Lecture Notes in Medical Technology: Lecture #11:

Enzymology
mt-lectures.blogspot.com

Enzymes are organic catalyst that functions to enhance the rates of biochemical reactions
from 106 to1012 times those of uncatalyzed reactions.
Characteristics of an enzyme
1. They are protein in nature.
2. They will only act on specific or particular substance or on closely related substance.
3. They are active only within a limited pH range.
4. They are destroyed easily by heat, acids or alkali.
5. They are present in small amount in the blood; therefore they cannot be measured in the
usual terms of mg/100 ml but must be measured in special unit.
6. They speed or hasten the chemical reactions without being used up.
7. They act in several ways affecting hydrolysis, oxidation, reduction, etc.
8. They cannot be standardized efficiently as they are hard to obtain in pure forms.
9. Blood enzymes continue to act at room temperature even at refrigerator temperature so
that the blood sample should be analyzed as soon as possible.
10.  They may be safely stored through freezer.
System of naming enzymes
1. Most enzymes obtained their names by adding the suffix “ase” to the substrate that they
act on.
            protease acts on protein
            uricase acts on uric acid
2. An enzyme is also named after its function rather than its substrate
e.g.       hydrogenase – brings about the removal of hydrogen
            oxidase – bring about oxidation
3. Some enzymes still maintain their original non–descriptive names
Terms associated with enzymes
1. Holoenzymes – an active substance formed by combination of coenzyme and
apoenzyme.
a. Apoenzyme – the protein portion is subject to denaturation, in which the enzyme loses its
activity.
b. Cofactors – these are non–protein substances added in the enzyme–substrate complex
before enzyme activity can be manifested.
(1)  Coenzyme – an organic molecule that hastens enzymatic reaction but undergoes a
change or is consumed to another product; the dialyzable portion of the haloenzymes.
Examples:       NAD – nicotinamide adenine dinucleotide
                        NADP – nicotinamide adenine dinucleotide phosphate
(2)  Activators – an inorganic ion that modifies reaction catalyzed
2. Isoenzymes – enzymes present in an individual with similar enzymatic activity but differ
in their physical, biochemical and immunologic characteristics.
3. Metalloenzymes – enzymes whose metal ions are intrinsically part of the molecule
4. Proenzymes – inactive precursors of enzymes, also referred to as zymogen .
5. Substrates – substances acted upon by enzymes which are specific for each of their
particular enzymes.
1. The amount of energy required to energize the substrate is known as the energy of
activation (Ea). In many laboratory procedures, this energy is supplied by heat. However,

since heat beyond normal body temperature (37oC) is injurious to cells, organisms must use
a catalyst in the form of an enzyme to provide the Ea sufficient to accelerate the reaction.
2. nzymes function as biochemical catalyst by lowering the energy of activation, thus
allowing the reaction to proceed at normal body temperature and at pace compatible with
life.
3. Ezymes accomplish this task by attaching to the reacting substrate molecules, forming
an enzyme–substrate (ES) complex.
4. This ES complex brings the substrate molecules into proper alignment with the enzymes
so that its catalytic activity can be exerted and the product can be formed. Once catalysis
occurred, the enzyme remains unchanged and is free to catalyze other reactions.
The general reaction can be written as,
E + S ------------> ES -----------> P + E
                                    ES        =          Enzyme–substrate complex
5. One of the unique features of enzymes is its ability to exhibit substrate specificity.
Types of specificity enzyme exhibit
a. Absolute specificity – catalyzes only one specific reaction with one specific substrate
b. Group specificity – broader number of substrates or similar structural group can react
with the enzyme
c. Bond specificity – enzyme which act on certain types of bonds such as peptide bonds of
proteins or glyocosidic bonds of carbohydrates.
d. Stereospecific – react only with certain optical isomer.
Theories of enzyme specificity
a. Emil Fisher’s Lock and Key theory
It is based on the rigid enzyme molecule into which the substrate fits. The shape of the key
(substrate) must fit into the lock.
b. Kochland’s induced fit theory
It is based on the attachment of a substrate to the active site of an enzyme, which then
causes conformational changes in the latter. This is the more acceptable theory since the
protein molecule is flexible enough to allow conformational changes and it somehow
explains the influence of hormones on enzymatic activity.
Factors affecting enzyme activity
1. Substrate concentration

