Laboratory #
Enzymes
Laboratory #
Enzymes- Liver
Skills= 16
Objectives:
Upon completion of this exercise, the student will be able to:
1. List three (3) general conditions under which cytoplasmic enzymes diffuse into the plasma.
2. Compare/contrast fixed time and kinetic methods of enzyme assay.
3. Identify the substrates acted upon and products produced by enzymes.
4. Identify and state the purpose of the reagents used in the procedures.
5. State reference ranges for enzymes of clinical significance.
6. Perform AST, ALT, Alkaline Phosphatase and GGT enzyme procedures as instructed.
7. Determine acceptability of the run by analyzing and evaluating control results.
Materials:
1. Clinical Chemistry analyzer (For on-site ACC Students- the Alera)
2. Pipets
3. Reagents
4. Sample cups
5. Sample seglets
6. QC materials (Level 1 and Level 2)
7. Lecture notes: Part Two Enzyme Measurement and Clinical Significance
References:
Alera Package inserts
Enzyme Activity:
At normal body temperature, cellular chemical reactions, particularly those for the oxidation or transformation of organic compounds would occur very slowly. While raising the temperature would increase the speed of the reaction, living cells cannot be subjected to high temperatures because of the delicate nature of the protoplasm.
Biological systems, however, developed a number of catalysts that enable the necessary metabolic reactions to occur at body temperature. Enzymes are the functional proteins that catalyze biological reactions. Enzyme catalysts are not used up in the process and do not change the equilibrium point of the reaction; they merely accelerate the rate of reaching equilibrium.
The velocity of the enzyme reactions is affected by a number of different factors including: concentration of reactants, pH, temperature, ionic strength, and the presence of inhibitors. Essentially, any substance that alters the configuration of an enzyme affects its activity.
Enzymes are present in all body cells. Enzymes catalyze all essential reactions: oxidation, reduction, hydrolysis, esterification, synthesis, and molecular conversions necessary for vital activities.
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Laboratory #
Enzymes
Enzymes can be secreted, or they can be produced from the disintegration of cells during normal breakdown and replacement. Some enzymes appear in body fluids in much higher concentrations following injury or death of tissue cells. In addition, altered membrane permeability that may occur during inflammation may be sufficient to permit the diffusion of cytoplasmic enzymes from cells into body fluids. Although serum is the usual fluid taken for enzyme measurement, other body fluids may be chosen under appropriate circumstances.
The concentration of enzyme in serum or plasma cannot be measured readily in terms of ug per liter because it is difficult to determine in a direct manner the manner of a specific enzyme per unit volume of fluid. Instead, the rate at which the enzyme catalyzes a reaction with substrate is measured under specific conditions. Three ways have been used to measure enzyme rate:
1. Measuring decrease in substrate
2. Increase in product
3. Change in cofactor
The enzyme activity may be measured by two different methods: fixed time and continuous monitoring/ kinetic assays.
Fixed time assays utilize a fixed time for the reaction and express the enzyme activity as the amount of substrate transformed by a specified volume of serum under the particular conditions of the test. Incubation times are often long, which as a consequence, lead to enzyme inactivation, product inhibition, and suboptimal concentration of the substrate after a time. The rate of enzyme activity decreased with time after an early period of linearity.
In the continuous monitoring or kinetic assays, enzyme concentration is determined through the observation of the enzyme’s rate of activity over a short period of time.
Because the reaction time is usually short, there is little danger of enzyme inactivation.
Also, continuous monitoring permits multiple readings for the determination of the rate.
If, during the multiple readings, a decrease in enzyme reaction rate is noted, the technician should suspect high enzyme activity causing substrate depletion. 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 if a colored product is generated from a non-colored substrate.
Procedures:
Aspartate Aminotransferase- AST
Purpose:
Aspartate aminotransferase is found in practically every tissue of the body, including red blood cells. It is in particularly high concentration in cardiac muscle and liver, immediate in skeletal muscle and kidney, and in lower concentration in other tissues. The
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Enzymes measurement of the serum AST level is helpful in the diagnosis of hepatocellular disease, and in skeletal muscle disorders. Increased levels are also found following pulmonary embolism. Following myocardial infarction, the serum activity of AST begins to rise about 6 to 12 hours after myocardial infarction and usually reaches its maximum value in about 24 to 48 hours. It usually returns to normal by the fourth to sixth day after the infarction. It is a much less specific indication of myocardial infarction than a rise in
CK., since many other conditions can cause a rise in serum AST.
Principle:
AST catalyzes the transfer of an amino group from glutamic acid to oxaloacetic acid with the formation of alpha-ketoglutarate and L-aspartate. In the Alera assay, the reaction mixture contains an excess of malate dehydrogenase. In the presence of reduced nicotinamide adenine dinucleotide (NADH), the malate dehydrogenase converts oxaloacetic acid to malic acid with the oxidation of NADH to nicotinamide adenine dinucleotide (NAD). NADH absorbs strongly at 340 nm, whereas NAD does not. Therefore the rate of conversion of NADH to NAD can be determined by monitoring the decrease in absorbance bichromatically at 340-647 nm.
The conversion rate is a function of the activity of AST.
Note: Please refer to lecture notes for reaction.
