ENZYMES

ENZYMES
¨ biologic proteins that catalyze biochemical
reactions without altering the equilibrium point of
the reaction or being consumed or changed in
composition
Enzyme Nomenclature
¨ Practical or Trivial Name
¨ Systematic Name
Practical/Trivial name:
¨ According to the name of the substrate with the
addition of the suffix “ase”
Examples:
¤ Enzymes acting on lipids – lipase
¤ Enzymes acting on proteins - protease
Practical/Trivial name:
¨ According to the type of reaction they catalyzed.
Examples:
¤ Transfer of amino group from substrate to another -
transferase
¤ Transfer to phosphate group from a high energy
phosphate compound to its substrate - kinase
¤ Effect of hydrolysis on phosphate esters – phosphatase
¤ Removal of hydrogen atoms from its substrate -
dehydrogenase
 Systematic Name
¨ According to the numerical designation given by
the Enzyme Commission (E.C.)
Examples:
¤ E. C. 1. 1. 1. 7 for lactate dehydrogenase
¤ E. C. 3. 2. 1 .1 for amylase
¤ E. C. 2. 6.1. 2 for alanine aminotransferase
¨ The first number defines the class to which the
enzyme belongs, while the next two numbers
indicate subclass and sub class to which the
enzyme is assigned. The last number Is a specific
serial number to each enzyme In its sub-class.
GENERAL CLASSIFICATION OF
ENZYMES:
1. Oxidoreductases - removal or addition of
electrons (reduction-oxidation ["redox"] reaction.)
Examples:
(a) oxidase - cytochrome oxidase
(b) dehydrogenase
lactate dehydrogenase (LDH)
malate dehydroenase (MDH)
isocitrate dehydrogenase (ICD)
GENERAL CLASSIFICATION OF
ENZYMES:
2. Transferase - catalyze the transfer of a
chemical group from one substrate to another
Examples:
(a) aspartate aminotransferase (AST)
(b) alanine aminotransferase (ALT)
(c) cretine kinase (CN) or
creatine phosphokinase (CPK)
(d) gamma glutamyl transferase (GGI)
(e) ornithine carbamyl transferase (OCT)
GENERAL CLASSIFICATION OF
ENZYMES:
3. Hydrolase - hydrolyze the splitting of a bond by the addition of water
(hydrolysis reaction)
Examples:
(a) esterases
acid phosphatase (ACP)
alkaline phosphatase (ALP)
cholinesterase (CLS)
lipase (LPS)
(b) peptidases
trypsin (PTS)
pepsin (PPS)
leucine aminopeptidase (LAP)
(c) glycosidase
amylate, kAMS)
amylo 1,6 glycosidase
galactoxsdases
GENERAL CLASSIFICATION OF
ENZYMES:
4. Lyases - remove groups from substrate without
hydrolysis, leaving only double bonds in the
molecular structure of the product.
Examples
(a) aldolases
(b) glutamate decarboxylase
(c) pyruvate decarboxyiase
(d) tryptophan decarboxylase.
GENERAL CLASSIFICATION OF
ENZYMES:
5. Isomerases - catalyzes the intramolecular
rearrangement of the substrate compound.
Examples:
(a) glucose phosphate isomerase
(b) ribose phosphate lsomerase
GENERAL CLASSIFICATION OF
ENZYMES:
6. Ligases (Synthetases) - joins two substrate molecules
together using the energy released from
hydrolyzing a pyrophosphate bond to a high-
energy phosphate compound.
TERMS ASSOCIATED WITH ENZYMES:
¨ Holoenzyme -an active substance formed by
combination of a coenzyme (cofactor) and
apoenzyme.
¨ Apoenzyme - the protein portion subject to
denaturation, in which the enzyme loses its activity.
Catalytically inactive protein when cofactor is
removed. They are heat labile and dialyzable.
¨ Isoenzyme - enzymes present !it an individual with
similar enzymatic activity but differ in their physical
biochemical and immunologic characteristics
TERMS ASSOCIATED WITH ENZYMES:
¨ Metalloenzyme - enzyrne whose metal ions are
intrinsically part the molecule such as catalases and
cytochrome oxidase.
¨ Proenzyme - inactive precursor of enzymes, also
referred to as zymogens
¨ Substrates - substances acted upon by the
enzymes which are specific for each of their
particular enzyme.
¨ Cofactors - these are non-protein
substance/compounds needed by an enzyme
before enzymatic activity can be manifested.
Cofactors are thermostable and dialyzable.
Cofactor
¨ Organic molecule - Coenzyme.
n It hastens enzymatic reaction but undergoes a change or
is consumed to another product.
n Examples:
¤ NAD – nicotinamide Adenine dinucleotide
¤ NADP - nicotinamide Adenine dinudeotide
phosphate
Cofactor