The more substrate molecules available, the more likely they are able to attach to the active
sites of the enzymes, thus enabling the enzymes to exert its activity at maximum velocity.
However, eventually a maximum rate is reached and any further increase in substrate
concentration will not increase the velocity. At maximum velocity, the substrate
concentration is sufficiently high so that all enzyme molecules have their active site
engaged. As soon as a product exits the active site, another substrate molecule enters. The
enzyme at maximum velocity is said to be saturated.
Types of reaction order:
a. First order kinetics – velocity is directly proportional to substrate
b. Zero order kinetics – maximum velocity is reached and any additional increase in
substrate will not alter the velocity.
Michealis Menten hypothesis
An enzyme–substrate complex can either dissociates back to E +S or breakdown to product
(P) or free the enzyme.
The velocity of reaction is increased with an increased enzyme concentration and is
decreased with lower enzyme concentration
Reaction rate increases with temperature up to the optimum temperature which gives its
maximal reaction. With every 10oC increase, the activity may increase 50 – 100. Some
enzymes double their activity for every 10oC increase. Animal enzymes are destroyed above
60oC.
4. Hydrogen ion concentration (pH)
At its optimum pH, the enzyme will be most active. Deviation from this level will lower the
reaction, inactivate or destroy the enzyme. Buffers are incorporated into the reaction
mixture when measuring enzyme activity to prevent extreme fluctuation in pH during the
enzymatic reaction.
Substances known as activators increase the rate of an enzymatic reaction. Activators are
usually small molecules or ion such as metal ion. Common enzyme activators include ions
such as Zn2+, Mg2+, Fe2+ and Mn2+. One mechanism of action of activators is to provide an
electropositive active site that attracts negatively charged groups of substrate.
Other activators serve a structural function and aid in stabilizing the tertiary and quaternary
structure of the enzyme. Regardless of their mechanism, activators must be present for
those enzymes with such a requirement to enable their optimum enzymatic activity.
In contrast to activators, inhibitors selectively bind to different sites of the enzyme molecule
producing a varying effect on the velocity of the reaction. It has three types:
a. Competitive inhibitors
(1)  These are similar to normal substrate molecule and compete with the substrate for
binding to the active site of a specific enzyme.
(2)  Can be reversed by increasing the concentration of the substrate.
(3)  Example: sulfonamides
b. Non–competitive inhibitors
(1)  Occurs when a substance binds to the enzymes at a site other the active site. This
binding causes a conformational change in the structure of the enzyme in such a way that
the active site is altered and is no longer receptive to the substrate.
(2)  It can be reversible depending on the type of bond formed.
(3)  Example: Lead and Mercury
c. Uncompetitive inhibitors
(1)  It occurs when an inhibitor binds to the ES complex to form an enzyme– substrate–
inhibitor complex that does not yield products.
(2)  Increasing the substrate concentration actually increases the inhibition by providing
more complexes to which the inhibitor can bind.
Measurement of enzyme activity
1. End poind method / Fixed incubation / Two–point assay
a. The reaction is allowed to incubate for pre–determined period. An absorbance reading is
taken at the end of that time from which the enzyme activity is calculated.
a. Based on the assumption that the reaction progress is linear and follows zero – order
kinetics. With no means of verifying that the reaction is linear, an error is more likely to go
undetected, resulting in erroneous values.
2. Multiple point method
a. Makes several absorbance readings during the progress of the reaction, enabling the
verification of a linear reaction. This means of analysis is more practical with the availability
of microprocessor in modern computerized chemical analyzers. Any deviation from linearity
can be immediately determined.
3. Continuous monitoring method
a. Uses a recording spectrophotometer to trace the progress of the reaction over a period of
several minutes. The slope of the linear portion of the curve is used to determine enzyme
activity. Any deviations from linearity can be immediately observed from the trace
recording.
B. Progress curve in enzyme activity
a. Termed as the period of equilibrium, wherein serum enzymes and reaction mixture where
brought together.
b. Enzyme activity should not be measured at this phase since the reaction rate is not yet in
zero–order kinetics.
a. In this phase, the rate of product formation is constant and product concentration
increases linearly with respect to time. The enzymatic reaction now exhibits zero–order
kinetics and represents the phase during which the enzyme activity should be measured.
a. A progressive decline in product formation occurs at this stage as it enters first order
kinetics.
b. Factors resulting in reduced velocity:
(1)  Decreasing substrate concentration
(2)  Establishment of equilibrium
(3)  Progressive inactivation of the enzyme as the buffer becomes insufficient to control the
pH.
c. This phase is also unsuitable for measurement of enzyme activity because in first order
kinetics, the enzyme is not functioning at maximum efficiency.
C. Units for measuring enzyme activity
1. International Units (I.U. or U)
Equivalent to the amount of enzyme that catalyzes the conversion of 1 micromole of
substrate per minutes under controlled condition.
Equivalent to the amount of enzyme that catalyzes the conversion of 1 mole of substrate
per second under controlled condition.
Classification of enzymes
In an attempt to establish a systematic process for enzyme nomenclature, the Commission
on Enzymes was establish in 1956 under the direction of International Union of
Biochemistry. From the work of this original committee and subsequent recommendations,
a completely revised version of enzyme nomenclature was published in 1972.
The numerical designation for each enzyme consist of four numbers separated by periods,
e.g. 1.9.3.1
The first number defines the class to which the enzyme belongs. The enzymes are assigned
to one of the 6 classes characterized by the type of reaction they catalyze.
The second figure gives more specific information about the enzyme. In the tranferase
group, the second figure indicates the nature of the group transferred.
The third figure of the code gives more specific details about such things as the type of
acceptor molecules for an oxidoreductase of the precise chemical type of group transferred
by a transferase.
The fourth figure is the serial number of the enzyme in the class as indicated by the third
element.
A. Oxidoreductase – catalyze the oxidation–reduction reactions between two substrates.
a.       Cytochrome oxidase                                           EC 1.9.3.1
a. 3–hydroxybutyrate dehydrogenase                 EC 1.1.1.30
b. Lactate dehydrogenase                                      EC 1.1.1.27
c. Glucose–6–phosphate dehydrogenase                        EC 1.1.1.49
d. Malate dehydrogenase                                       EC 1.1.1.37
e. Isocitrate dehydrogenase                                  EC 1.1.1.42
f.  Glutamate dehydrogenase                                 EC 1.4.1.2
B. Transferase – catalyze the transfer of a group other than hydrogen between two
subunits.
a. Gamma glutamyl transferase                             EC 2.3.2.2
b. Aspartate aminotransferase                              EC 2.6.1.1
c. Alanine aminotransferase                                  EC 2.6.1.2
d. Creatinine kinase                                                EC 2.7.3.2
e. Ornithine–carbamoyl transferase                     EC 2.1.3.3
C. Hydrolase – catalyze the hydrolytic cleavage of compounds
a. Acid phosphatase                                                EC 3.1.3.2
b. Alkaline phosphatase                                        EC 3.1.3.1
c. Cholinesterase                                                     EC 3.1.1.8
d. Triacylglycerol acylhydrolase (lipase)         EC 3.1.1.3
a. Leucine aminopeptidase                                    EC 3.4.1.1
d. 5’–nucleotidase                                                   EC 3.1.3.5
a. Alpha–amylase                                                    EC 3.2.1.1
b. Amylo–1–6–glucosidase                                     EC 3.2.1.33
c. Glucosidase                                                          EC 3.2.1.20
d. Galactosidase                                                       EC 3.2.1.23
D. Lyases – catalyze the removal of groups from substrates without hydrolysis, leaving
double bonds in the product.
b. Glutamate decarboxylase                                   EC 4.1.1.15
c. Tryptophan decarboxylase                               EC 4.1.1.28
E. Isomerase – catalyze the interconversion of isomers.
a. Glucose–6–phosphase isomerase                     EC 5.3.1.9
b. Ribose–5–phosphate isomerase                       EC 5.3.1.6
F. Ligases – catalyze the joining of two molecule coupled with the hydrolysis of a
pyrophosphate bond in ATP or similar component.
a. Carbamoyl–phosphate synthetase                    EC 6.3.5.5
b. Acetyl–CoA carboxylase                                                 EC 6.4.1.2
******  LACTATE DEHYDROGENASE  ******
A hydrogen transfer enzyme catalyzing the oxidation of L–lactate to pyruvate with the
mediation of NAD as the hydrogen acceptor. The reaction is reversible, which strongly
favors the reverse reaction which is the reduction of pyruvate to lactate. Specifically, it is
important in the Embden–Meyerhoff metabolic pathway of glycolysis.

