cells. Examples are those enzymes involved in blood clotting (e.g., thrombin), fibrinolysis (e.g., plas-min), and complement activation, as well as cholinesterase... 

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