¨ Metal ion - Activator
¨ In such, the metal ion may serve as:
¤ a bridge to hold the substrate and enzyme together
¤ the primary catalytic center
¤ stabilizing agent In the conformation for catalytic activity.
Examples:
Amylase Cl , Br-
LDH Zn2
LipaseCa++
ENZYME KINETICS:
A. An enzyme (E) catalyses a reaction by combining with its
substrate (S) to create an enzyme—substrate complex (ES).
The enzyme-substrate complex according to Michaelis and
Menten can either dissociate back to E + S or breakdown to
product (P) and free enzyme (provided that the product has
a low affinity for the enzyme).
¨ THE MICHAELIS-MENTEN EQUATION GIVES THE
MEANS TO DETERMINE TOTAL ENZYME
CONCENTRATION IN SERUM AND OTHER BODY
FLUIDS
¨ Accurately describes virtually all single-substrate
enzyme-catalyzed reactions and many bisubstrate
reactions in which the concentration of one substrate
is constant throughout the course of the reaction.
Michaelis-Menten Constant
!!"# (#)
¨ V= %!&(#)
Where:
V = Velocity of the reaction
Vmax = Maximum velocity
S = Substrate concentration
Km = Michaelis-Menten constant
Types of Specificity
¨ Absolute Specificity – enzymes combine with only
one substrate and catalyzes only one corresponding
reaction
¨ Group Specificity – enzymes combining with all
substrates containing a particular chemical group
¨ Bond Specificity – enzymes are specific to chemical
bonds
¨ Stereoisomeric Specificity – enzymes that
predominantly combine with only one optical isomer
of a certain compound
Enzyme Specificity
¨ 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 conform into the lock (enzyme).
Koshland'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 enzyme. This theory Is
more acceptable because the protein molecule Is
flexible enough to allow conformational changes
and also allow some explanation on the influence of
hormones on enzymatic activity.
Types of Reaction Order:
1. Zero Order Reaction - is the rate of reaction linear
with time, independent of concentration of substrate
and directly proportional to enzyme concentration.
2. First Order Reaction - the rate of reaction is
determined by the concentration of substrate.as well
as of enzymes (the rate of reaction changes
continuously with time as the substrate is consumed.
Factors Affecting Enzyme Reactions:
¨ Enzyme concentration. - An increase in the
concentration of enzyme produces an increase in
the rate of reaction, provided that the other
conditions remain the same and that a constant
but excess amount of substrate Is present.
Meaning, if the amount of enzyme is doubled, the
reaction proceeds twice as fast.
Factors Affecting Enzyme Reactions:
¨ Substrate Concentration - An increase in the
concentration of substrate produces also an
increase in the rate of reaction, provided all other
conditions are kept constant However, the rate of
the reaction reaches a maximal value at a
particular concentration of substrate, and higher
concentrations of substrate do not result in
increased rate of reaction (Saturation kinetics).
Factors Affecting Enzyme Reactions:
¨ Temperature - The rate of any chemical reaction is
usually increased 2-3 times for every I0 degrees
Celcius rise in temperature.
Factors Affecting Enzyme Reactions:
¨ Hydrogen Ion Concentration or pH - Enzymatic
reactions proceed at their fastest rate at an
optimum pH and are considerably slowed or even
stopped at higher or lower pH values.
ENZYME INHIBITION:
¨ Competitive Inhibitor - These are substances that
compete with the substrate for enzyme binding
because they are chemically analogous to the
substrate and bind to the active sites of enzymes
ENZYME INHIBITION:
¨ Non- competitive Inhibitor. - These are substances
that do not resemble the substrate and bind to the
enzyme in areas other than the active site
ENZYME INHIBITION:
¨ Uncompetitive Inhibition – inhibits enzyme by
binding to the enzyme substrate complex
ENZYME INDUCTION:
¨ This phenomenon states that a certain enzyme has
the ability to adapt to their biochemical systems
Types of Enzyme Assays
¨ Endpoint Analysis
¤ Reaction is initiated by addition of substrate
¤ Reaction is allowed to proceed for a period of time