1.      Sulfhydryl reagents – e.g. mercuric ions

LD is ubiquitous cytoplasmic enzyme found in nearly all cells of the body with highest
activity in brain, erythrocytes, white blood cells, kidney, liver, lung, lymph nodes, myocardium,
platelets and skeletal muscles.
LD occurs as a tetramer consisting of four polypeptide chains. Each chain or subunit maybe
one of the two types and is designated H (heart) or M (muscle). Combinations of these
subunits can result in any one of the five isoenzymes:
Distribution of LD isoenzymes
a. LD–1 and LD–2 – fast moving fractions and are heat stable, found mostly in the
myocardium and erythrocytes; also found in renal cortex.
b. LD–3 – found in a number of tissues predominantly in the white blood cells and brain
c. LD–4 and LD–5 – slow moving and are heat labile, found mostly in the liver and skeletal
muscle; also seen in ileum and skin.
d. Each isoenzyme is broken down as follows:
a. An optimum pH is required to catalyze the inteconversion of NAD+ and NADH at 340 nm.
The basic reaction can thus proceed:
Forward: (L–lactate -------------------->   pyruvate) pH 8.3 – 8.9

Reverse:   (P–pyruvate ----------------->  lactate) pH 7.1 – 7.4

b. Wrobleuski – La Due method
The method employs the pyruvate–lactate reaction and is followed by measuring the rate
absorbance decrease at 340 nm and at 25oC as NADH is oxidized to NAD.
Pyruvate remaining in the solution after incubation in the presence of NADH by reacting it
with 2, 4,dinitrophenylhdrazine to form the corresponding phenylhydrazone which has a
golden–brown color at alkaline pH.
NADH is coupled to the reduction of a tetrazolium salt (2–p–iodophenyl–3–p– nitrophenyl–
5–phenyl tetrazolium chloride) with phenazine methosulfate (PMS) serving as intermediate
electron carrier forming bright red color with maximum absorption at 500 nm.
(1)  Substrate – product relationship
(3)  Differential chemical inhibition of LD activity
(1)  Heat denaturation at 60oC for 30 minutes.
(2)  Ion exchange chromatography using diethylaminoethyl (DEAE)
(3)  Hydroxybutyrate substrate substitution
Specimen collection and handling
1. Serum is the specimen of choice however; heparinized plasma may also be used.
2. Oxalates inhibit LD activity
3. Ascorbic acid can also decrease LD activity.
4. Increased values can be obtained from hemolyzed samples and if not immediately
separated from cells.
5. Serum for LD analysis should be kept at 25oC and should be analyzed within 24 hours of
collection.
6. LD–4 and LD–5 are sensitive to cold exposure.
A. Highest LDH value is seen in
2.      Extrinsic carcinomatosis
B. Moderate elevations (2 – 4 fold) is seen in
1.      Myocardial infarction
5.      Infectious mononucleosis
C. Elevated LDH activity have been reported in the urine of patients with the following
disease:
1.      Carcinoma of kidney or bladder
2.      Malignant hypertension
1.      Forward reaction:     100 – 226 U/L
2.      Reverse reaction:      90 – 320 U/L
3.      Newborn to 2 y/o:    450 U/L above
****** GLUCOSE–6–PHOSPHATE DEHYDROGENASE  ******
Glucose–6–phosphate dehydrogenase (G–6–PD) is an oxidoreductase that catalyzes the
first step in the pentose phosphate pathway of glucose metabolism. Specifically, it
catalyzes the oxidation of glucose–6–phosphate to 6–phosphogluconate with the
production of NADPH. NADPH is necessary to reduce glutathione, an important reducing
substance that protects hemoglobin from oxidative denaturation.
1. Highest activity is seen in immature erythrocytes and decrease with cell maturation.
2. Minimal activity is seen in serum, adrenal cortex, lymph nodes, thymus and spleen.
3. Adrenal glands and mammary gland (especially during lactation) also has highest
activity.
1. All heavy metals except cupric and zinc ions.
2. Inhibition can be counteracted by EDTA but not by sulfhydryl agents like cysteine.
4.      Bentler and Mitchells
Glucose–6–PD deficiency is a genetically determined disorder that results in an inadequate
supply of NADPH and ultimately inadequate levels of reduced glutathione.  Beutler Test is a
screening procedure for G–6–PD deficiency.
Lack of adequate levels of reduced glutathione results in oxidative destruction of
hemoglobin and various components of the cell membrane, rendering the red cell
susceptible to hemolysis
2.      RBC:                0.117 – 0.143 U/L
******  SORBITOL DEHYDROGENASE  ******
a. Found mainly in the liver.
b. Involved in the conversion of sorbitol to fructose
c. Normal serum contains little SDH but levels are markedly elevated in sera of patients with
acute hepatitis.
d. Little SDH activity has been found in sera of patients with obstructive jaundice or in
patients with acute myocardial infarction.
e. Determination of SDH should therefore provide valuable diagnostic assistance in the
differences between hepatitis on one hand and obstructive jaundice and myocardial
infarction on the other hand.
******  AMINOTRANSFERASE  ******
The aminotransferase, commonly referred to as transaminase, constitute a class of
enzymes that catalyze the transfer of an amino group from an alpha–amino to an alpha–
keto acid forming a new amino acid and a new keto acid.
Two important members of this group are:
1. Aspartate aminotransferase – formerly known as Serum Oxaloacetic Acid Transferase
(SGOT)
2. Alanine aminotransferase – formerly known as Serum Pyruvic Acid Transferase (SGPT)
The aminotransferase are relatively stable enzyme. They will maintain their activity for
several days if the serum is stored in a refrigerator.
A.   Aspartate aminotransferase
AST catalyzes the transfer amino group from L–glutamate or L–aspartate to alpha
oxaglutarate or oxaloacetate.
L–aspartate + alpha–oxaglutarate  -------------->    oxaloacetate + L–glutamate
Although the reaction is reversible, the equilibrium favors the formation of aspartate. The
coenzyme pyridoxal–5’–phosphate (P5P) binds to AST and thus serves as a necessary
prosthetic group for full catalytic activity.
This enzyme is found in great concentration in the following organs and tissues:
1. Spectrophotometric method
Serum is incubated with aspartic acid and alpha glutamic acid. Oxaloacetic acid, as it is
formed is made to react with malic dehydrogenase in the presence of NADH. Oxaloacetic
acid is transformed to malic acid by malic dehydrogenase and the NADH is oxidized to NAD.
NADH shows strong absorption of ultraviolet rays at 340 nm while NAD has no specific
absorption.

