Overview 2

 Enzymes- are specific biologic proteins that catalyze

biochemical reactions without altering the equilibrium
point of the reaction or being consumed or changed in
composition
 Catalyzed reactions are frequently specific and essential
to physiologic functions, such as the hydration of carbon
dioxide, nerve conduction, muscle contraction, nutrient
degradation, and energy use
 In all body tissues, enzymes frequently appear in the
serum following cellular injury or sometimes in smaller
amounts from degraded cells.
 Plasma or serum enzyme levels are often useful in the
diagnosis of particular diseases.
GENERAL PROPERTIES 3

Active site often a water-free cavity, where the

substance on which the enzyme acts (substrate)
interacts with particular charged amino acid
residues.
Allosteric site is a cavity other than the active site
and allows binding to regulator molecules.
 GENERAL PROPERTIES 4

 Even though a particular enzyme

maintains the same catalytic
function throughout the body, that
enzyme may exist in different forms
within the same individual.
(ISOENZYMES)
 The different forms may be
differentiated from each other
based on certain physical
properties such as electrophoretic
mobility, solubility, or resistance to
inactivation.
 Definition of Terms 5
 An ISOFORM- results when an enzyme
is subject to posttranslational
modifications.
 Isoenzymes and Isoforms contribute
to heterogeneity in properties and
function of enzymes.
 A non-protein molecule, called a
COFACTOR may be necessary for
enzyme activity.
 Inorganic cofactor, such as chloride
or magnesium ions are called
ACTIVATOR
Definition of Terms 6

 A coenzyme is an organic cofactor, such

as nicotinamide adenine dinucleotide
(NAD). When bound tightly to the enzyme,
the coenzyme is called a prosthetic group.
 The enzyme portion (apoenzyme) with its
respective coenzyme, forms a complete
and active system, holoenzyme
 Some enzymes, mostly digestive enzymes,
are originally secreted from the organ of
production in a structurally inactive form
called a proenzymes or zymogen
 7
ENZYME CLASSIFICATION AND
NOMENCLATURE
International union of biochemistry
 The IUB system assigns a systematic name to
each enzyme, defining the:
 substrate acted on,
 the reaction catalyzed,
 the name of any coenzyme involved in the reaction.
 Trivial recommended name is also assigned by
the IUB system
 The IUB system identifies each enzyme by an EC
numerical code containing four digits separated
by decimal points. The first digit places the
enzyme in one the following six classes
ENZYME CLASSIFICATION AND 8
NOMENCLATURE
 The IUB system identifies each enzyme by an EC
numerical code containing four digits
separated by decimal points.
 The first digit places the enzyme in one the
following six classes
 The second and third digits of the EC code
number represent the subclass of the enzyme,
respectively, divisions that are made according
to criteria specific to the enzymes in the class.
 The final number is the serial number specific to
each enzyme in a subclass.
 Classes of Enzymes 9
 Oxidoreductases- catalyzes an oxidation-reduction
reaction between two substrates.
 Tranferases- catalyze the transfer of a group other than
hydrogen form one substrate to another
 Hydrolases- catalyze hydrolysis of various bonds
 Lyases- catalyzed removal of groups from substrate
without hydrolysis; the production contains double
bonds
 Isomerases- catalyze the interconversion of geometric,
optical or positional isomers
 Ligases- catalyze the joining of two substrate molecules,
coupled with breaking of the pyrophosphate bond in
adenosine triphosphate (ATP) or a similar compound.
 ENZYME KINETICS 10

 A chemical reaction may occur spontaneously if the free

energy or available kinetic energy is higher for the reactants
than for the products
 The reaction then proceeds toward the lower energy if a
sufficient number of the reactant molecules posses enough
excess energy to break their chemical bonds and collide to
form new bonds.
 The excess energy, called activation energy, is the energy
required to raise all molecules in 1 mole of a compound at a
certain temperature to the transition state at the peak of the
energy barrier.
 At the transition state each molecule is equally likely to either
participate in product formation or remain an unreacted
molecule
 Reactants possessing enough energy to overcome the
energy barrier participate in product formation
 ENZYME KINETICS
11
 One way to provide more energy for a reaction is
to increase the temperature and thus increase
intermolecular collisions, however, this does not
normally occur physiologically.
 Enzymes catalyze physiologic reactions by
lowering the activation energy level that the
reactant (substrates) must reach for the reaction
to occur
 The reaction may then occur more readily to a
state of equilibrium in which there is no net forward
 The extent to which the reaction progresses
depends on the number of substrate molecules
that pass the energy barrier
12
Enzyme, Substrate and 13

Product Relationship
 The general relationship among enzyme,
substrate, and productivity may be
represented as follows