¤ Measurement is done at the end of the reaction

¤ Disadvantage: underestimation of the “true” enzyme

activity and linearity of reaction cannot be observed
Types of Enzyme Assays
¨ Multi-point and Kinetic Assay
¤ Change in concentration of the indicator substance at
several intervals
¤ Continuous measurement of change in concentration as
function of time
Types of Enzyme Assays
¨ Use of Coupled Reactions:
¤ Enzymatic activity is measured by coupling the activity
with colorimetric reaction
UNITS FOR EXPRESSING 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.
¨ Katal Unit (K.U.)
¤ Equivalent to the amount of enzyme that catalyzes
the conversion of 1 mole of substrate per second
under controlled conditions.
MEANS OF MEASURING ENZYME
ACTIVITY
¨ Change in Coenzyme Concentration
¨ Increase in Product Concentration
¨ Decrease in Substrate Concentration
PITFALLS IN ENZYME ASSAYS
¨ Hemolysis cause falsely elevated values due to the
release of enzymes from red blood cells.
¨ Serum rather than plasma is the preferred
specimen due to the adverse effects of
anticoagulants on enzyme activity.
¨ Lactescent or milky serum causes variable
absorption by the spectrophotometer.
¨ Storage
Storage of Samples
¨ Most enzymes are stable at 6oC for at least 24 hrs
¨ Few enzymes are inactivated at refrigerator temp
(LD 4 and 5)
Quality Control Program for Enzyme
Assays
¨ Strict adherence to zero-order kinetics
¨ Proportionality with increments of sample
¨ Use of pooled frozen serum or stable reference
materials as controls
¨ Replicate measurements to evaluate precision of
assays
SPECIFIC ENZYMES
Phosphatases
¨ Characterized by its ability to hydrolyze a large
variety of organic phosphate esters with the
formation of an alcohol and a phosphate ion
¨ Hydrolases
¨ Class 3
A. Alkaline Phosphatase
¨ Alkaline Orthophosphoric Monoester
Phosphohydrolase

¨ Reference Value: 30-90 U/L

Alkaline Phosphatase (EC 3.1.3.1) or
ALP
¨ ALP is an enzyme involved in the cleavage of
phosphate containing compounds in alkaline pH. It
facilitates movement of substances across cell
membranes.
¨ Several isoenzymes exist which include:
¤ Placental isoenzyme
¤ Intestinal isoenzyme
¤ Liver isoenzyme
¤ Bone isoenzymes.
Isoenzymes
CHEMICAL INHIBITION TEST
HEAT
ISOENZYME
FRACTIONATION Phenylalanine
Urea Levamisole
Rgt.
1 Liver ALP -- -- -- Inhibited

2 Bone ALP Most heat labile -- Inhibited Inhibited

3 Placental Most heat stable Inhibited -- --

ALP
4 Intestinal -- Inhibited -- --
ALP
Carcinoplacental ALP
¨ Regan ALP – lung, breast, ovarian and
gynecological cancers; bone ALP co-migrator; most
heat stable, inhibited by phenylalanine reagent
¨ Nagao ALP – adenocarcinoma of the pancreas and
bile duct, pleural cancer, variant of Regan; inhibited
by L-leucine and phenylalanine
¨ Kasahara ALP – hepatoma/hepatocellular
carcinoma
Methods of Determination
¨ Bowers and Mc Comb(continuous-monitoring
technique) – pH 10.15; 405 nm
p-nitrophenylphosphate p-nitrophenol +
phosphate ion
 METHODS SUBSTRATE END PRODUCTS
• Bodansky