2. Colorimetric method (Reitman and Frankel)

The method measures the amount of oxaloacetic acid produced under fixed condition by
the reddish–brown hydrazone it forms with 2,4 dinitrophenylhydrazine in alkaline solution.

            Advantages of colorimetric method over the spectrophotometric methods are:

2.      Small amount of serum is needed.
3.      Does not require a special kind of spectrophotometer.
4.      Easier to control temperature
Temperature is a problem in spectrophotometric method. AST increases by 7% from each
1oC rise in temperature
1. Myocardial infarction
3. Moderate increase occurs in other liver disease like
b.  Neoplasm of the liver
4.  Certain forms of muscular dystrophy
B.   Alanine aminotransferase
ALT along with P5P catalyzes the transfer of an amino group from L–glutamate or L –
alanine to alpha ketoglutarate or pyruvate
Alanine + alpha – oxoglutarate  --------> pyruvate + L – glutamate
Although the reaction is reversible, the equilibrium of the ALT reaction favors formation of
alanine. Both reactions proceed to the right in vivo to provide a source of nitrogen for the
urea cycle.
This enzyme is distributed in skeletal muscle, kidney, heart, pancreas, lungs, spleen and
erythrocytes but is predominantly a liver–specific enzyme. Although a mitochondrial ALT
exists in tissue, it has not been demonstrated in normal human serum.
Methods of determination
1. Spectrophotometric method
The pyruvic acid produced by the substrate alanine and alpha – ketoglutarate acid are
converted to lactic acid by lactic dehydrogenase in the presence of NADH. The conversion
of NADH to NAD is followed by a decrease in absorbance at 340 nm.
2.  Colorimetric method (Reitman and Frankel)
The pyruvic acid formed in the reaction is measured by the reddish–brown hydrazone it
forms with 2,4,dinitrophenyl–hydrazine in alkaline solution.
AL T in serum is incubated with buffered substrate, DL alanine and alpha– ketoglutaric acid
at pH 7 at 37oC for 30 minutes. The pyruvic acid reacts with with DNPH to form pyruvate
dinitrophenylhydrazone which presents a brown color upon addition of an alkaline solution.
The procedure is similar to AST except for
b. Incubation period which is ½ hour instead of 1 hour.
c. The substance that is formed is pyruvic acid which reacts with DNPH.
Measures the disappearance of NADH at 340 nm and proceeds as follows
Alanine + alpha–ketoglutarate  -------> pyruvate + glutamate

pyruvate + NADH ----------------------->   lactate + NAD+

4. Fluorescence method (Henry Pollard)
The disappearance of NADH in the proceeding coupled enzymatic reaction is monitored as
a decrease in fluorescence
1. Primarily used for the diagnosis of acute liver injuries like viral hepatitis