E + S = ES = E + P

 Where E is enzyme, S is substrate, ES is

enzyme-substrate complex and P is product
 The ES complex is a physical binding of a
substrate to the active site of an enzyme.
14
 Specificity
15
 Different enzymes are specific to substrates in different
extents respects.
 Absolute specificity - meaning that enzyme combines with
only one substrate and catalyze only the one corresponding
reaction.
 Group specificity - they combine with all substrate containing
a particular chemical group, such as a phosphate ester.
 Bond specificity – specific to chemical bonds
 Stereoisometric specificity - refers to enzymes that
predominantly combine with only one optical isomer of a
certain compound.
 In addition an enzyme, an enzyme may bind more than
one molecule of substrate, and this may occur in a
cooperative fashion.
 Binding of one substrate molecule, therefore may facilitate
binding of additional substrate molecules
 16
FACTORS THAT INFLUENCE
ENZYMATIC REACTIONS
SUBSTRATE CONCENTRATION
 Michaelis and Menten hypothesized the role substrate
concentration in formation of the enzyme-substrate
 First-order kinetics - the reaction rate is directly proportional to
substrate concentration.
 Zero-order kinetics -the reaction rate depends only on enzyme
concentration
17
 18
ENZYME CONCENTRATION
 As long as the substrate concentration exceeds the
enzyme concentration, the velocity of the reaction
is proportional to the enzyme concentration.
 The higher the enzyme level, the faster the reaction
will proceed because more enzyme is present to
bind with the substance
pH
 Changes in pH may denature an enzyme or
influence its ionic state, resulting in structural
changes or a change in the charge on an amino
acid residue in the active site
 Enzyme operates within a specific pH range and
maximally at a specific pH.
 Most physiologic enzymatic reactions occur in the
pH range of 7.0 to 8.0
 pH by means of appropriate buffer solutions
 Temperature
19
 increasing temperature usually increases the rate of a
chemical reaction by increasing the movement of
molecules.
 this is the case with the enzymatic reactions until
temperature is high enough to denature the protein
comparison of the enzyme.
 For each 10⁰ increase in temperature, the rate of the
reaction will approximately double until, of course the
protein is denatured
 the optimal temperature is usually close to that of the
physiologic environment of the enzyme (37C)
 Rate of denaturation increases as the temperature
increases and is usually significant at 40⁰ to 50⁰C.
 Temperature
20
 Low temperatures renders enzymes reversibility
inactive, many serum or plasma specimens for
enzyme measurement are refrigerated or frozen
to prevent activity loss until analysis.
 Storage procedures may vary from enzyme to
enzyme because of individual stability
characteristics. Repeated freezing and thawing
however, tends to denature protein and should
be avoided
 Enzymes should be analyzed under strictly
controlled temperature conditions. Incubation
temperatures should be accurate within 0.1 C.
 Usually attempt to establish an analysis
temperature for routine enzyme measurement of
25⁰, 30⁰ or 37⁰C.
 Cofactors 21
 Some common coenzymes (organic cofactors) are
nucleotide phosphate and vitamins.
 Coenzymes serve as second substrate for
enzymatic reactions.
 Example NAD as a cofactor may be reduced to
nicotinamide adenine dinucleotide phosphate
(NADP) in a reaction in which the primary substrate
is oxidized.
 Increasing coenzyme concentration will increase
the velocity of an enzymatic reaction in a manner
synonymous with increasing substrate
concentration
 When quantitating an enzyme that requires a
particular cofactor, that cofactor should always be
provided in excess so that the extent does not
depend on the concentration of the cofactor.
INHIBITORS 22
Enzymatic reactions may not progress
normally if a particular substance, and
inhibitor, interferes with the reaction

 Competitive inhibitors physically bind to

the active site of an enzyme and
complete with the substrate for the
active site.
 With substrate concentration significantly
higher than the concentration of the inhibitor,
the inhibition is reversible because the
substrate is more likely than the inhibitor to
bind the active site and the enzyme has not
yet destroyed.
23
 INHIBITORS 24

 Non-competitive inhibitor- binds an enzyme at a

place other than the active site and may be
reversible
 if the inhibitor binds the enzyme independently from the
substrate, increasing substrate concentration from the
substrate concentration does not reverse the inhibition

 Uncompetitive inhibition-
 is another kind of inhibition in which the inhibitor binds to the
ES complex
 increasing substrate concentration results in more ES
complexes to which the inhibitor binds and thereby
increases the inhibition.
 Enzyme-substrate-inhibitor complex does not yield a product
25
Measurement of Enzyme activity
26
 Because enzyme are usually present in
very small quantities in biologic fluids
and often difficult to isolate from similar
compounds, a convenient method of
enzyme quantitation is measurement of
catalytic activity.
 Activity is then related to concentration.
 Common methods might
Photometrically measure :
 an increase in product concentration
 a decrease in substrate concentration
 a decrease in coenzyme concentration or
 an increase in the concentration of an
altered coenzyme
 Methods of Measuring
Enzymatic Reaction 27
 Fixed-timed method, the reactants are combined, the
reaction proceeds for a designated time, the reaction is
stopped (usually by inactivating the enzyme with a weak
acid) and a measurement is made of the amount of
reaction that has occurred.
 Reaction is assumed to be linear over the reaction time; the larger the
reaction, the more enzyme is present