Reference Range : 5-30 U/L
Alanine Aminotransferase- ALT
Purpose:
The concentration of ALT in tissues is not nearly as great for AST. It is present in moderately high concentration in the liver, but is low in cardiac and skeletal muscle and other tissues. It is primarily used in the clinical setting to diagnose liver disease and to resolve some ambiguous increases in serum AST in cases of suspected myocardial infarction, when the CK test are not available. When both AST and ALT are elevated in serum, the liver is the primary source of the enzymes. If the serum AST is elevated while the ALT remains within normal limits, myocardial infarction is suspected.
Principle:
Alanine aminotransferase in serum converts the L-alanine and alpha-keto-glutarate in the reaction to L-glutamate and pyruvate. In the Alera assay, the pyruvate that is formed reacts with reduced nicotinamide adenine dinucleotide (NADH) in the presence of lactate dehydrogenase to form lactic acid and oxidized nicotinamide adenine dinucleotide (NAD). The rate of conversion of the reduced cofactor to the cofactor can be determined by monitoring the decrease in absorbance bichromatically at 378 nm-
505 nm. This rate of conversion from the reduced cofactor to the cofactor is a function of the activity of ALT.
Reference Range: 6-37 U/L
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Laboratory #
Enzymes
Alkaline Phosphatase-ALP
Purpose:
Serum alkaline phosphatase enzyme levels are of interest in the diagnosis of hepatobiliary disorders and bone disease associated with increased osteoblastic activity. Moderate levels may be seen in several conditions that do not involve the liver or bone.
Principle:
The Alera Alkaline Phosphatase procedure is a modification of the kinetic measurement introduced by Bowers and McComb. Alkaline phosphatase in serum catalyzes the hydrolysis of colorless p-nitrophenyl phosphate to p-nitrophenol and inorganic phosphate. In an alkaline solution, p-nitrophenol is in the phenoxide form and has a strong absorbance at 408 nm. The rate of increase in absorbance, monitored at 408 nm is directly proportional to the alkaline phosphatase activity in the sample.
Note: Please refer to lecture notes for reaction.
Reference Range: 30-90 U/L
Gamma- Glutamyltransferase- GGT
Purpose:
Increased levels of GGT activity in serum are believed to be a more specific indicator of hepatobiliary disease than the levels of ALP, ALT or AST. GGT is also increased in alcohol consumption.
Principle:
GGT in serum catalyzes the transfer of the gamma-glutamyl group from L-gammaglutamyl-3-carboxy-4-nitroanilide to the glyclyglycine in the reagent. The product: 5amino-2-nitrobenzoate, absorbs strongly at 408 nm. The rate of increase in absorbance, monitored bichromatically at 408nm/486 nm is directly proportional to the GGT activity in the sample.
Reference Range:
– Male: 10-34 U/L
– Female: 9-22 U/L
Sample Collection :
Specimens for liver enzyme analysis should be clear, unhemolyzed serum. Specimens showing bacterial contamination should not be analyzed.
Quality Control :
Two levels of controls should be run every day of patient testing, prior to reporting any patient results. In addition, controls are also run when new bottles of reagent are
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Laboratory #
Enzymes loaded, when recalibration is performed, or when major instrument maintenance is performed.
Procedure:
1. The instructor will deliver a specimen and requisition to each student.
2. An appointed group will perform daily maintenance and quality control on the
Alera.
3. Each student will log the daily QC onto his/her report sheet.
4. Each student will program his/her sample demographics into the Alera, then load the sample on the instrument.
5. Following analysis, each student will review the printout, looking for critical values, and flags.
6. If no follow-up is needed, the student will attach the printout with his/her name and date to the report sheet and turn into instructor.
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Laboratory #
Enzymes
Student Name:______________
Date:____________________
Laboratory: Liver Enzymes
Report Form
Skills= 16
1. Log the lot number, expiration date, control range, and your results from running the QC in the table. Indicate whether you are “in control”. (10 points)
Lot/Exp
Date
Test
Quality Control
Alera Results Controls’ Range of
Expected Results
AST
In control?
Yes/No
Level 1:
ALT
GGT
ALP
Level 2: AST
ALT
GGT
ALP
**Note: include units
2. Based on the above information, can you run your patient? If not, why? ( 1 point)
3. Attach your patient printout to your report form. Make sure your name, and today’s date are written on the printout. ( 5 points)
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MLAB 2401

Laboratory #
Enzymes
Laboratory #: Liver Enzymes
Study Questions
Points= 15
Student Name:_______________ Date:_______________________
1. List the three general conditions, as stated in the lab that would cause cytoplasmic enzymes to diffuse into the plasma resulting in increased concentration. (1.5 points)
2. According to the information presented in the lecture and lab, complete the below table: (2 points)
Incubation time Danger of Inactivation
(Y/N)
Fixed Time
Continuous Monitoring/
Kinetic
3. According to the information presented in lecture and lab, complete the following table: (3 points)
Old initials New initials
SGOT
What do they stand for?
What do they stand for?
SGPT
4. Using your lecture notes, write the reaction catalyzed by ALT. Label the substrate and the products. ( 2 points)
5. Using your lecture notes, write the reaction catalyzed by AST. Label the substrate and the products.(2 points)
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Laboratory #
Enzymes
6. What are the references values and units for: (1 point each) a. AST:_________________ b. ALT:_________________ c. Alkaline Phosphatase:__________________ d. GGT:____________________
7. Compare the expected values for ALT and AST following myocardial infarction.
(1 point)
8. What enzyme is used to indicate alcohol consumption? (0.5 points)
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