CHAPTER 9: Diagnostic Enzymology
Compiled by: Mandy A. Delfin, RMT, MSMT
CHAPTER 9
DIAGNOSTIC ENZYMOLOGY
COMPILED BY:
MANDY A. DELFIN, RMT, MSMT
o
PROPERTIES OF
ENZYMES
o
Enzymes are
organic or protein
catalysts.
Components:
1. APOENZYME: Protein portion which is mainly responsible for
enzyme action.
2.
COFACTOR (Enzyme partner): Acts with and is essential to enzyme
activity
o
o
Parts:
1.
2.
ACTIVE SITE: where
the substrate fits in
ALLOSTERIC SITE.
where effector
(regulates the activity
of the enzyme)
molecules or
modifiers
3.
Types of Cofactor:
a.
Coenzyme: non-protein organic substance which is
dialyzable, thermostable and loosely attached to the
protein part.
b.
A prosthetic group: an organic substance which is
dialyzable and thermostable which is firmly attached to
the protein or apoenzyme portion.
c.
A metal-ion-activator: these include K+, Fe++, Fe+++, Cu++,
Co++, Zn++, Mn++, Mg++, Ca++, and Mo+++.
HOLOENZYME: The functional unit and it is made up of
APOENZYME + COFACTOR
PAGE 1
VH REVIEW CENTER
KORONADAL, CITY SOUTH COTABATO
CHAPTER 9: Diagnostic Enzymology
Compiled by: Mandy A. Delfin, RMT, MSMT
CLASSIFICATION OF ENZYMES
Theories that explain the high degree of specificity of an enzyme
A.
1.
Chemical reaction catalyzed
Group
1 Oxidoreductases
Catalyzed reaction
Redox reactions
Examples
LDH, G6PD, GLDH
2 Transferase
Transfer of functional
group
Hydrolysis of various
bonds
Removal of groups from
substrates without
hydrolysis and the
resulting product
contains double bonds
interconversion of
geometric, optical, or
positional isomers
Joining of 2 substrate
molecules utilizing ATP
AST, ALT, CK, GGT,
Glutathione S transferase
ALP, ACP, AMS, LPS,
CHS, digestive enzymes
ALD, decarboxylases
3 Hydrolase
4 Lyase
5 Isomerases
6 Ligase
B.
substrate fits. The shape of the k
ey (substrate) must conform to the
lock (enzyme)
2.
Glucose phosphate
isomerase
Glutathione synthetase
1.
One of the properties of enzymes that makes them so important as
diagnostic and research tools is the specificity, they exhibit relative to
the reactions they catalyze
Absolute specificity: Acts on only one substrate and one reaction.
2.
Group specificity: acts only on molecules that have specific
functional groups.
3.
Linkage specificity: Acts on a chemical bond.
4.
Stereo chemical specificity: acts on a steric or optical isomer.
Kochland’s Induced Fit Theory:
Based on the attachment of the
substrate to the active site of an
enzyme, which then causes
conformational changes in the
enzyme. This theory is more
acceptable because the protein
molecules are flexible to allow
conformational changes.
Factors Affecting Enzyme Activity
Specificity of Enzymes:
1.
Emil Fisher’s Lock and Key
Theory: Based on the rigid
enzyme molecule into which the
2.
Temperature:
o The rate of an enzyme-catalyzed reaction increases as the
temperature is raised.
o
1 or 2 degrees may introduce changes of 10 to 20% in the results.
However, it will be denatured at temperatures above 40C
o
Storage of enzymes at 5C or below is generally the most suitable
but some enzymes lose their activity when frozen
pH
o
Extremely high or low pH values result in complete loss of enzyme
activity. Note that pH is also a factor in the stability of enzymes
o
The optimum (most active) pH value will vary greatly from one
enzyme to another.
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VH REVIEW CENTER
KORONADAL, CITY SOUTH COTABATO
CHAPTER 9: Diagnostic Enzymology
Compiled by: Mandy A. Delfin, RMT, MSMT
3.
4.
ENZYME CONCENTRATION
o
The higher the enzyme level, the faster the reaction will proceed
because more enzymes are present to bind with the substrate.
o
As long as the substrate concentration exceeds the enzyme
concentration, the velocity of the reaction is proportional to the
enzyme concentration.
o
The Michaelis constant Km is defined as the substrate
concentration at 1/2 the maximum velocity.
o
Using this constant and the fact that Km can also be defined as:
o
Michaelis
constants have
been determined
for many of the
commonly used
enzymes.
§
The size of Km tells us several things about an enzyme.
SUBSTRATE CONCENTRATION
o
If the amount of the enzyme is kept constant and the substrate
concentration gradually increased, the reaction velocity will
increase until it reaches a maximum.
Substrate saturation: the concentration at which it reaches
its maximum rate and all the active sites are full.
Turnover Number: Number of substrate molecules
converted to product per second per enzyme molecule under
conditions of optimum temperature and pH.
o
After this point, increased in substrate concentration will not
increase the velocity (maximum velocity)
o
If maximum velocity has been reached, the entire available enzyme
has already been converted to ES, the enzyme substrate complex
o
Using this maximum velocity and equation:
§
Michaelis developed a set of mathematical expressions to
calculate enzyme activity in terms of reaction speed from
measurable laboratory data.
o
Small Km: Maximum velocity is reached at relatively low
substrate concentrations.
o
Large Km: Requires high substrate concentrations to achieve
maximum reaction velocity.
o
The substrate with the lowest Km is ASSUMED to be the
Enzyme's natural substrate.
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VH REVIEW CENTER
KORONADAL, CITY SOUTH COTABATO
CHAPTER 9: Diagnostic Enzymology
Compiled by: Mandy A. Delfin, RMT, MSMT
5.
PRESENCE OF ANY INHIBITORS OR ACTIVATORS
ENZYME NOMENCLATURE
1.
Competitive Inhibitor.
o Competes with the substrate for enzyme. A high substrate
concentration will overcome the effect of the inhibitor.
a)
b)
Non-competitive Inhibitor
o Does not resemble the substrate and bind to the enzyme in
areas other than the active site.
c)
Uncompetitive Inhibitor
o Binds to the enzyme-substrate complex. Increasing the
substrate concentration results in more ES complexes where
the inhibitor binds, increasing inhibition process.