• Shinowara
Inorganic phosphate +
Beta-glycerophosphate
• Jones glycerol

• Reinhart

• King and Armstrong Phenylphosphate Phenol

• Bessey, Lowry and
Brock p-nitrophenol or yellow
p-nitrophenyl phosphate
nitrophenoxide ion
• Bowers and Mc Comb

• Huggins and Talalay Phenolphthalein

Phenolphthalein red
diphosphate
• Moss Alpha-naphthol phosphate Alpha-naphthol
• Klein, Babson and Reid Buffered phenolphthalein
Free phenolphthalein
phosphate
Measurement
¨ Assays to measure ALP activity use p-nitrophenyl
phosphate substrate at an alkaline pH.
¨ Activators for this enzyme are zinc, magnesium. and
other cations,
¨ Chelators (such as EDTA, citrate, oxalate) can falsely
lower activity.
¨ Activity of enzyme increases slightly on storage due
to loss of inhibitors.
¨ ALP is relatively stable at 4oC for up to one week.
¨ Optimum pH: 8.6-10
Increased ALP is seen on the ff.
conditions:
¨ Osteitis deformans/Paget’s disease
¨ Osteomalacia
¨ Rickets
¨ Osteoblastic bone tumors
¨ Bone Cancer
¨ Sprue
¨ Hyperparathyroidism
¨ Obstructive jaundice
¨ Hepatitis and cirrhosis
ALP Elevations
¨ Liver and bone diseases (most common causes)
¨ Hepatic causes including cholestasis.
¨ Osteosarcoma, tumor metastates to bone (increases
osteoblastic activity; ii turn, increases bone ALP activity)
¨ Diabetes, renal failure
¨ Germ cell tumors (such as seminoma, dysgerminoma) causes
elevated placenta – like isoenzymes; as well as serous
carcinoma of the ovary, nonseminomatous germ cell tumors,
endometrial carcinoma, and leukemia.
Decreased ALP
¨ After blood transfusions or cardiopulmonary
bypass (transiently decrease)
¨ Malnutrition
¨ Hypophosphatemia (prolonged, severely low
levels)
¨ Zinc deficiency (prolonged, severely low levels.
¤ Zinc is a necessary cofactor in ALP activity.
B. Acid Phosphatase
¨ Catalyzes same reaction made by ALP
¨ Active at pH 5.0
¨ Diagnostic significance: detection of prostatic
carcinoma
¨ Tissue sources: prostate (major source), RBC,
platelets and bone
¨ Reference values: 2.5-11.7 (total ACP male)
0-3.5 ng/mL (prostatic ACP)
Acid Phosphatase (EC 3.1.2.3) or
ACP
¨ ACP is a hydrolytic enzyme secreted by a number
of cells.
¨ There are several isoenzymes of ACP, each with
tissue specificity. Isoenzymes may be fractionated
into five bands,
ACP Isoenzymes
¨ Band 1, the major source is prostate gland.
Prostatic ACP activity is inhibited by tartrate.
¨ Band 2 and 4 isoenzymes are from granulocytes.
¨ Band 3 is the major form present in plasma. This
isoenzyme (band 3) is derived from platelets,
erythrocytes, and monocytes.
¨ Band 5 is found mainly in osteoclasts. This
isoenzyme, (band 5) is resistant to tartrate
inhibition.
Methods of Determination
METHODS SUBSTRATE END PRODUCTS
1. Gutman and Gutman Phenyl PO4 Inorganic PO4
2. Shinowara PNPP P-nitrophenol
3. Babson, Read and
Alpha-naphthyl PO4 Alpha-naphthol
Phillips
4. Roy and Hillman Thymolphthalein monoPO4 Free thymolphthalein
Tartrate-inhibited ACP (prostatic
isoenzyme)
¨ Prostatic Cancer
¨ Benign prostatic hyperplasia
¨ Prostatic infarction.
¨ Urinary tract obstruction, carcinoid tumors of
the rectum, and prostatic massage
¨ Medico-legal cases. ACP also has
implications in suspected rape. Positive ACP
is evident in vaginal swab if semen is present
for the first 12 hours up to four days from
the incident.
Tartrate-resistant ACP (bone
isoenzyme)
¨ Active osteoclast-mediated bone resorption
¨ Gaucher's cells
¨ Hairy cell leukemia
 Chemical Inhibition Technique