2. Maybe elevated in myocardial infarction

******  CREATININE KINASE  ******
CPK catalyzes the reversible phosphorylation of creatine by adenosine triphosphate. The
equilibrium position for the reaction is dependent on the hydrogen ion concentration.
  Creatinine + ATP  -------------> creatinine phosphate + ADP
1.      Magnesium ions – required for maximum activity
2.      Calcium – to a lesser degree.
1. The largest distribution of CK is found in skeletal muscle. The remaining tissue sources
in order of descending activity are brain, rectum, stomach, bladder, colon, uterus, prostate,
small intestine and kidney. Negligible amounts can be detected in the liver, placenta and
thyroid tissue.
2.  It has the following isoenzymes
a. CK–1 or CK–BB – found predominantly in the brain and CNS; however, small quantities
are found in numerous tissues, including the lungs, prostate, uterus and gastrointestinal
tract. It migrates most rapidly toward the anode on electrophoresis.
b. CK–2 or CK–MB – is confined almost exclusively to cardiac muscle tissue. It is the
second most rapid moving isoenzyme.
c. CK–3 or CK–MM – is the predominant isoenzyme in both skeletal and cardiac muscle.
d. CK–mito – rarely seen in normal human serum but its presence signals a grave prognosis
for the patient because it indicated extensive tissue damage with release of mitochondrial
content
e. CK–IgG complex or CK type I
f.  CK–IgA complex or CK type II
a.  Forward reaction:           pH 9.0
Creatinine  ----------------> creatinine phosphate
b.  Reverse reaction:                        pH 6.8
Creatinine  phosphate ----------------> creatinine
Rosalki and oliver method (reference method)

Tanzer and Gilvarg method

a.  ADP + PEP -----------> ATP + Pyruvate
b.  Pyruvate + NADH ------------> pyruvate + NAD+
Creatinine reacts with ninhydrin (triketohydrinoene hydrate) solution to produce fluorphoe
Creatinine is made to react with diacetyl and alpha napthol to produce a pink end product.
a. ATP + creatinine --------->ADP + phosphocreatinine

b. Phosphocreatinine ----------> creatinine + inorganic phosphorous

(1)  CK bands are separated by applying an electromotive force at pH 8.6
(2)  The relative amount of CK activity present in each band is determined by incubating the
gels with a substrate mixture.
(3)  The fluorescence NADPH generated by each band is proportional to the amount of CK
activity in each band.
(4)  To detect the amount of NADPH generated colorimetrically, addition of phenazine
methosulfate and tetranitrozolium dye is coupled in the reaction and purple formazan is
produced.
(5)  To determine the relative percentage of CK activity in each band, the gels are then
scanned by a densitometer.
(1)  Rapid, has good sensitivity, requires only a very small amount of sample.
(2)  Allows the technician to visualize the band and any isoenzyme variant.
(1)   A semiquantitative method
b. Ion exchange chromatography – considered as the reference method
1. CK is heat labile, when serum is heated at 56oC for 15 minutes, there is total loss of
activity.
2. CK is unstable in serum so that 45% of the original activity is lost in 4 hours at room
temperature. Full activity can be restored by the addition of suitable sulfhydryl compound
like cysteine and Cleland’s reagent.
******  ORNITHINE CARBAMOYL TRANSFERASE  ******
This enzyme catalyzes the reversible conversion of ornithine to citrulline which is intimately
involved in the synthesis of urea. The enzyme is found mostly in the liver and only 1% of this
is found in the intestine.
The enzyme activity is markedly increased in acute viral hepatitis and other form of hepatic
necrosis
Slight elevation of OCT is observed in
7.      Intestinal obstruction
 Ornithine + carbamoyl phosphate ------------>citrulline + H3PO4
            The citrulline formed is measured colorimetrically
******  GAMMA GLUTAMYL TRANSFERASE  ******
Gamma glutamyl transferase (GGT) is an enzyme belongings to the class of transferases. It
is membrane–associated enzyme whose important function involves the transfer of the
gamma glutamyl residue from glutathione and other gamma glutamyl peptides to amino
acids or small peptides to form the gamma glutamyl amino acids and cysteinyl–glycine.
Gamma–glutamyl–p–nitronilide + glycylglycine ------>    gamma–glutamyl–glycylglycine +
p– nitroaniline 
1. It has 85–93% sensitivity in the differential diagnosis of hepatic versus biliary disorders.
2. Five times above the upper reference range seen in biliary tract disorder in which
cholestasis is the primary pathologic process such as biliary obstruction.
3. Two to five times above the reference range is seen in hepatic disease owing to
hepatocellular dysfunction such as viral hepatitis and cirrhosis.
4. Because GGT is a microsomal enzyme, its synthesis is induced by alcohol and other
drugs, making it a sensitive indicator of alcohol abuse.
5. Many drugs also induced the synthesis of GGT. These include phenobarbital,
antidepressants, anticonvulsants such as phenytoin and some contraceptive pills
containing estrogen.
1. Spectrophotometric method
Gamma–glutamyl–p–nitroaniline -----------> p–nitroaniline
The absorbance is measured at 405 nm
******  PHOSPHATASE  ******
These are a group of related enzymes, low specificity are characterized by their ability to
hydrolyze a large variety of organic phosphate esters with the formation of an alcohol and
phosphate ion. They attack only monoesters of phosphoric acid. 
Clinically, there are three types of phosphatase:
1. Alkaline phosphatase (of serum, liver, bone, lungs, spleen, leukocytes and intestines) –
with optimal activity at pH 9.8
2. Acid phosphatase (of serum, prostate, liver) – with optimal pH at 4.9 – 5.0.
3. Red cell phosphatase – with optimal pH 5.9 – 6.0.
Probable function of the phosphatase is the transfer of the phosphate groups from a color
substrate to an acceptor compound containing an OH group.
A. Serum alkaline phosphatase
Serum phosphatase is the generic name for a group of enzymes that display maximum
activity at pH 9.0 to 10.5. ALP functions to liberate inorganic phosphate from an organic
esters with the concomitant production of alcohol.
H2O + R–HPO4 ---------------> R–OH + H2PO4