Type of Inhibition
Competitive
Noncompetitive
Uncompetitive
Change in Km
Increased
No change
Decrease
Change in Vmax
No change
Decreased
Decreased
According to the name of the substrate with the addition of the suffix
“ase”.
o
o
2.
Enzymes acting on lipids:
lipase
Enzymes acting on protease: protease
According to the type of reaction they catalyze
a) Transferase Transfer of amino group from substrate to another:
b)
Kinase Transfer to phosphate group from a high energy phosphate
compound to its substrate
c) Phosphatases: Effect of hydrolysis on phosphate esters
d)
3.
Dehydrogenase: Removal of hydrogen atoms from its
substrate
According to the numerical designation given by the Enzyme Comission
(E.C)
o
o
o
E.C. 1.1.127 for lactate dehydrogenase
E.C. 3.2.1.1. for amylase
E.C. 2.6.1.2. for alanine amino transferase
•
The first number defines the class to which the enzyme
belongs
•
the next two numbers indicate the subclass to which the
enzyme is assigned.
•
The last number is a specific serial number to each
enzyme in its sub-class.
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VH REVIEW CENTER
KORONADAL, CITY SOUTH COTABATO
CHAPTER 9: Diagnostic Enzymology
Compiled by: Mandy A. Delfin, RMT, MSMT
ENZYME MEASUREMENT
o
Enzymes are measured in terms of their activity rather than in terms of
their concentration.
1.
Substrate Measurement. This starts with a high substrate
concentration.
2.
Product Measurement. This starts with zero initial product level. It
is more accurate.
3.
Coenzyme Measurement. This measure the increase or decrease in
coenzyme.
•
Katal Unit (K.U): Equivalent to the amount of enzyme that catalyzes
the conversion of 1 mole of substrate per second under controlled
conditions
SOURCES OF ERROR IN ENZYME MEASUREMENTS:
1.
Hemolysis: There is release of certain enzymes from red cells causing
falsely elevated values.
2.
Anticoagulants: Many anticoagulants cause adverse effects on enzymes
especially enzyme inhibition; therefore, serum is preferred over plasma
(in general).
3.
Lactescence or Milky Serum: It may result in variable absorbance
readings in spectrophotometry.
UNITS FOR EXPRESSING ENZYME ACTIVITY
•
Enzyme concentration is measured in terms of their activity not in terms
of their absolute value.
•
Enzyme activity is expressed in units which usually represent any of the
means of measuring enzyme activity
•
International Unit (I.U or U): Equivalent to the amount of enzyme
that catalyzes the conversion of 1 micromole of substrate per
minute under controlled conditions.
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VH REVIEW CENTER
KORONADAL, CITY SOUTH COTABATO
CHAPTER 9: Diagnostic Enzymology
Compiled by: Mandy A. Delfin, RMT, MSMT
ENZYMES OF CLINICAL SIGNIFICANCE
MUSCLE ENZYMES
Enzymes in this category include, CK, lactate dehydrogenase,
aldolase and glycogen phosphorylase.
A.
CREATINE KINASE (CK) or Creatine Phosphokinase (CPK)
ATP:Creatine-N-phosphotransferase
E.C.2.7.3.2
Tissue sources:
Major Sources: Skeletal muscle, heart muscle, and brain tissue.
§
§
Other tissue sources: Bladder, placenta, gastrointestinal tract, thyroid,
uterus, kidney, lung, prostate, spleen, liver and pancreas.
Methods of Measurement:
§
Analytically the reverse reaction is preferred for it proceeds about six
times faster than the forward reaction.
§
Reaction Catalyzed
General Reaction:
Creatine phosphate + ADP
ATP + Glucose
HK
Glucose-6-phosphate
§
Physiologic Function:
o Generally associated with ATP regeneration in contractile or
transport systems that occurs in muscle cells, where it is involved in
the storage of high energy Creatinine phosphate.
o
Every contraction cycle of muscle result in Creatinine phosphate
use, with the production of ATP. This results in relatively constant
levels of muscle ATP.
Creatine + ATP
Glucose-6-phosphate + ADP
G6PDH
NADP
Optimum pH:
Forward Reaction: (Cr +ATPà ADP +CrP) : 9.0
Backward Reaction: (CrP +ADP à ATP + Cr): 6.7
CK
6-phosphogluconate
NADPH + H
Methodologies:
1.
Oliver Method: Monitors the change in absorbance as NADP is
reduced to NADPH.
2.
Rosalki Method: Improved the method of Oliver by adding:
o AMP: inhibit adenylate kinase
o Cysteine: activate CK. stabilize the enzyme by maintaining its –
SH type cross linkages. (dithiothreiol or mercaptoethano)
3.
Szasz et.al Method: Optimized the assay by adding
o N-Acetylcysteine: activate CK
o EDTA: bind to calcium and to increase the stability of the
reaction mixture
o Adenosine phosphate in addition of AMP t: inhibit AK
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VH REVIEW CENTER
KORONADAL, CITY SOUTH COTABATO
CHAPTER 9: Diagnostic Enzymology
Compiled by: Mandy A. Delfin, RMT, MSMT
Considerations
§
o
Patient Preparation: Exercise and intramuscular injections cause
CK elevations.
o
Serum sample: Hemolyzed specimen is not used since it contains
cellular products and intermediates like adenylate kinase, ATP and
Glucose 6 phosphate dehyhdrogenase which can cause an
elevated activity.
o
CK-BB: (CK-1)
CK-MB: (CK-2)
CK-MM: (CK-3)
o
o
Male
Female:
46-300 U/L
34-180 U/L
Clinical Significance:
PRONOUNCED ELEVATION
(5 OR MORE TIMES NORMAL)
Duchenne’s muscular dystrophy
Polymyositis
Dermatomyositis
Myocardial infarction
MILD OR MODERATE ELEVATION
(2-4 TIMES NORMAL)
Myocardial infarction, severe ischemic
injury
Severe exercise, trauma, surgical
procedure, intramuscular injections
Delirium tremens, alcoholic myopathy
Pulmonary infarction
Pulmonary edema (some patients)
Hypothyroidism
Acute agitated psychoses
•
CK activity demonstrates an inverse relationship with thyroid
activity
•
In myocardial infarction, CK will rise 4-6 hours after the onset of
pain, peaks at 18-30 hours and returns to normal on the 3rd day.