RBC ACP Prostatic ACP

Copper (Cu2+) + -
Tartrate - +
Copper Resistant: Prostatic ACP
Tartrate Resistant: RBC ACP
ACP Elevations
¨ Moderate elevation of Total ACP
¤ Female Breast CA
¤ Paget’s disease

¤ Hyperparathyrodism

¨ Non-prostatic ACP elevations

¤ Neimann-Pick disease
¤ Gaucher’s disease
¤ Myelocytic leukemia
Aminotransferases
¨ Catalyzes the transfer of an amino group of one
amino acid to a hydrocarbon to form a different
amino acid
¨ Aminotransferases are of two types:
¤ Aspartate Aminotransferase (EC 2.6.1.1) or AST
¤ Alanine Aminotransferase (EC 2.6.1.2) or ALT
¨ Cofactor à pyridoxal-S'-phosphate or P-5’-P
ALT
¨ L-alanine + alpha ketoglutarate « pyruvate +

glutamate
AST
¨ L-aspartate + 2-oxoglutarate « oxaloacetate +

glutamate
Specimen Stability
¨ The half-life of AST is 17+ 5 hours while ALT has a
half-life of 47 +10 hours.
¨ Specimen
¤ AST is stable in serum at refrigerator temperature for up
to three weeks, indefinitely if frozen. ALT has the same
stability but markedly decreases with freezing.
¤ Specimens for AST and ALT are stable in whole blood for
up to 12 to 24 hours, but increase with time due to
release from red blood cells.
¨ Optimum pH: 7.4
AST (Aspartate Aminotransferase)
¨ Involved in the transfer of an amino group between
aspartate and α-ketoacids with the formation of
oxaloacetate and glutamate
¨ Has 2 isoenzymes fractions: cytoplasm and
mitochondrial
¨ Major tissue source: cardiac tissue, liver and skeletal
muscles
¨ Other sources: kidney, pancreas and RBC
¨ Reference values: (5-37 U/L)
Method of determination
¨ Karmen Method – pH 7.5; 340 nm
¨ Uses malate dehydrogenase and monitors the