 Continuous monitoring or kinetic assays- multiple

measurements, usually of absorbance change, are made
during the reaction, either at specific time intervals (usually
every 30 to 60 seconds) or continuously by a continuous-
recoding spectrophotometer.
 These assays are advantages over fixed-time methods because the
linearity of the reaction may be more adequately verified
 Continuous measurement are preferred because any deviation from
linearity is readily observable
Calculation of enzyme activity
 When enzyme are quantitated relative to their
28
activity rather than a direct measurement of
concentration, the units used to report enzyme levels
are activity units.
 Specific methods developers frequently established
their own units for reporting results and often named
the units after themselves (i.e Bodansky and King
units)
 EC defined the international unit (IU) as the amount
of enzyme that will catalyze the reaction of 1 µmol of
substrate per minute under specified conditions of
temperature, pH substrate and activators
 Enzyme concentration is usually expressed in units per
liter (IU/L)
 The mole is the unit for substrate concentration, and
the unit of time is the second.
 Enzyme concentration can also be expressed as
katals per liter (kat/L) (1.0IU= 17 nkal)
ENZYMES OF 29
CLINICAL SIGNIFICANCE

• Tissue Source
• Diagnostic Significance
• Assay Method
• Source Of Error
• Reference Range
Creatine Kinase 30
 CK is an enzyme with a molecular weight of
approximately 82, 000 that is generally
associated with ATP regeneration in
contractile or transport systems where it is
involved in the storage of high-energy
creatine phosphate.
 Contraction cycle of muscle results in
creatine phosphate use, with the production
of ATP.
 This results in relatively constant level of
muscle ATP.
CK
Creatine + ATP --- Creatine phosphate +ADP
 Tissue source
31
 CK is widely distributed in tissue with highest
activities found in skeletal muscle, heart
muscle and brain tissue
 CK is present in much smaller quantities in
other tissue sources, including the bladder,
placenta, gastrointestinal tract, thyroid,
uterus, kidney, lung, prostate, spleen, liver
and pancreas
 Diagnostic significance 32
 Because of the high concentration of CK in
muscle tissue CK levels are frequently elevated
in disorders of cardiac and skeletal muscle.
 The CK level is considered a sensitive indicator
of acute myocardial infarction (AMI) and
muscular dystrophy, partially the Duchenne
type.
 Levels of CK also vary with muscle mass and
therefore may depend on gender, race,
degree of physical conditioning and age
 Elevated CK levels are also occasionally seen in
central nervous system disorders such as central
nervous system shock (CK-BB elevated).
 Serum CK levels and CK/progresterone ratio
have been useful in the diagnosis of ectopic 33
pregnancies
 Serum CK levels have also been as an early
diagnostic tool to identify patients with
Vibrio vulnificus infections
 CK occurs as a dimer consisting of two
subunits that can be separated readily into
three distinct molecular forms.
 Three isoenzymes have been designated as
CK-BB ( brain type) CK-MB (hybrid type)(CK-
2) and CK-MM (muscle type)(CK-3) on
electrophoretic separation CK-BB will
migrate fastest toward the anode an is
therefore called CK-1
 CK-MB (CK-2) and finally by CK-MM (CK-3) exhibiting34
the slowest mobility
 Values for the MB isoenzyme range from
undetectable to trace (<6% of total CK)
 CK-BB is present in small quantities in the sera of
healthy people
 CK-MM is the major isoenzyme fraction found in
striated muscle and normal serum
 Majority of CK activity in heart muscle is also
attributed to CK-MM with approximately 20%
 Normal serum consist of approximately 94% to 100%
CK-MM.
 Normally, CK-MB 1 is the predominant isoform in the
plasma and the CK-MB2/CK-MB1 ratio is ≤1.
However, following an AMI, the CK-MB2 isoform
becomes elevated in the plasma and the ratio
increases, and ratios of >1.5 are considered to be
diagnostic for myocardial damage
 Mild to strenuous activity may contribute to 35
elevated CK levels as may intramuscular
injections. In physical activity, the extent of
elevation is variable
 The quantity of CK-BB in the tissue is usually small.
The small quantity, coupled with its relatively short
half-life (1-5 hours) results in CK-BB activities that
are generally low and transient and not usually
measurable when tissue damage occurs.

 CK-MB
 Following myocardial infraction the CK-MB levels
begin to rise within 4 to 8 hours peak at 12 to 24
hours, and return to normal levels within 48 to 72
hours.
 Increased quantities are not entirely
specific for AMI but probably reflect 36
some degree of ischemic heart damage
 The speficity of CK-MB levels in the
diagnosis of AMI can be increased if
interpreted in conjunction with LDH
isoenzymes and/or troponins and if
measured sequentially over a 48-hour
period to detect the typical rise and fall
of enzyme activity seen in AMI
 Non-enzyme proteins (troponin I and
troponin T) have been used as a more
specific marker of myocardial damage.
These proteins are released into the
bloodsteam earlier and persist longer
than CK and its isoenzyme CK-MB
37
Atypical Creatine Kinase Isoenzymes
38
 Macro- CK appears to migrate to a position
midway between CK-MM and CK-MB.
 largely comprised of CK-BB complexed with
immunoglobulin. In most instances, the
associated immunoglobin is IgG, although a
complex with IgA is also common
 The term macro-CK has also been used to
describe complexes of lipoproteins with CK-
MM
 The incidence of macro-CK in sera ranges
from 0.8% to 1.6%. currently, no specific
disorder is associated with its presence,
although it appears to be age and sex
related, occurring frequently in women older
than age 50.
 Mitochondrial CK(CK-Mi) is bound to the
exterior surface of the inner membranes of 39
muscle, brain and liver.
 CK-Mi is not present in normal serum and is
typically not present following myocardial
infraction.
 The incidence of CK-Mi ranges from 0.8%-
1.7%.
 For it to be detected in serum, extensive
tissue damage must occur, causing
breakdown of the mitochondrion and cell
wall.
 CK-Mi has been detected in cases of
malignant tumor and cardiac abnormalities.
Methods used for measurement of 40
CK isoenzymes