CK is the most specific enzyme indicator for Myocardial infarction.
Brain type
Heart hybrid
Striated muscle type
§
CK isoenzyme variants
macro-CK
complex of the M subunit, the B subunit
CK-Mt
oligomeric isoenzyme present in greater amounts
in the mitochondrion.
§
Methods of CK isoenzyme determination:
Anticoagulants: oxalates and fluorides inhibit its action of CK
Reference Value:
Isoenzymes:
1.
Electrophoresis: Separated with the use of agar, agarose, or
cellulose acetate. Bands are visualized by incubating the support
with a concentrated CK assay mixture using the reverse reaction.
2.
Immuno-inhibition (measures the catalytic activity): To measure the
CK-MB fraction
a) an anti-CK M is used to inhibit both CK-MM and the single
subunit of CK-MB
b) The B subunit of CK-MB is measured
c) The combined activity of CK-BB, AK and macro CK is
determined in a separate reaction
d) The value of CK-MB therefore is calculated by subtracting
the obtained B subunit to the CK-BB isoenzymes, AK and
macro CK.
3.
Immunoassay (measures the mass concentration): Makes use of
“Sandwich Technique”
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VH REVIEW CENTER
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CHAPTER 9: Diagnostic Enzymology
Compiled by: Mandy A. Delfin, RMT, MSMT
B.
LACTATE DEHYDROGENASE (LD)
E.C 1.1.1.27
L-lactate: NAD+ oxidoreductase
It is a hydrogen transfer enzyme that catalyzes the oxidation of Llactate to pyruvate with the mediation of NAD+ as a hydrogen acceptor.
Optimum pH:
Forward Reaction: (LàP): 8.8-9.8
Reverse Reaction (PàL): 7.4-7.8
Tissue Sources:
o Major Sources: heart, liver, skeletal muscle, kidney, RBC’s
o Minor Sources: lungs, smooth muscles, and brain.
Methods of Determination:
o
An (LàP) continuous monitoring is considered the reference
method
1.
Wrobleuski-Cabaud: Pyruvate formed in the forward reaction
is reacted with 2, 4 DNPH to produce a golden brown
phenylhydrazone at an alkaline pH.
Wrobleuski andLa Due: employs the reverse reaction and
measures the decrease in absorbance as NADH is consumed.
3.
Wacker et.al.: Uses the Là P reaction with the formation of
NADH
Considerations
Reaction Catalyzed:
The reaction is reversible, and the reaction equilibrium strongly
favors the reduction of pyruvate to lactate (PàL) -- the reverse reaction.
2.
§
Specimen: Serum is preferred over plasma for plasma samples maybe
contaminated with platelets which contain high concentration of LD.
Hemolyzed samples should not be used for Red cells contain 150
times more LDH than serum
§
Storage: Room temperature is ideal for storage of LDH particularly
LD4 and LD5, for these isoenzymes are cold labile and its activity is lost
at -25C
Reference Value:
o Forward Reaction: 125-220 U/L
o Reverse Reaction: 297-537 U/L
Isoenzymes:
Isoenzyme
LDH 1 (HHHH)
14-26% (a-HBD)
LDH 2 (HHHM:
29-39%
LDH 3 (HHMM)
20-26%
Characteristics
Fast moving fractions are heat stable, found mostly in
the myocardium and erythrocytes; also found in the
renal cortex.
LDH 4 (HMMM)
8-16%
LDH 5 (MMMM)
6-16%
Slow moving and are heat labile found mostly in the
liver and skeletal muscles; also, seen in the ileum of the
skin
Found in several tissues predominantly in the white
blood cells and brain, Lungs, spleen, pancreas
PAGE 8
VH REVIEW CENTER
KORONADAL, CITY SOUTH COTABATO
CHAPTER 9: Diagnostic Enzymology
Compiled by: Mandy A. Delfin, RMT, MSMT
Clinical Significance:
PRONOUNCED
ELEVATION
(5 OR MORE TIMES
NORMAL)
Megaloblastic anemia
Widespread
carcinomatosis,
especially hepatic
metastases
Systemic shock and
hypoxia
Hepatitis
Renal infarction
C.
MODERATE
ELEVATION
(3-5 TIMES NORMAL)
Myocardial infarction
Pulmonary infarction
Hemolytic conditions
Leukemias
Infectious
mononucleosis
Delirium tremens
Muscular dystrophy
SLIGHT
ELEVATION
(UP TO 3 TIMES
NORMAL)
Most liver diseases
Nephrotic
syndrome
Hypothyroidism
Cholangitis
In myocardial Infarction: LD increases 8-12 hours after the onset of
pain, peaks at 48-60 hours and remains elevated for 10-14 days.
Levels of LD isoenzymes in certain conditions.
Conditions
Findings
Normal serum
LD2 > LD1 > LD3 ? LD4 > LD5
Acute MI
LD1> LD2 (called “flipped”)
Acute renal infarct
Hemolysis
Normal CSCF
LD1 > LD2 > LD3 > LD4 > LD5
Hydrocephalus and seizures
LD2 > LD1
Bacterial meningitis
LD5 > LD4 > LD3 > LD2 > LD1
o
ALDOLASE (ALD)
D-Fructose-1,6-bisdiphosphate D-glyceraldehyde-3-phosphate-lyase
E.C. 4.1.2.13
Reaction Catalyzed
§
Splits D-fructose diphosphate to D-glyceraldehyde phosphate and
dihydroxy acetone phosphate, an important reaction in the
glycolytic breakdown of glucose to lactate.