Inhibitors:            PO4, Zn++, Ba++, SO4, oxalates

1. Liver – the rapidly moving isoenzymes; 50 – 70% inactivated by heat; 0 – 10% inactivated
by L – phenylalanine; strongly inhibited by urea.
2. Bone – the 2nd fastest moving isoenzyme; the most susceptible to heat inactivation; 0 –
10% inactivated by L – phenylalanine; strongly inhibited by urea.
3. Intestine – the 3rd fastest moving isoenzyme; 50 – 60% inactivated by heat; 75% inhibited
by L – phenylalanine; moderately inhibited by urea.
4. Placenta – overlap with bone and liver bands in PAGE electrophoresis; resistant to heat
inactivation, 75% inhibited by phenylalanine; moderately inhibited by urea.
5. Regan isoenzyme – also known as carcinoplacental isoenzyme because of its similarities
to the placental fraction. It is the result of ectopic production from malignant tissue. It is the
most heat resistant of the isoenzymes, resisting heat denaturation at 65oC for 30 minutes,
but has low sensitivity since it is detected only in 3 – 15% of cancer patient.
6. Nagao isoenzyme – associated with carcinoma of the pleural cavity, pancreas and bile
duct
(1)  Cholestatic liver disease
(2)  Hepatobiliary disease
(3)  Infectious mononucleosis
(7)  Primary hepatocellular carcinoma
(8)  Secondary metastatic liver carcinoma
(1)  Acute and chronic pancreatitis
(2)  Chronic renal failure
(4)  Intraabdominal bacterial infection
(6)  Benign transient hyperphosphatemia
(7)  Drugs: estrogens, progesterone, chlorpromazine
(1)  Paget’s disease (osteitis deformans) – excessive bone destruction
Serum is added to sodium glycerophosphate that has been buffered to pH 8.6 to ensure
optimum condition of pH and electrolyte content. The mixture is incubated at 37oC for
exactly one hour. The phosphatase in serum decomposes sodium glycerophosphate during
this incubation period and liberates phosphorous.
Phosphorous determination is then run on the incubated serum and on non– incubated
serum which consists of adding trichloroacetic acid to both specimen to precipitate the
proteins, adding molybdic acid to form a phosphate complex, adding a reducing agent to
reduce the complex and finally measuring the depth of color in a colorimeter
The alkaline phosphatase units are then determined by subtracting the phosphorous value
of the non–incubated specimen form the phosphorous value of incubated specimen.
b. Bessey –Lowry Brock method
Serum is added to p–nitrophenyl phosphate that has been buffered to pH 10.3 to ensure
optimum conditions for reaction. The mixture is incubated at 37oC for exactly 30 minutes.
During this period, phosphate hydrolyzes p – nitrophenylphosphate and liberates p–
nitrophenol. The nitrophenol color intensity is measured in a colorimeter.
p–nitrophenyl phosphate + H2O ----------> p–nitrophenyl + H3PO4
c.  Klein, Babson and Reed method
Phenolphthalein monophosphate + H20 -------------->          phenolphthalein + H3PO4
Phenolphthalein formed is determined colorimetrically by its red color in alkaline solution
Advantages over p–nitrophenyl phosphate
(1)  Absorption peak of phenolphthalein is much further from that of bilirubin and
hemoglobin thus interference from this compound is reduced.
(2)  For a given enzyme concentration, much more color is produced by phenolphthalein,
thus increasing sensitivity of this method.
Phenolphthalein diphosphate is used as a substrate. The substrate gives linear reaction
kinetic and is useful and sensitive substrate for assaying substrate.
The substrate 4–methyl umbelliferyl phosphate is hydrolyzed to 4–methyl umbelliferone
which displays fluorescence at 360 nm.
f.  King and Armstrong method
Phenylphosphate is used as substrate. The rate of reaction is followed by measuring the
phenol formed. Phenol formed is measured using Folin Ciocalteau or amino antipyrine
(King–King) or by reaction with a diazo reagent.
The method employing the Folin Ciocalteau reagents requires 30 minutes incubation period
followed by deproteinization of the incubation mixture when antipyrine is used as the
chromogenic reagent, deproteinization is not needed and a 15 minute reaction period is
sufficient.
d.      Immunochemical method
Precaution in quantitation
1. The type of buffer present affects the rate of enzymatic activity. The buffers used are
barbital, glycine, piperazine, MAP, Tris.
2. In the post–absorptive state, elevations of ALP occur in individuals who are of blood
group B or O.
Acid phosphatase (ACP) is a non–specific heterogenous group of phosphatase that belong
to the class of hydrolase enzymes. It catalyzes the same reactions as ALP but functions in
an acidic environment. The optimum pH ranges from 4.5 to 7.0
ACP catalyzes the hydrolysis of several orthophosphoric monoesters to yield the
corresponding alcohol and inorganic phosphate as seen in the following equation.
H2O + R–HPO4 ---------------> R–OH + H2PO4
ACP has greatest concentration in prostate, liver, kidney, erythrocytes, platelets and
osteoclastic cells of bone. Since ACP activity in the prostate is more than 100 times greater
than in other tissues, it is primarily for prostate disorders that ACP measurement are
requested.
ACP assays suffer from insensitivity in detecting carcinoma that has not metastasized and
are not completely specific for the prostate. Measurement of prostate ACP (PAP) levels has
been proven to be more sensitive.
a. Granulocytic leukemia
b. Acute / chronic lymphocytic leukemia
f.  Primary thrombocythemia
Exactly the same procedure as the Bodansky alkaline phosphatase procedure except that
the buffer solution is an acid buffer of pH 5.0 instead of 8.6.
b. King–Armstrong method
The same procedure for ALP except that the buffer solution is acidic (pH 5.0)
c. Bessey–Lowry–Brock method
Same as for ALP except the pH is at 4.8 and serum blanks are not incubated. The p–
nitrophenol liberated during the incubation period is measured. The non– incubated serum
blanks are also measured.
d. Roy and Hillman method
The substrate thymolphthalein monophosphate is hydrolyzed by the prostate ACP to yield
thymol with the formation of blue color which is measured spectrophotometrically at 595
nm.
e. Babson, Read and Philips
The substrate alpha napthyl phosphate is acted upon by ACP to produce alpha napthol
which is brown in color and can be determined spectrophotometrically.
a.  Differential substrate
d.  Immunologic techniques
(2)  Counterimmunoelectrophoresis
(3)  Fluorescent immunoassay
Precaution in quantitation
1. ACP is unstable at temperature above 37oC.
2. 50% of the prostatic enzymes are particularly labile and over 50% activity may be lost in
one hour at room temperature. To prevent this, 0.01 ml of 20% acetic acid per ml serum is
added to stabilize the enzyme.
3. Hemolyzed serum should not be used because RBC contains high level of ACP.
4. Prostatic enzyme is strongly inhibited by tartrate while red blood cell acid phosphatase is
inhibited by formaldehyde and cupric ions.
******  CHOLINESTERASE  *****
Cholinesterase catalyzes the hydrolysis of choline esters to form choline and fatty acids as
shown in the following reactions:
Acetylcholine + H2O ----------------> choline + acetic acid
Two types of cholinesterase
1. True cholinesterase / Acetylcholinesterase (AChE) – found primarily in the red blood
cells and central nervous system and function in the hydrolysis of acetylcholine at the
neuromuscular junction to allow nerve conduction.
2. Pseudocholinesterase (ChE) / Non – specifc cholinesterase – found primarily in the liver
and white matter of the brain and accounts for the enzyme activity measured in serum or
plasma. Its physiologic role is not known.
1. As a measure of exposure of some organophosphorous compounds that are found in
many insecticides and nerve gases.
2. Detection of abnormal variants of ChE that cannot hydrolyze succinyl choline, a muscle
relaxant administered during the induction of anesthesia for surgical procedures.
3. Indication of synthetic capacity of the liver.
1. Base on the following reaction
Butyrylcholine ------------> thiocholine + butyric acid