 electrophoresis
 ion exchange chromatography
 immunoinhibition methods.
 Point of care assay system for CM-MB are
available but not as widely used as those for
troponins
 CK-MB electrophoresis has been the reference.
 Assay enzyme activity 41

 CK Catalyzes both forward and reverse

reactions involving phosphorylation of creatine
or ADP.
 The reverse reaction proposed by Oliver
modified by Rosalki is the most commonly
performed method in the clinical laboratory
reaction
 The optimal pH for the reverse reaction is 6.8; for
the forward reaction, it is 9.0
Reference range 42

 Male – 46-171 U/L (37C)

 Female – 34-145 U/L (37C)

 CK-MB <6% total CK

 The higher the values in males are attributed to increased
muscle mass.
Source of error 43
 Hemolysis of serum samples may be source
of elevated CK activity.
 Serum should be stored in a dark place
because CK is inactivated by light.
 Activity can be restored after storage in the
dark at 4⁰C for 7 days or at -20⁰C for 1 month
when the assay is conducted using a
sulfhydryl activator.
 Because of the effect of muscular activity
and muscle mass on CK levels, it should be
noted that people who are physically well
trained tend to have elevated baseline
levels and the patients who are bedridden
for prolonged periods may have decreased
CK activity
 44
Lactate dehydrogenase
 LDH is an enzyme that catalyzes the interconversion of
lactic and pyruvic acids.
 It is a hydrogen-transfer enzyme uses the coenzyme NAD+

Rxn:
Lactate + NAD+ → Pyruvate + H+
 Tissue source 45

 LDH is widely distributed in the body.

 Highest activities of LDH are found in the
heart, liver, skeletal muscle, kidney and
erythrocytes
 lesser amounts are found in the lung,
smooth muscle and brain
Diagnostic significance 46
 Because of its widespread activity in numerous body tissue, LDH
is elevated in a variety of disorders.
 Increased levels found in cardiac, hepatic, skeletal muscle, and
renal diseases, as well as in several hematologic and neoplastic
disorders.
 The highest levels of total LDH are seen in pernicious anemia and
hemolytic disorders
 In AMI, LDH levels begin to rise within 12 to 24 hours reach peak
levels within 48 to 72 hours and may remain elevated for 10 days.
 When coupled with α-fetoprotein and human chorionic
gonadotropin, LD has been found to be an important serological
marker for diagnosis, staging, recurrence, and monitoring of
germ cell tumors. In particular, marked elevations can also be
observed in most patients with acute lymphoblastic leukemia.
 The enzyme can be separated into five major fractions, each
comprising four subunits.
Isoenzymes 47

 Two different polypeptide chains,

arrangements to yield the five major
isoenzyme fractions
 LDH-1, migrates most quickly toward
the anode, followed in sequence by
the other fractions, with LDH-5
migrating the slowest
 In the sera of healthy individual, the
major isoenzyme fraction is LDH-2
followed by LDH-1, LDH-3 , LDH-4, and
LDH-5
48
 Cardiac tissue and red blood cells contain a
higher concentration of LDH-1. 49
 LDH is not specific to cardiac tissue and therefore
is not a preferred marker of diagnosis of AMI.
 LDH-1/LDH-2 ratios greater than 1 also may be
observed in hemolyzed serum samples.
 Elevations of LDH-3 occur most frequently with
pulmonary disorders and carcinomas
 The LDH-4 and LDH-5 isoenzymes are found
primarily in liver and skeletal muscle tissue
 LDH-5 levels have greatest clinical significance in
the detection of hepatic disorders, particularly
intrahepatic disorders
 LDH-6 is alcohol dehydrogenase. In 50
reported studies, LDH-5, if elevated
concurrently with the appearance of LD6
signifies a grave prognosis and impending
death. (Probably representing hepatic
congestion due to cardiovascular disease)
 It is suggested, therefore that LDH-6 may
reflect liver injury secondary to severe
circulatory insufficiency
 Analysis of LDH isoenzymes can be
accomplished by electrophoresis by
immunoinhibition or chemical inhibition
methods.
 Assay for enzyme activity
51
 LDH catalyzes the interconversion of lactic and
pyruvic acids using the coenzyme NAD+
 The reaction can proceed in either a forward
(lactate [L] or reverse (pyruvate [P] direction.
 Both reactions have been used in clinical assays.
 The rate of the reverse reaction is approximately
three times faster, allowing smaller volumes and
shorter reaction times
 However the reverse reaction is more susceptible to
substrate exhaustion and loss of linearity.
 The optimal pH for the forward reaction is 8.3 to 8.9;
for the reverse reaction, it is 7.1 to 7.4
 Sources of error 52
 Erythrocytes contain an LDH concentration approximately
100 to 150 times that found in serum therefore any degree
of hemolysis should render a sample unacceptable for
analysis.
 LDH activity is unstable in serum regardless of the
temperature at which is it stored.
 If the sample cannot be analyzed immediately, it should
be stored at 25⁰C and analyzed within 48 hours.
 LDH-5 is the most labile isoenzyme.
 Loss activity occurs more quickly at 4⁰C than 25⁰C. serum
samples for LDH isoenzyme analysis should be stored at
25⁰C and analyzed within 48 hours of collection
Reference range 53