Optimum pH: 6.8 - 7.2
Methods of Determination:
§
§
Employs the forward reaction and coupled with two other enzyme
reactions.
§
Triose phosphate isomerase: ensure rapid conversion of all
GLAP to DAP
§
Glycerol-3-Phosphate dehydrogenase: to reduce DAP to
glycerol-3-phosphate with NADH acting as a hydrogen donor.
§
The Decrease in NADH concentration is then measured
Historical methods: Pinto, Kaplan and Van Dreal, Sibley and
Lehninger
PAGE 9
VH REVIEW CENTER
KORONADAL, CITY SOUTH COTABATO
CHAPTER 9: Diagnostic Enzymology
Compiled by: Mandy A. Delfin, RMT, MSMT
Considerations:
D.
§
Storage: Enzyme activity is stable at ambient temperature for up to
48 hours and at 4C for several days.
§
Sample: Hemolysis should be avoided since the red cells contain
10 times as much ALD as Serum.
GLYCOGEN PHOSPHORYLASE
1,4-alpha-D-glucan:orthosphate
EC 2.4.1.1
Reaction Catalyzed: Regulates carbohydrate metabolism by mobilizing
glycogen. It catalyzes the first step in glycogenolysis in which glycogen
is converted to glucose-1-phosphate.
Reference Range: 2.5-10 U/L
4x higher in neonate and 2x higher in children
Physiological function: Provides energy supply required for muscle
contraction.
Clinical Significance:
Severe elevations
Muscle
degeneration
Viral hepatitis
Isoenzymes:
Moderate elevations
Gangrene, Megaloblastic anemia,
Metastatic liver CA, Granulocytic leukemia,
Psychosis, Trichinosis
Adult skeletal muscle, myocardium
Liver
Brain, myocardium
Reference Range: Up to 7 ug/dL
Isoenzymes:
Aldolase A
Aldolase B
Aldolase C
GP-MM
GP-LL
GP-BB
Found predominantly in skeletal muscles
Found in the liver, kidney and WBC
Found in the brain tissues
Clinical Significance: GP-BB is significantly more sensitive than CK and
CK-MB for AMI diagnosis during the first 3-4 hours after the onset of
chest pain. Thus maybe an important marker for the early diagnosis of
AMI.
Methods of Determination
1.
2.
3.
Electrophoresis
Coupled assay system
Imunoenzymometric assay
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KORONADAL, CITY SOUTH COTABATO
CHAPTER 9: Diagnostic Enzymology
Compiled by: Mandy A. Delfin, RMT, MSMT
CARDIAC PROFILE
1.
§
Usually requested to establish baseline values and requested for several
sampling in 3 to 8-hour intervals over a 12 to 24-hour period. Frequently
blood is drawn every 3 hours for analysis during the first 12-hour period.
§
Laboratory testing used to assess AMI includes
a. Cardiac troponin T or I
b. CK-MB,
c. Myoglobin
Troponin
Tissue location: Troponins T, I, and C form a complex of three
§
proteins that bind to filaments of skeletal muscle and cardiac to
regulate muscle contraction.
§
§
§
Markers of Myocardial Infarction
§
Test
onset
peak
duration
CK
Troponin
LDH
Myoglobin
3-12H
3-12H
6-12H
1-4H
18-24H
18-24H
24-48H
6-7 H
36-48H
Up to 10 days
6-8 days
24 H
2.
In many institutions, once the cardiac troponin appears elevated,
additional sampling and testing is halted, and the elevated cardiac
troponin is considered diagnostic for AMI.
3.
Clinical significance: cTnT or cTnI (cardiac troponin T or cardiac
troponin I) is used as an AMI indicator because of specificity and
early rise in serum concentration following AMI.
Test methodology: Immunoassay
Reference ranges:
cTnT<0.03 ng/mL
cTnI<0.40 ng/mL
Myoglobin
§
Tissue location: Found in skeletal and cardiac muscles
§
Clinical significance: Increased in skeletal injuries, muscular
dystrophy, and AMI. It is not tissue specific. it is better used as a
negative predictor in the first 2-4 hours following chest pain.
§
Test methodology: Immunoassay
§
Reference ranges:
Male, 30-90 ng.mL
female, <50 ng/mL
Creatine Kinase and CK-MB and other Cardiac Enzymes
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CHAPTER 9: Diagnostic Enzymology
Compiled by: Mandy A. Delfin, RMT, MSMT
LIVER ENZYMES
Methods of Determination:
Enzymes in this category include alanine and aspartate
aminotransferases, glutamate dehydrogenase, ALP, 5’-nucleotidase, γglutamyltransferase, serum cholinesterase and glutathione S-transferase.
1.
Continuous monitoring method (Karmen et.al):
2.
Historical Methods:
A.
ASPARTATE AMINOTRANSFERASE (AST)
Serum Glutamate Oxaloacetate (SGOT)
L-aspartate-2-oxoglutarate aminotransferase
E.C. 2.6.1.1.
Reaction Catalyzed:
a)
Reitman and Frankel : The oxaloacetate formed is determined by
the reddish brown hydrazone it produces with 2,4,
Dinitrophenylhydrazone in alkaline medium
b)
Babson et al Method: The oxaloacetate formed is treated with
daizonium salt (Azoene Fast Violet B) to produce a violet colored
product
Reference Value: 5-30 U/L at 37C
o
o
o
Involves the transfer of an amino group between Aspartate and
keto acids that is necessary in the synthesis and degradation of
amino acids.
The ketoacids formed by the reaction are ultimately oxidized by the
tricarboxylic acid cycle to provide energy.
Coenzyme: Pyridoxal-5’-phosphate
Tissue sources:
o
o
Clinical Significance:
Myocardial infarction: 4-10 X the upper limit of normal. 6-8 hours
o
after the onset of pain and peak on the 24 – 36 the hour. These
usually normalize on the 4th or 5th day.
Major: Sources: Cardiac tissue, liver, and skeletal muscle
Minor Sources: kidney, pancreas, and erythrocytes.
