Download Unit: Enzymes II

UNIT: Enzymes II (Kinetic/Rate Reaction)
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Task
1.
2.
3.
4.
Review of kinetic assays
Overview of selected medically significant enzymes
Isoenzymes
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.
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UNIT: Enzymes II (Kinetic/Rate Reaction) (continued)
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.
2.
3.
4.
Bodansky method – Serum is incubated with B-glycerophosphate for one (1) hour.
King-Armstrong method – Serum is incubated with disodium phenyl phosphate for 30
minutes.
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).
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
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UNIT: Enzymes II (Kinetic/Rate Reaction) (continued)
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:
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UNIT: Enzymes II (Kinetic/Rate Reaction) (continued)
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
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UNIT: Enzymes II (Kinetic/Rate Reaction) (continued)
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.
2.
3.
4.
Electrophoresis produces 1-4 bands (fast liver, liver, bone, and intestine)
Heat-inactivation. Serum heated to 56°C for 15 minutes will lose any ALP activity due to
bone isoenzyme.
Chemical inhibition. High concentrations of urea readily inhibits bone isoenzyme, while liver
has intermediate resistance, and placental is most resistant.
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.
2.
Substrate preference. Selection of a substrate preferred by the prostatic ACP isoenzyme.
! thymolphthalein monophosphate
! % naphthyl phosphate (preferred for continuous monitoring procedures.
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 (CK1 ), CK-MB (CK2 ) and
CK-MM (CK3 ). The MM isoenzyme is found primarily in skeletal and cardiac muscles but low
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UNIT: Enzymes II (Kinetic/Rate Reaction) (continued)
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, LD1 ,
and LD2 ). 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
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UNIT: Enzymes II (Kinetic/Rate Reaction) (continued)
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 ()A340 ) 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 A340 sharply increases and is proportional to the CPK activity.
Supplies and Equipment
1.
2.
3.
A narrow-bandwidth spectrophotometer capable of transmitting & detecting light at 340 nm.
Conventional or automatic pipets
3.0 mL (volumetric)
50 uL
2.0 mL (serologic)
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:
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UNIT: Enzymes II (Kinetic/Rate Reaction) (continued)
a.
b.
c.
d.
Reconstitute 1 vial with 3.0 mL water, cap and invert several times to dissolve contents.
Pipet 1.5 mL of the solution into a small test tube.
Pipet 0.05 mL of serum and mix.
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
1000
TCF
5
6.22
0.1
=
=
=
=
=
=
volume (mL) of reaction mixture
conversion of micromolar units/mL to micromolar units/L
(Temperature Correction Factor) 1.0 at 25°C
conversion of )A per 5 min to )A per min
millimicromolar absorptivity for NADPH at 340 nm
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 1.52.
To correct International Units/L at 25°C to International Units/L at 37°C, multiply by 2.17.
2.
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.
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UNIT: Enzymes II (Kinetic/Rate Reaction) (continued)
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
Males
Females
Serum Units/mL International Units/L (25°C)
3 - 11
14 - 55
2-8
8 - 40
Performance Characteristics
Linearity of reaction rate has been observed with a )A per 5 minutes as high as 0.350.
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
Kinetic Enzyme Report Form
Enzyme being tested
Reaction temperature
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
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UNIT: Enzymes II (Kinetic/Rate Reaction) (continued)
Example Calculations
Quality Control
Your Results
Controls’ range of expected results.
Level 1 ID______________
Level 2ID_______________
Accepting Patient Results?
Reason
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In control? Yes / No
UNIT: Enzymes II (Kinetic/Rate Reaction) (continued)
Name
Date
Kinetic Enzyme Report Form
Enzyme being tested
Reaction temperature
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
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UNIT: Enzymes II (Kinetic/Rate Reaction) (continued)
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UNIT: Enzymes II (Kinetic/Rate Reaction) (continued)
Name
Date
Study Questions
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)
$ glycerophosphate
Bodansky method
disodium phenylphosphate
King-Armstrong method
p-nitrophenolphosphate
Bessey-Lowry-Brock method
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
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UNIT: Enzymes II (Kinetic/Rate Reaction) (continued)
8.
List at least three (3) ways that can be used to identify isoenzyme fractions. (3 pts)
!
!
!
!
!
9.
electrophoretic mobility
resistance to chemical inactivation
resistance to thermal inactivation
ion exchange
immunological
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
13.
CK2
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
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