 Total LDH : 100 – 225 U/L For Forward

 80-280 U/L For Reverse

 Newer Reference! 125-220 U/L (37 C)

Aspartate Aminotransferase 54

 Aspartate aminotransferase (AST) is an enzyme

belonging to the class transferases.
 It is commonly referred to as a transminase and is
involved in the transfer of an amino groups
between aspartate and ᾳ-keto acids.
 The older terminology, serum glutamic-
oxaloacetic transaminase (SGOT or GOT) may also
used.
 Pyridoxal phosphate functions as coenzyme.
AST

Aspartate + a-keta-glutarate → Oxaloacetate + glutamate

Tissue source 55

 The highest concentrations are found in

cardiac tissue, liver and skeletal muscle,
with smaller amounts found in the kidney,
pancreas and erythrocytes
 Diagnostic significance 56
 The clinical use of AST is limited mainly to the
evaluation of hepaticellular disorders and skeletal
muscle involvement.
 In AMI, AST levels begin to rise within 6 to 8 hours,
peak at 24 hours and generally return to normal
within 5 dayss
 Following congestive heart failure, AST level also
may be increased probably reflecting liver
involvement as a result of inadequate blood
supply to the organ
Diagnostic significance 57

 AST levels are highest in acute hepatocellular

disorders. In viral hepatitis, levels may reach 100
times the normal levels
 Skeletal muscle disorders, such as the muscular
dystrophies and inflammatory conditions also
cause increase in AST levels.
 Exists as two isoenzyme fractions located in the
cell cytoplasm and mitochondria.
 The cytoplasmic isoenzyme is the predominant
form occurring in serum.
 Assay for enzyme activity 58

 Assay method for AST are generally based

on the principle of the Karmen Method,
which incorporates a coupled enzymatic
reaction using malate dehydrogenase
(MD) as the indicator reaction and
monitors the change in absorbance at 340
nm continuously as NADH is oxidized to
NAD
 Source of error 59

 Hemolysis should be avoided because it

can dramatically increase serum AST
concentration.
 AST activity is stable in serum for 3 to 4
days at refrigerated temperatures

Reference Range:

AST: 5 to 35 U/L
 Alanine Aminotransferase 60

 Transfer of an amino group for alanine to ἀ-

ketoglutarate with the formation of glutamate and
pyruvate.
 The older terminology was serum glumatamic pyruvic
transaminase (SGPT, or GPT)
Rxn:
ALT

Alanine + a-ketoglutarate → pyruvate + glutamate

Tissue source 61

 ALT is distributed in many tissues, with

comparatively high concentrations in the liver.
 It is considered the more liver specific enzyme of
the transferases
 Diagnostic significance 62
 Clinical applications of ALT assays are confined mainly to
evaluation of hepatic disorders.
 Higher elevations are found in hepatic disorders than in
extrahepatic or intrahepatic obstructive disorders.
 In acute inflammatory conditions of the liver, ALT
elevations are frequently higher than those of AST and
tend to remain elevated longer as a result of the longer
half-life of ALT in serum (16 and 24 hours respectfully)
 Cardiac tissue contains a small amount of ALT activity,
but the serum level usually remains normal in AMI unless
subsequent liver damage has occurred.
 ALT levels have historically been compared with the levels
of AST to help determine the source of an elevated AST
level and to detect liver involvement concurrent with
myocardial injury.
 Assay for enzyme activity 63
 Using LDH as the indicator enzyme which catalyze
the reduction of pyruvate to lactate with the
simultaneous oxidation of NADH.
 The change in absorbance at 340 nm measured
continuously is directly proportional to ALT activity.

Alanine + ᾳ-ketoglutarate ----→ pyruvate +

glutamate pyruvate + NADH + H -----→ Lactate +
NAD
Source of Error 64

 ALT is stable for 3 to 4 days at 4C.