Thiocholine + 5,5–dithosis–(2–nitrobenzene) -------------->                 5–thio–nitrobenzoic acid

The assay is performed with or without dibucaine. The atypical variant of ChE is detected by
its greater resistance to inhibition by dibucaine.
Lipase hydrolyzes glycerol esters of long chain fatty acid tricglycerides. LPS preferentially
hydrolyzes the ester bonds of carbon 1 and 3 of the triglyceride molecule.
Triacylglycerol ----------------> 2–monoglyceride + 2 fatty acids
1. Sulfhydryl compounds like cystine and thioglycollic acid 
2. Bile salts – promote the formation of stable and finely dispersed emulsion of fat in water
3. Calcium – removes fatty acids liberated in the reaction by forming insoluble calcium
salts.
LPS activity has been observed in pancreas, intestinal mucosa, stomach, leukocytes and
adipose tissue. However, only pancreatic lipase is of clinical significance. Its main function
is to hydrolyze dietary triglycerides that have been emulsified by bile acids, thus aiding in fat
absorption in digestive tract.
Increase levels are seen in:
2.      Acute alcohol poisoning
3.      Accidental or surgical trauma to the abdomen
1. Cherry–Crandall method
Lipase in incubated serum and in the presence of a phosphate buffer catalyzes the
hydrolysis of triolein (olive oil) with subsequent formation of oleic acids. The acids are then
quantitated with the standard base using thymolpthalein indicator. The differences between
the amount of standard base that neutralize the acid in the test and in the control represent
the lipase activity present in serum.
Instead of olive oil as a substrate, a lipase substrate is used and titration is carried to the
end point using N/10 NaOH as titrating agent and thymolphthalein as indicator
******  LEUCINE AMINOPEPTIDASE  *****
Leucine aminopeptidase is a proteolytic enzyme which catalyzes the hydrolysis of the N –
terminal amino acid of peptides during intestinal protein digestion
           L–leucinamide + H2O -------------> Leucine + NH3
Increased levels are seen in
2.      Carcinoma of the pancrease
3.      Carcinoma of the bile duct
1.  Based on the following reaction
a. Leucine–p–nitroanilide ---------->  p–nitroaniline
b. L–leucyl–beta–napthylamide + H2O ---------> leucine + beta–napthylamine
The amount of beta–napthylamine is proportional to the amount of LAP activity. The beta
napthylamine being colorless is diazotized with nitrous acid and then reacted with a dye
base, N–(l–napthyl)–ethylenediamine hydrochloride to form a puplish colored complex azo
dye that is proportional to the amount of beta–napthylamine formed.
2. Other suitable substrate are
e.       Alamyl – napthylamide
3. Optimal pH: 7.2 – 7.5
******  5’ – NUCLEOTIDASE  *****
5’–nucleotidase (5’–NT) catalyzes the hydrolysis of most ribonucleoside 5’–
monophosphate and deoxynucleoside 5’–monophosphate to the corresponding nucleoside
and orthophosphate. It is found predominantly in the liver.
Adenosine–5’–monophosphate + H2O ----------> adenosine + H3PO4
1. Based on the following reaction
5’–adenosine monophosphate + H2O ---------> adenosine + H3PO4
Adenosine + H2O -----------> inosine + NH3
************  AMYLASE  ***********
Amylases are group of enzymes belonging to class hydrolase which can split
polysaccharides like starch and glycogen. It is a calcium–requiring metalloenzymes.
1. Beta–amylase (bacterial amylase) – can act only at the terminal reducing ends of a
polyglucan chain splitting off a section of a two glucose limits (maltose) at a time.
2. Alpha–amylase (animal amylase including man) – also referred to as endoamylase
because they can attack alpha 1,4 linkage in a random manner anywhere in the polyglucan
chain. Large polysaccharide molecules are rapidly broken down into smaller units.
Characteristics of alpha amylase:
1. Active at pH 6.9 – 7.0
2. Active at temperature as high as 50oC but it customarily assayed at 37–40oC.
3. Full activity is displayed only in the presence of a variety of univalent anions like
chlorides, bromides, nitrates, chlorates, or monohydrogen phosphatase.
4. Molecular weight of amylase is 45,000.
5. The size is small enough to pass through glomerular filter.
6. Enzyme is stable, negligible activity is lost at room temperature in the course of one week
and at refrigerator temperature over a two month period. All common anticoagulants except
heparin inhibits amylase activity.
Two isoenzyme of alpha–amylase
1. Salivary amylase – fast moving, secreted also by lungs, bone and thyroid. Sixty percent of
activity is derived from this isoenzyme.
2. Pancreatic amylase – slow moving, secreted exclusively in the pancreas. Accounts for
40% activity in serum. The P3 fraction is the most elevated in acute pancreatitis.
Methods of determination
1. Saccharogenic methods
The course of the enzyme reaction is followed by measuring the quantity of reducing sugars
formed. Reducing sugars determined on PFF of the of reaction mixture.
Enzyme activity is evaluated by following the decrease in substrate (starch) concentration
rather than measuring the product.
The time required for amylase to completely hydrolyze all starch present in a reaction
mixture is measured. The end point is reached when there is absence of any material
capable of forming the blue starch iodine color.
Serial dilution of the enzyme preparation is added to a fixed quantity of starch and the
dilution is found which is just able to hydrolyze all the starch present in a fixed time.
c.  Amylometric procedure
Measures the amount of starch hydrolyzed in a fixed period of time using the blue starch
iodine color as the measure for quantitating starch.
Maltotetraose + H2O    ----------> 2 maltose
     Maltose + Pi   -------------------> glucose + B–glucose–1–P
B–glucose–1–P -----------------> glucose–6 – P
Glucose–6–P + NAD -----------> gluconate–6 –P + NADH + H+
MP – maltose phosphorylase
B–PGM–B – posphoglucose mutase
G–6–PD – glucose–6–phosphate–dehydrogenase 
Precautions in specimen collection
1. Opiates and morphine causes false elevation in value.