Optimum pH:
o 7.4
§
o
Increased levels are seen in hepatocellular disease, chronic alcohol
abuse and hepatoxicity.
o
Decreased AST levels are seen in pregnant women
Consideration in AST Assays:
a)
b)
c)
Patient Preparation: Muscle trauma like intramuscular
injections, exercise or surgical operation Increases AST activity
Storage: Serum is the best specimen and stable for 48 hours
at 4C.
Sample:
§
Hemolyzed samples must not be used
§
Alcohol contamination Lowers AST values
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VH REVIEW CENTER
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CHAPTER 9: Diagnostic Enzymology
Compiled by: Mandy A. Delfin, RMT, MSMT
B.
ALANINE AMINO TRANSFERASE
Serum Pyruvate Transaminase (SGPT)
E.C. 2.6.1.2
Reaction Catalyzed
Considerations in ALT Assays:
a)
Sample: Slight hemolysis does not interfere (Ciulla)
b)
Storage:
o ALT activity should be assayed on the day of sample collection
since activity is lost at room temperature, 4C and -25C.
o
Stability is maintained at -70C.
Reference Value: 6-37 IU/L 37C
o
Catalyzes the transfer of the amino group of Alanine to alpha
ketoglutaric acid to form pyruvic acid and glutamic acid. Pyridoxal
phosphate acts as a coenzyme
Tissue sources:
§
§
Clinical Significance:
o
o
o
Major source: liver (considered to be the more liver-specific
enzyme)
Minor source: kidneys, heart, skeletal muscle and the pancreas. It is
Optimum pH: 7.4
Methods of Determination:
1.
Continuous monitoring (Walker et.al Method)
2.
Historical Methods:
a)
Reitman and Frankel Technique
b)
Henry and Pollard method: Based on the declining
absorbance of pyruvate (brown in color) as it is converted to
lactate (colorless)
More specific in detecting diseases in non-alcoholic asymptomatic
patients.
Increased levels prompt diagnosis of acute hepatitis.
Was also used to screen donors of blood to exclude patients with
hepatitis C. Levels > 55 U are excluded. However, this is no longer
used today because of the availability of immunoassays for
hepatitis C antibodies
AST/ALT Ratio (De Ritis Ratio)
§
§
§
Very high in alcoholic or toxic liver disease (3-4:1).
Low in acute or chronic viral hepatitis where ALT is high.
If ratio is <1, indicates acute hepatocellular injury (due to high ALT).
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VH REVIEW CENTER
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CHAPTER 9: Diagnostic Enzymology
Compiled by: Mandy A. Delfin, RMT, MSMT
C.
GAMMA GLUTAMYL TRANSFERASE (GGT)
γ-glutamyl peptide: amino acid γ-glutamyltransferase
EC 2.3.2.2
Reaction Catalyzed: Catalyze the hydrolytic cleavage of peptides to form
amino acids or smaller peptides.
Tissue distribution:
§
Major Source: liver (canaliculi of hepatic cells and epithelial cells lining
biliary ductules)
§
Minor Sources: kidneys, pancreas, intestine
.
Methods of Determination:
General principle:
§
The free p-nitoraniline is measured eat 405-420.
§
The Szasz Assay makes use of Gamma-L-glutmayl-2-carboxy-4nitroanilide, a compound similar to L-glutamyl-p-nitroanilide. One
product of the reaction, 5 amino-2-nitrobenzoate is measured at 420
nm. This provides better absorbance and improved sensitivity.
§
Historical methods: Orlowski, Dimora and Kulhanek, Rosalki and Tarlow
Reference Value:
o Females: up to 38 U/L
o Males: up to 55 U/L
Clinical Significance:
o
Increased levels in all hepatobiliary diseases in intra- and post
hepatic biliary tract obstruction, with levels increasing to 5-30 times
the upper reference.
o
GGT activity is induced by drugs (e.g., phenobarbital and
phenytoin) and by alcohol consumption (Indicator of alcohol
consumption)
o
GGT levels are normal in the presence of bone disease and during
pregnancy in contrast to alkaline phosphatase, where levels would
be elevated.
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VH REVIEW CENTER
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CHAPTER 9: Diagnostic Enzymology
Compiled by: Mandy A. Delfin, RMT, MSMT
D.
ALKALINE PHOSPHATASE: (ALP)
Alkaline Orthophosphoric Monoester Phosphohydrolase
E.C. 3.1.3.1
METHODS FOR ALKALINE PHOSPHATASE ACTIVITY MEASUREMENT
Reaction Catalyzed: Catalyze the liberation of inorganic phosphate from
organic phosphate ester with concomitant production of an alcohol.
Optimum pH: 8.6 –10
Physiologic Importance: Facilitates the transfer of metabolites across cell
membranes associated with lipid transport and the calcification process in
osseous tissues.
Tissue sources: liver, bone, spleen, kidney, intestines, placenta.
Methods of Determination: Chromogenic or “self-indicating substrates”
§
Hydrolysis of nitrophenyl phosphate (4-NPP) or P-nitro phenyl
phosphate with 4- Mg++
§
The enzyme reaction is continuously monitored by observing the
formation of the 4-nitrophenoxide ions which is yellow in color.
§
Considered as the reference method
Method
Bodans (Kay
and
Bodansky)
ShinowaraJonesReinhart
KingArmstrong
Besseylowry-Brock
Substrate
Βglycerophosphate
End Products
Inorganic
phosphate
Comments
Long incubation
time
Βglycerophosphate
Organic
phosphate
High blank values;
long incubation time
Phenylphosphate
Phenol
p-nitrophenyl
phosphate
BowersMcComb
p-nitrophenyl
phosphate
KleinBabsonReed
Higgins and
Talalay
Moss
Buffered
phenolphthalein
phosphate
Phenolphthalein
diphosphate
α-naphthyl
phosphate
p-nitrophenyl or
yellow
nitrophenoxide
p-nitrophenol
or yellow
nitrophenoxide
Free
phenolphthalein
Endpoint; requires
protein removal
Endpoint or kinetic;
rapid
Uses phosphateaccepting buffer
Phenolphthalein
α-naphthol
Considerations
o Specimen Preservation: If specimen is frozen and thawed ALP levels will
increase by 1% per hour and a 10% increase after 96 hours. This is due
to the breakdown of ALP lipoprotein complex.
o
Anticoagulant Interference: EDTA inactivates ALP due to the Chelation
of magnesium ions
o
Activators of ALP: Methyl amino propanol, Dietholamine, Tris
(hydroxymethyl) amino methane, Ethyl amino Ethanol
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KORONADAL, CITY SOUTH COTABATO
CHAPTER 9: Diagnostic Enzymology
Compiled by: Mandy A. Delfin, RMT, MSMT
Reference Value:
Differential characteristics of selected ALP isoenzymes.