 It is relatively unaffected by hemolysis

Reference Range:
ALT: 7-45 U/L
 Alkaline phosphate 65
 Catalyze the hydrolysis of various phosphate
monoesters at an alkaline pH.
 Consequently ALP is a nonspecific enzyme
capable of reacting with many different
substrates.
 ALP functions to liberate inorganic phosphate
from an organic phosphate ester with the
concomitant production of an alcohol.
 Optimal pH varies with the substrate used. The
enzyme requires Mg²+ as an activator.
 Tissue source 66

 ALP activity is present on cell surfaces in

most human tissue.
 The highest concentrations are found in
the intestine, liver, bone, placenta, spleen
and kidney
 Diagnostic significance 67

 Elevations of ALP are of most diagnostic

significance in the evaluation of hepatobiliary and
bone disorders.
 In the hepatobiliary disorders, elevations are more
predominant in obstructive conditions than in
hepatocellular disorders
 In bone disorders elevations are observed when
there is involvement of osteoblasts
 In biliary tract obstruction, ALP levels range from 3
to 10 times ULN.
 Increase are primarily a result of increased
synthesis of the enzyme induced by cholestasis.
 In contrast, hepatocellular disorders, such as hepatitis
and cirrhosis, show only slight increase, usually less 68
 Perhaps the highest elevations of ALP activity occur in
Paget’s disease (osteitis deformans).
 Other bone disorder include osteomalacia, rickets
hyperparathyroidism and oesteogenic sarcoma.
 In addition increased levels are observed in healing
bone fractures and during the periods of physiologic
bone growth.
 In normal pregnancy increased ALP activity averaging
approximately 1 ½ times ULN, can be between weeks
16 to 20. ALP activity increases and persists until the
onset of labor
 Elevations also may be seen in complications
of pregnancy such as hypertension, 69
preeclampsia, and eclampsia, as well as in
threatened abortion.
 ALP levels are significantly decreased in the
inherited conditions of hypophosphatasia.
 The major isoenzymes, which found in the
serum and have been most extensively
studied are those derived from the liver, bone,
intestine and placenta.
 Electrophoresis is considered the most useful
single technique for ALP isoenzyme analysis.
 A direct immunochemical method can also
be used
 The liver fraction migrates the fastest, followed
by bone, placental and intestinal fractions.
 Many hepatobiliary conditions cause elevations of this 70
fraction (liver), usually early in the course disease. The
fast-liver fraction has been reported in metastatic
carcinoma of the liver, as well as in other hepatobiliary
disease. It presence is regarded as a valuable indicator
of obstructive liver disease
 The bone enzyme increase due to osteoblastic activity
and is normally elevated in children during periods of
growth and in adults older than age 50
 Presence of intestinal ALP isoenzyme in serum depends
on the blood group who have B or O blood group and
individual
 increase in intestinal ALP occur after consumption of a
fatty meal
 Increased in levels are also found in patients undergoing
chronic hemodialysis
 Difference in heat stability is the basis of a
second approach used to identify the 71
isoenzyme source of elevations.
 These are based on the finding that placental
ALP is the most stable fraction, followed by
intestinal, liver, bone fractions in decreasing
order of heat stability
 A third approach to identification of ALP
isoenzymes is based on selective chemical
inhibition. Phenylalanine is one of the several
inhibitors that have been used .
 Phenylalanine inhibits intestinal and placental
ALP to a much greater extent than liver and
bone. It is impossible to differentiate placental
from intestinal ALP or liver from bone ALP
 In addition to the four major ALP isoenxzyme 72
fractions,certain abnormal fractions are
associated with neoplasm, the most
frequently seen are the Regan And Nagao
Isoenzymes.
 They have been referred to as
carcinoplacental alkaline phosphates
because of their similarities of the placental
isoenzyme
 The regan isoenzyme has been characterized
as an example of an ectopic production of
an enzyme by malignant tissue.
Carcinoplacental 73

Alkaline phosphates
 Various carcinoma, such as lung, breast, ovarian and
colon with the highest incidence in ovarian and
gynecologic cancer.
 Regan isoenyzme migrates to the same positions as the
bone fraction and is the most heat stable of all ALP
isoenzymes, resisting denaturation at 65⁰C for 30 minutes
 Nagao isoenzyme may be considered a variant of the
regan isoenzyme. Its electrophoretic heat-stability and
phenylalaline- inhibition properties are indentical to those
of the regan fraction
 Assay For Enzyme Activity
74
 A continuous monitoring technique based on
method devised by Bowers and McComb allows
calculation of ALP activity based on the molar
absorptivity of p-nitrophenol is hydrolyzed to p-
nitrophenol (yellow) and the increase in
absorbance at 405 nm which is directly
proportional to ALP.