2. Elevated triglycerides act as an inhibitor and falsely lower amylase levels when assayed
with starch – iodine methods.
1. Elevated levels (Hyperamylasemia)
b.      Perforate peptic ulcers
c.       Intestinal obstruction
d.      Ruptured ectopic pregnancy
e.       Salivary gland disease
f.        Adenocarcinoma of the lung, ovary, etc.
Macroamylasemia – occurs when amylase binds with an immunoglobulin (IgA or IgA),
forming a complex that is too large to be filtered by the glomerulus.
************  ALDOLASE  ************
This enzyme belongs to a class of enzyme called lyases. It is the enzymes that takes part in
the intermediary breakdown of glucose at the levels of fructose–1–6–diphosphate and
convert it into dihydroxyacetone phosphate and glyceraldehyde–3–phosphate.
fructose–1–6–diphosphate  dihydroxyacetone phosphate + glyceraldehyde–3–phosphate
The enzyme shows absolute specificity only for dihydroxyacetone (DHAP). The optimal pH
for this enzyme is broad from 7.0 – 9.6. Heavy metals (Cu, Ag, Fe) inhibits its activity and
should not be used. Chelating agents like EDTA do not counteract the inhibition. Its optimal
activity is at 46oC.
The enzyme is present in all cells of the body but differences are observed in the rates at
which the enzymes of various organs act on two substrate: fructose–1–6–diphosphate
(FDP) and fructose–1–6–phosphate (F–1–P)
This serum enzyme is quite stable. Activity is unchanged for 48 hours at room temperature
and remains unchanged for at least 3 – 4 weeks if the enzyme is refrigerated.
The level in red cells is about 150 times higher than that of serum; therefore hemolyzed
serum should not be used for analysis. The enzyme is also found in CSF and serous
effusions, but not in urine unless associated with proteinuria.
1. ALS A – found in highest concentration in muscle. Increase levels is seen in progressive
muscular dystrophy (Duchenne type, Erb’s paralysis)

2. ALS B – found predominantly in the liver. Increase levels are seen in acute and chronic
hepatitis, cirrhosis, liver cell carcinoma and metastatic liver carcinoma.
3. ALS C – found predominantly in brain tissue. It is useful marker for cell damage within
the CNS
1. Coupled enzymatic procedure
fructose–1,6–diphosphate ------------->   dihydroxyacetone phosphate + glyceraldehyde–3–
phosphate
glyceraldehyde–3–phosphate------------>  dihydroxyacetone phosphate
2 dihydroxyacetone phosphate + 2NADH ------------>  2 glyceraldehyde phosphate + 2NAD

TPI – triosephosphate isomerase

GD – glycerol phosphate dehydrogenase
Absorbance is measured at 340 nm.