Adults :
0-3 months;
3 months to 10 yrs:
10 years to puberty:
20-105 U/L
70-220 U/L
50-260 U/L
60-295 U/L
Isoenzymes
Liver
Bone
Intestine
Placenta
Regan (CA)
Clinical Significance:
PRONOUNCED
ELEVATION
(5 OR MORE TIMES
NORMAL)
Bile duct obstruction
(intrahepatic or
extrahepatic)
Biliary cirrhosis
Osteitis deformans
(Paget’s disease)
Osteogenic sarcoma
Hyperparathyroidism
MODERATE
ELEVATION
(3-5 TIMES NORMAL)
Granulomatous or
infiltrative diseases of
liver
Infectious
mononucleosis
Metastatic tumors in
bone
Metabolic bone
diseases (rickets,
osteomalacia)
SLIGHT ELEVATION
(UP TO 3 TIMES
NORMAL)
Viral hepatitis
Cirrhosis
Healing fractures
Pregnancy (placental
isoenzyme conspicuous)
Normal growth patterns
in children
Differential diagnosis of liver and bone ALP.
ALP
5’N
LAP
GGT
Liver Disease
Increased
Increased
Increased
Increased
Inhibition by
L-phenyl alanine
Heat (65°C for 15
(5 x 10-3 M)
min) or urea (3 M)
Stable
Intermediate
Stable
Labile
Labile
Intermediate
Labile
Stable
Labile
Stable
Order of
anodal
migration
1 (fastest)
2
4 (slowest)
3
3
ALP Isoenzyme Studies in Cancer Patients:
o
Regan Isoenzyme. (Carcinoplacental) Found in patients with lung
cancer and in some women with cancer of the ovary and breast
cancer. the most heat-stable of all ALP isoenzymes being stable at
65°C for 30 minutes and inhibited by Phenylalanine.
o
Nagao Isoenzyme. (Carcinoplacental) seen in patients with
adenocarcinoma. Similar properties to Regan but this variant is
inhibited by L-Leucine
o
Kasahara Isoenzyme. Referred as the Regan variant. This is
observed in patients with hepatoma and in patients with tumors of
the gastrointestinal tract.
Bone Disease
Increased
Normal
Normal
Normal
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VH REVIEW CENTER
KORONADAL, CITY SOUTH COTABATO
CHAPTER 9: Diagnostic Enzymology
Compiled by: Mandy A. Delfin, RMT, MSMT
E.
5’-NUCLEOTIDASE
5’-ribonucleotide phosphorylase;NTP
EC 3.1.3.5
Reaction Catalyzed: Phosphatase that acts only on nucleoside-5’phosphates, such as adenosine-5’phosphate (AMP) and adenylic acid,
releasing inorganic phosphate.
§
§
Formation of H202 is monitored at 510 nm by oxidation of a
chromogenic substance
The effect of ALP in the substrate is inhibited by βglycerophosphate
Considerations in 5’-NTP Assay: NTP activity in serum or plasma heparin is
stable for at least 4 days at 4C and 4 months at -20C.
Clinical Significance:
§
§
It reflects hepatobiliary disease with considerable specificity. It is
increased threefold to six fold.
It is only moderately increased in serum hepatitis and in
parenchymal cell diseases.
Reference Value: 3-9 U/L
Tissue sources: It is a glycoprotein widely distributed throughout the tissues
of the body and is principally localized in cytoplasmic membrane of
Optimum pH: 6.6 and 7.0
Methods of Determination:
Inosine Monophosphate
NTP
Inosine
Purine-nucleoside phosphorylase
Inosine
Hypoxanthine
Xanthine oxidase
Hypoxanthine
+
H2O
Uric acid +
H2O2
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VH REVIEW CENTER
KORONADAL, CITY SOUTH COTABATO
CHAPTER 9: Diagnostic Enzymology
Compiled by: Mandy A. Delfin, RMT, MSMT
PANCREATIC ENZYMES
Assays of Serum AMY, LPS, TRY, Chymotrypsin, and elastase 1 (E1), are
applied to the investigation of pancreatic disease.
A.
6.
Coupled Kinetic Method (Tietz)
AMYLASE (AMS)
Alpha-1-4glucan-4-glucano-hydrolase
E.C. 3.2.1.1
AMS is the smallest in size of the enzyme with a molecular weight
of 50,000. Because of its small size, it is readily filtered by the renal
glomerulus and appears in the urine.
Reaction Catalyzed: Catalyzes the breakdown of starch and glycogen to
produce glucose, maltose and dextrin. Requires calcium and chloride ions
for activation.
Tissue sources: acinar cells of the pancreas and the salivary gland
Methods of Determination:
1.
2.
Amyloclastic Method (Caraway ): Measures the decrease in substrate
concentration as the blue color of substrate solution decreases as
indicated by I2.
.
Saccharogenic Method: This measure the products based on their
reducing properties.
3.
Amylometric method: This measures the amount of starch hydrolyzed
within a specified time (similar to amyloclastic)
4.
Chromometric Methods: This measure the time required for complete
hydrolysis of a substrate.
5.
Chromolytic Methods: This measure the amount of soluble dye released
from an insoluble starch-dye complex.
Considerations
§
Mouth pipetting will contaminate the sample with salivary amylase
§
Lipemic specimen causes reduction in amylase activity
§
Citrate, oxalate, fluoride and EDTA all inhibit amylase activity.