Source Of Error
 Hemolysis may cause slight elevations.
 Diet may induce elevations in ALP activity of
blood group B and O individuals who are
secretors

Reference Range
 75
Acid phosphatase

 Acid phosphatase (ACP) belongs

to the same group of phosphates
enzymes as ALP and is a hydrolyzes
the same type of reactions.
 The major difference between ACP
and ALP is the pH of the reaction.
 ACP functions at an optimal pH
approximately 5.0
Tissue source 76
 ACP activity is found in the prostate, bone liver,
spleen, kidney, erythrocytes and platelets.
 The prostate is the richest source with many times
the activity found in other tissue
 77
Diagnostic significance
 ACP measurement has been used as an aid in the
detection of prostatic carcinoma, particularly
metastatic carcinoma of the prostate
 Total ACP determination is relatively insensitive
technique detecting elevated ACP level resulting from
prostatic carcinoma in the majority cases only when
the tumor has metastasized.
 Newer markers, such as prostate specific antigen (PSA)
are more useful screening and diagnostic tools
 Specific substrate for prostatic ACP is thymolpthaline
monophosphate.
 Chemical inhibition methods used to differentiate the
prostatic portion used tartrate as the inhibitor
 One technique with much improved 78
sensitivity over conventional ACP assays
is the immunologic approach using
antibodies that are specific for the
prostatic portion
 ACP assays have proved useful in
forensic clinical chemistry, particularly in
the investigation. Activity has been to be
associated with the osteoclasts
 Because of ACP activity in platelets,
elevations are observed when platelets
damage occurs, as in the
thrombocytopenic purpura
 Assay for enzyme activity 79
 Reaction products are colorless at the acid pH of
the reaction but the addition of alkali stops the
reaction and transforms the products into
chromogens which can be measured
spectrophotometrically
 Thymophthalein monosphosphate(Roy
technique) is the substrate of choice for
quantitative endpoint reactions
 Immunochemical methods: ᾳ-naphthyl
phosphate is preferred. Immunochemical
techniques for prostatic ACP use several
approaches including RIA
 Sources of error 80

 Serum should be separated from the red cells as

soon as the blood has clotted to prevent leakage
of erythrocyte and platelet ACP.
 Serum activity decreases within 1 to 2 hours if the
sample is left at room temperature without the
addition of a preservative.
 Decreased activity is a result of a loss of carbon
dioxide from the serum, with a resultant increase
in pH. With acidification ACP is stable for 2 days
at room temperature
Reference Range:
Prostatics ACP: 0 to 3.5 ng/mL
 81
Glutamyltransferase (GGT)
 ᵞ-glutamyltransferase (GGT) is an enzyme
involved in the transfer of the ᵞ-glutamyl residue
form ᵞ-glutamyl peptides to amino acids H²O,
and other small peptides .
 Established but is suggested that GGT is
involved in peptide and protein synthesis,
regulation of tissue glutathione levels and the
transport of amino acids across cell membrane
 Tissue source 82
 Activity is found primarily in tissue of the
kidney, brain prostate, pancreas, and liver.
 Clinical applications of assay, however are
confined mainly to evaluation of liver and
biliary system disorders.
 Diagnostic significance 83
 In the liver, GGT is located in the canaliculi of the
hepatic cells and particularly in the epithelial cells
lining the biliary ducts.
 Because of the locations, GGT is elevated in virtually all
hepatobiliary disorders, making it one of the most
sensitive of enzyme assays in these conditions
 GGT levels will be increase in patients receiving
enzyme inducing drugs such as warfarin,
Phenobarbital and phenytoin.
 Enzyme elevations may reach levels four times ULN
 Assays are useful in monitoring the effects of abstention
from alcohol and are used as such by alcohol
treatment centers
Diagnostic significance 84

 GGT level are also elevated in other conditions, such

as acute pancreatitis, diabetes mellitus and
myocardial infraction.
 The source of elevation in pancreatitis and diabetes
is probably the pancreas, the source of GGT in
myocardial infraction
 Assay for enzyme activity
 The ᵞ-glutamyl residue is transferred to glycylglycine
85
releasing p-nitroaniline, a chromogenic product with a
strong absorbance at 405 to 420 nm. The reaction which
can used as continuous- monitoring or fixed point
method, is outlined

Source of errors
 GGT activity is stable with no loss of activity for 1 week at
4⁰C.
 hemolysis does not interface with GGT levels because
the enzyme is lacking in erythrocytes

Reference range
GGT:
Males: 6-55 U/L (37C)
Female: 5-38 U/L (37C)
 86
Amylase
 Amylase (AMS) is an enzyme belonging to the
class of hydrolases that catalyze the breakdown
of starch and glycogen.
 AMS is therefore an important enzyme in the
physiologic digestion of starches
 Tissue source 87
 The acinar cells of the pancreas and the salivary
glands are the major tissue source of serum AMS.
 AMS is the smallest enzyme, with molecular weight of
50, 000 to 55, 000.
 Because of its small size, it is readily filtered by renal
glomerulus and also appears in the urine
 Digestions of starches begins in the mouth with
hydrolytic action of salivary AMS.
 Salivary AMS activity however is of short duration
because on swallowing it is inactive by the acidity of
the gastric contents.
 Pancreatic AMS the performs the major digestive
action of starches once the polysaccharide reach
the intestine
 Diagnostic significance 88