Clinical Significance:
§
It peaks in 2-12 hours. Levels may rise 4-8 X it will return to normal
within 3-4 days. * Larson, D.L. Clinical Chemistry, Fundamentals and
Laboratory Techniques
§
Elevated levels are also seen in mumps, perforated peptic ulcers,
appendicitis, ruptured ectopic pregnancy, dissecting aortic
aneurysm and biliary tract disease.
Reference Range: 31-107 U/L
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VH REVIEW CENTER
KORONADAL, CITY SOUTH COTABATO
CHAPTER 9: Diagnostic Enzymology
Compiled by: Mandy A. Delfin, RMT, MSMT
Considerations
B.
LIPASE(LPS)
Triacyl Glycerol Acylhydrolase
E.C. 3.1.1.3
o
Lipemic specimens cause a reduction in Lipase Activity
o
Use of olive oil as substrate precludes measurement of non-specific
esterases like the “clearing factor”.
o
Opiates and morphines increase LPS activity due to their spasticv
effects of the duodenal musculature and the Spinchter of Oddi.
Reaction catalyzed:
o
Hydrolyzes the ester linkages of fats to produce alcohols and fatty
acids.
Clinical Significance:
o
In acute pancreatitis, LPS increase 4–8 hours, it may rise 2-50X
and remains elevated for 8–14 days. Increased lipase activity
rarely lasts longer than 14 days
o
LPS is also elevated in pancreatic duct obstruction and tumors
of the pancreas.
Tissue Sources: pancreas
Optimum pH: 7.8 – 8.0
Reference Range: Up to 38
Laboratory Methods:
Titrimetric.
a) Cherry-Crandall method: Lipase hydrolyzes triglycerides (tri-olein) in
olive oil. After an overnight incubation, the fatty acid released is
titrated with NaOH using phenolphthalein as the indicator.
b) Henry-Sobel-Berkman method (modified Cherry-Crandall), a pH
meter or a Thymolphthalein indicator is used. The incubation period
is reduced to 16 hours.
Turbidimetric. This uses olive oil as substrate. The decrease in turbidity
as the hydrolysis progresses is measured. Adaptation of this principle
includes the Shihabi-Bishop and the Sigma-Tietz methods.
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VH REVIEW CENTER
KORONADAL, CITY SOUTH COTABATO
CHAPTER 9: Diagnostic Enzymology
Compiled by: Mandy A. Delfin, RMT, MSMT
OTHER MEDICALLY IMPORTANT ENZYMES
A.
ACP Inhibitors
ACID PHOSPHATASE (ACP). Acid Orthophosphoric monoester
phosphohydrolase E.C. 3.1.3.2
Reaction Catalyzed: Belongs to the same group of phosphatase enzymes as
ALP and is a hydrolase that catalyzes the same type of reactions.
Optimum pH:
Inhibits specific prostatic ACP
Inhibits Red cell ACP
Considerations in ACP assays
4.9 to 5
o
Due to release of ACP from platelets during clotting ACP is slightly
higher than plasma. Hemolyzed specimens must be avoided since
red cells contain ACP, same with chylous serum, which presents
analytical difficulty.
o
Serum from all specimens should be immediately separated from
red cells and stabilized by the addition of sodium monohydrate
citrate. (10 mg/ml of serum)
Tissue Sources: Prostate gland (richest source) erythrocytes, platelets, liver,
spleen milk and bone marrow.
Methods of Determination
Method
Bodansky
Substrate
βglycerophosphate
Gutman &
Gutman KingArmstrong
Hudson
Phenylphosphate
Babson & Reed
α-naphthol
Bessey-LowryBrock
Roy
PNPP
Rietz,-Guilbault
PNPP
Thymolphthalein
monophosphate
4-methyl
umbeliferone
phosphate
Product
Inorganic
phosphate and
glycerol
Phenol
Comments
Lengthy assay
and nonspecific
Non-specific
p-nitrophenol
(yellow)
α-naphthol
Non-specific;
rapid
Complicated;
less sensitive
p-nitrophenol
(yellow)
Thymolphthalein
(blue)
4-methyl
umbelliferone
Clinical Significance:
o
Elevated levels of ACP are seen in patients with metastatic carcinoma of
the prostate particularly the tartrate inhibitable ACP
Moderate Elevation of Total ACP
Paget’s disease
Female breast cancer
Hyperparathyroidism
o
More specific
for prostatic
from due to
the specific
substrate
Fluorescent;
some
improved
sensitivity
L-tartrate ions
Formaldehyde and cupric anions
Non-prostatic ACP elevation
Neimann-Pick disease
Gaucher’s disease
Myelocytic Leukemia
ACP assays have proven useful in forensic clinical chemistry, particularly
in the investigation of rape. Vaginal washings are examined for seminal
fluid-ACP activity is presumptive evidence of rape in such cases.
Reference Value:
Total ACP:
Prostatic ACP
Males
Females:
Males:
Females:
2.5-11.7
0.3-9.2
0.2-5.0
0.0-0.8
U/L
U/L
U/L
U/L
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VH REVIEW CENTER
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CHAPTER 9: Diagnostic Enzymology
Compiled by: Mandy A. Delfin, RMT, MSMT
B.
GLUTAMATE DEHYDROGENEASE (GLD)
L-glutamate : NAD(P) oxidoreductase
E.C. 1.4.1.3
C.
CHOLINESTERASE
Type
Reaction Catalyzed: Catalyzes the removal of hydrogen from L-glutamate to
form the corresponding keto amino acid that undergoes spontaneous
hydrolysis to 2-oxoglutarate. GLD is a zinc containing enzyme
Acetylcholinesterase
Acetyl Choline acetyl
hydrolase
EC 3.1.1.7
Pseudocholinesterase,
Acylcholineacylhydrolase
EC 3.1.1.8
Common
name
RBC
cholinesterase
plasma
cholinesterase
Substrate
Source
acetylcholine
blood and
neural
synapses
butyrylcholine
liver
Reaction Catalyzed:
Tissue Sources: is a mitochondrial enzyme in the liver, heart muscles,
kidneys
Methods of Determination: Continuous-Monitoring method: Measures the
rate of the decrease in absorbance at 340 NADH + H is converted into NAD.
Considerations in GD Assay:
o
o
Oxamate is added in the reverse to inhibit LD activity.
Its activity in serum is stable at 4C for 48 hours and at -20C for
several weeks.
Reference Value
o Male: up to 8 U/L
o Female: up to 6 U/L
Clinical Significance:
o
§
Catalyze the hydrolysis of the neurotransmitter acetylcholine into
choline and acetic acid, a reaction necessary to allow a cholinergic
neuron to return to its resting state after activation.
Methods of Determination: Measures. increase in absorbance is measured
at 410 nm.
Substrates for PChE:
§
•
Butyrylcholine
•
Propionythiocholine (Eliman Technique)
•
Benzylcholune (Michel< Kalow and Genest)
Increased in serum of patients with hepatocellular damage:
§ Fourfold to fivefold: Chronic hepatitis
§ Twofold: Cirrhosis
§ Very large rise: Halothane toxicity and other hepatoxic agents
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VH REVIEW CENTER
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CHAPTER 9: Diagnostic Enzymology
Compiled by: Mandy A. Delfin, RMT, MSMT
Considerations Serum is the sample of choice. Enzyme activity in serum is
stable if specimen is kept in the refrigerator, and for several years if stored at
-20C. Hemolysis interfere with the test (Ciulla)
Clinical Significance:
o
Increased G6PD is seen in myocardial infarct and megaloblastic
anemia.
o
G6PD Assay
1.
Ascrobate Cyanide Test.
§
As a test of liver function
§
As an indicator of possible insecticide poisoning
o
§
For the detection of patients with atypical forms of the enzyme who are
at risk of prolonged responses to certain muscle relaxants used in
surgical procedures.
Blood is incubated with a solution of sodium cyanide and sodium
ascorbate, hydrogen peroxide is generated from the coupled
oxidation of ascorbate and hemoglobin.
o
Cyanide inhibits catalase, hydrogen peroxide is available to oxidize
hemoglobin, and the brown color of methemoglobin is discernable.
o
This occurs more rapidly in G6PD-deficient cells than in normal cells.
D. GLUCOSE 6-PHOSPHATE DEHYDROGENASE (G6PD)
EC 1.1.1.49
§
Tissue source: Found in erythrocytes, adrenal glands, thymus, lymph
nodes, spleen
§
Physiologic Function:
o
Maintains NADPH in reduced form this is required to generate
sufhydryl-containing proteins e.g., glutathione (from oxidized to
reduced state).
o
In reduced form glutathione protects hemoglobin from oxidation
by oxidizing agents (oxidants) e.g., drugs (primaquine which is antimalarial), superoxide radicals, hydrogen peroxides present in the
cell.
Decreased G6PD results in decreased NADPH and decreased
glutathione. Red cells exposed to oxidants undergo hemolysis due
to the oxidation of hemoglobin and damage to cell membranes.
o
o
2.
Fluorescent Spot Test. (recommended by the International Committee
for Standardization in Hematology)
o Whole blood is added to a mixture of glucose-6-phopshate, NADP,
saponin (to lyze the red cells), and a buffer.
o A spot of this mixture is placed on filter paper and observed for
fluorescence with UV lamp.
o If G6PD is present, NADP is converted to NADPH. NAPH fluoresces
but not NADP. Results are available after 30-60 minutes.
3.
Colorimetric Test.
§
Basically, the same as the fluorescent spot test. However, the
NADPH produced is used to reduce tetrazolium dye to a violet
formazan product if the enzyme is present.
G6PD deficiency is an inherited sex-linked trait that may result in
drug-induced hemolysis.
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VH REVIEW CENTER
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CHAPTER 9: Diagnostic Enzymology
Compiled by: Mandy A. Delfin, RMT, MSMT
G. Miscellaneous Enzymes:
E.
Leucine Amino Peptidase
§
Reaction Catalyzed:
o
o
§
2.
Angiotensin-Converting Enzyme (ACE).
This enzyme exhibits a naphthylamidase activity. The substrate
for the enzyme LAP is acyl β naphthylamide (ABN).
The enzyme attacks the free amino end of the peptide chain
releasing the amino acid which carries the group. It is rich in
the pancreas.
Goldbarg-Rutenburg method. The β-naphthylamide
produced by the enzymatic activity is allowed to react with
ethylene diamine dihydrochloride to form a blue product
which is measured spectrophometrically.
Clinical Significance:
o
F.
OCT (ornithidine carbamoyl trasferase) and ID (Iditol
dehydrogenase). It is found almost exclusively in the liver. It is an
excellent marker for liver diseases (rarely used).
Laboratory Methodology
o
§
1.
LAP increases in hepatobiliary disease, pancreatic cancer (if
obstructive jaundice and metastasis to liver are present), and
last trimester of pregnancy. It is highest in obstructive biliary
disease paralleling ALP levels. It is normal in bone diseases.
Other Glycolytic Pathway Enzymes
1.
Phosphohexose isomerase (PHI): It is increased in metastatic
cancer.
2.
Isocitrate Dehydrogenase (ICD): It is increased in primary liver
disease, MI with congestive heart failure. It highest concentration is
seen in the in central veins of the liver (sensitive index of
centrilobular necrosis)
o
It has peptidyl dipeptide hydrolase activity. Its substrate is
benzoyl glycyl histidyl leucine.
o
The released hippuric acid is measured. The main sources are
the lungs, testis, and brain (for Alzheimer’s use CSF).
o
It is increased in liver disease, active sarcoidosis leprosy, and
Gaucher’s disease. It is normal in inactive diseases and
granulomatous diseases of the lungs e.g., tuberculosis and
berylliosis.
3.
Guanase. This is used for hepatic disease.
4.
β-glucoronidase. This is a biochemical clue to neoplastic diseases
and hepatitis.
5.
Alcohol dehydrogenase. This is used also for hepatic disease.
6.
Pepsinogen. This reflects function and disease of stomach. It is
increased in peptic ulcer and decreased in pernicious anemia.
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