 The diagnostic significance of serum and urine AMS

measurements is in the diagnostic of acute
pancreatitis
 in acute pancreatic serum AMS levels begin to rise 2
to 12 h, and return to normal levels within 3 to 5 days.
 Other disorders causing an elevated serum AMS level
include salivary gland lesions, such as mumps and
parotitis and other intra-abdominal diseases, such as
perforated peptic ulcer
 In acute pancreatitis there is typically an increase in P-
type activity with P3 also have been detected in
cases of renal failure and therefore, is not entirely
specific for acute pancreatitis
 Assays for enzyme activity
89
 The four main approaches are categorized as
amyloclast, saccharogenic, chromogenic and
continuous monitoring
 Amyloclastic methods AMS is allowed to act on a
starch substrate to which iodine has been attached
 AMS hydrolyzes the starch molecule into smaller units
iodine is released and a decrease occurs in the initial
dark-blue color intensity of the starch iodine
 The sacchrorogenic method uses a starch substrate
that is hydrolyzed by the action of AMS to its
constituents carbohydrate molecules that have
reducing properties. The amount of reducing sugars is
then measured where the concentration is
proportional
 Chromogenic methods used a starch substrate to
which a chromogenic dye has been attached,
forming an insoluble dye substrate complex
Source of error 90

 AMS in serum and urine is stable

 Little loss of activity at room temp for 1 week
or at 4C for 2 months
 Plasma triglyceride suppress or inhibit serum
AMS activity
 Opiates and pain relief before sampling can
increase AMS levels

Reference Range:
Serum: 30-220 U/L (37 C)
Urine: 6.5-48 IU/h
Lipase 91

•Function:
•Hydrolyzes ester linkages of fats in triglycerides.
•Produces alcohols and fatty acids, with a preference for
positions 1 and 3 of the triglyceride molecule.
•Reaction:
•Catalyzes the partial hydrolysis of dietary triglycerides in
the intestine, producing 2-monoglyceride and long-chain
fatty acids.
•Conditions for Activity:
•Requires substrate in emulsion for activity.
•Reaction rate is accelerated by colipase and bile salt.
•Under alkali conditions, can act on all three positions of
triglyceride.
Lipase 92
•Tissue Source:
•Primarily found in the pancreas, also present in the
stomach and small intestine.
•Diagnostic Significance:
•Clinical assays used for the diagnosis of acute
pancreatitis.
•Elevated levels persist for about 8 days.
•More specific for pancreatic disorders compared to
amylase (AMY).
•Isoenzymes:
•Three known isoenzymes: L1, L2 (pancreatic lipases), and
L3 (carboxyl-ester lipase).
•Changes in L2 levels have significant diagnostic value.
•Assay for Enzyme Activity:
•Methods include titrimetric, turbidimetric, and
colorimetric analyses.
•Measures liberated fatty acids or glycerol production.
Glucose-6-Phosphate 93

Dehydrogenase
•Function:
•Maintains NADPH in reduced form in erythrocytes.
•Essential for anabolic pathways and maintaining
reduced glutathione levels.
•Deficiency Consequences:
•Inherited sex-linked trait.
•Results in inadequate NADPH supply, leading to
inability to maintain reduced glutathione levels.
•Exposure to oxidizing agents can cause hemolysis.
•Clinical Manifestations:
•Drug-induced hemolytic anemia, especially with
oxidant drugs like primaquine.
•Severity of hemolysis related to drug concentration.
Glucose-6-Phosphate 94

Dehydrogenase
•Diagnostic Significance:
•Used to diagnose G-6-PD deficiency.
•Elevated levels reported in myocardial
infarction (MI) and megaloblastic anemias.
•Assay for Enzyme Activity:
•Assayed using red cell hemolysate for
deficiency and serum for evaluation of
enzyme elevations.
•Reference Range:
•G-6-PD activity normal range: 7.9–16.3 U/g
Hgb.
Drug-Metabolizing 95

Enzymes:
•Function:
•Transform xenobiotics into inactive, water-soluble
compounds for excretion.
•Can activate prodrugs, convert xenobiotics into toxic
compounds, or extend elimination half-life.
•Act in either phase I (e.g., cytochrome P-450
enzymes) or phase II reactions (conjugation reactions).
•Phase I Reactions:
•Catalyze hydroxylation, oxidation, dealkylation,
dehydrogenation, reduction, deamination, and
desulfonylation reactions.
•Often mediated by cytochrome P-450 (CYP 450)
enzymes.
 Drug-Metabolizing 96

Enzymes:
•CYP 450 Enzymes:
•Superfamily of isoenzymes involved in over 50% of drug metabolism.
•Classified into families (e.g., CYP 1, 2, 3, 4) and isozymes with genetic
variants.
•Responsible for metabolizing drugs and biosynthesizing endogenous
compounds.
•Genetic variants can lead to different metabolizer phenotypes (e.g.,
ultrametabolizers, poor metabolizers).
•Endogenous Compound Biosynthesis:
•CYP 450 families (e.g., CYP 5, 7, 27) involved in thromboxane
synthesis, bile acid hydroxylation, vitamin D3 inactivation, and steroid
hormone synthesis.
•Other Metabolizing Enzymes:
•NAT, UGT1A1, GST, TPMT also have genetic variants affecting
function.
•NAT2 fast/slow acetylator phenotypes impact isoniazid metabolism.
•UGT1A1 deficiency leads to hyperbilirubinemia.
•TPMT variants affect response to thiopurine drugs.