change in absorbance AST

Aspartate + α-ketoglutarate oxaloacetate +
glutamate
MD
Oxaloacetate + NAD + H malate + NAD
Clinical Significance
(Increased AST activity)
¨ In the evaluation of myocardial infarction,
hepatocellular disorders and skeletal muscle
involvement
¨ MI à AST level is usually 4-10 times the upper limit
of normal
¨ Diagnosis of chronic alcohol abuse and drug
hepatotoxicity
¨ Pulmonary infarction, pericarditis, acute hepatitis
Clinical Significance
(Decreased AST activity)
¨ Decreased level is seen during pregnancy
ALT (Alanine aminotransferase)
¨ Has enzymatic activity similar to AST
¨ Highest concentration is in the liver
¨ Other sources: kidney, pancreas, RBC, heart,
skeletal muscles, lungs
¨ Reference Values: 6-37 U/L
Method of determination
¨ Coupled Enzymatic reaction: pH 7.5; 340 nm
¨ Reitman-Frankel Method
ALT
Alanine + α-ketoglutarate pyruvate + glutamate
pyruvate + NAD + H lactate + NAD
LD
Measurement
¨ Aminotransferase activity measurement is done
by coupled enzymatic reactions, using NADH as
the final reaction product.
¨ Reagents with NH4 will give falsely increased
ALT and AST owing to the conversion of NADH to
NAD by the ammonium ion.
¨ International Federation of Clinical Chemistry
(IFCC) recommended that methods should include
P-5'-P in the reagents.
Diagnostic significance
¨ Significant in the evaluation of hepatic disorders
¤ Diagnosis of acute or chronic viral hepatitis à ALT
increases to a greater degree than AST
¨ Monitors the course of hepatitis treatment and the
effect of drug therapy
Aminotransferase levels are altered in:
¨ Hepatocyte injury (increase in AST, and ALT but to
a lesser degree)
¨ Muscle injury (increase in both enzymes)
¨ Kidney infarcts (increase in both enzymes)
¨ Renal failure (falsely lowered)
Creatine Kinase (EC 2.7.3.2) or CK
¨ Involved in the reversible phosphorylation of
creatine by ATP
¨ CK is an enzyme is involved in energy storage of
tissues.
¨ In the course of active muscle contraction. ATP is
used up and creatine phosphate is converted by CK
to creatine and ATP. This process allows continued
contraction.
¨ During periods of rest, ATP is converted to creatine
phosphate by CK to serve as energy reservoir.
¨ Creatine kinase requires magnesium as a cofactor.
CK Isoenzymes
¨ CK1 or CK-BB (found predominantly in the brain and
smooth muscles)
¨ CK2 or CK-MB (normal muscle contains 14% to 20% of
CK-MB; in skeletal muscle, CK-MB comprises 0% to 1%
of total CK in type 1 fibers, and 2% to 6% of total CK
in type 2 fibers).
¨ CK3 or CK-MM. (also found in skeletal muscles)
¨ Macro-CK (an oligomer present in mitochondria and is
seldom released into circulation)
¨ Reference Value: 15-160 U/L Male
15-130 U/L Female
6% of total CK à CK-MB
CK Isoenzymes
¨ CK-BB à most rapidly moving isoenzyme
¨ CK-MB à hybrid
¨ CK-MM à slowest and most common form
Diagnostic significance
¨ AMI
¨ Duchenne’s Muscular dystrophy à highest elevation
of total CK
Measurement
¨ Electrophoresis is the method of choice. All
isoenzymes can be measured at one time because
of technical difficulties, it has been seldom used.
¨ Immunoinhibition assays for CK-MB uses antibodies
against the CK-M subunit. And residual CK activity
is measured.
¨ Mass immunoassay is the most commonly used
method for measuring CK-MB. It may use two
different antibodies or by using the "Conan"
monoclonal antibody, specific for CK-MB.
Methods of Determination
¨ Tanzer-Gilvarg Assay (Forward Direct method) – pH
9.0 @ 340 nm
CPK
Creatine + ATP Creatine PO4 + ADP
PK
ADP + phosphoenopyruvate Pyruvate + ATP
LD
Pyruvate + NADH Lactate + NAD
Methods of Determination
¨ Oliver-Rosalki method (Reverse/indirect method) –
pH 6.8 @ 340 nm; most commonly used; faster
reaction
CPK
Creatine PO4 + ADP Creatine + ATP
HK
ATP + Glucose ADP + glucose-6-PO4

G-6-PD
Glucose-6-PO4 + NADP 6-phosphogluconate
+ NADPH
Considerations in CK Assays:
¨ CK is light sensitive
¨ Anticoagulants (Oxalates and Fluoride) à inhibits
CK action
¨ CK in serum is very unstable and is rapidly lost
during storage à activity can be regenerated by
adding substances with –SH groups (cysteine,
dithiothreitol, mercaptoethanol)
¨ Exercise and IM injections causes CK elevations
CK levels are increased in:
¨ Progressive muscular dystrophy
¨ Poliomyelitis
¨ Acute psychosis
¨ Alcoholic myopathy
¨ Delirium tremens
¨ Hypothyroidism
¨ Malignant hyperthermia
¨ Acute cerebrovascular disease
¨ Trichinosis and dermatomyositis
Clinical Significance:
¨ Increases in CK MB may be due to:
¤ Cardiac or skeletal muscle damage
¤ Chronic myopathies
¤ Chronic renal failure
¤ Acute respiratory exertion
Clinical Significance:
¨ CK-BB is increased in:
¤ Smooth muscle injury (intestinal ischemia)
¤ Malignancies (prostate cancer, small cell carcinoma
of the lung, and intestinal malignancies).
Clinical Significance:
¨ Macro-CK2 is present in
¤ Malignancies
¤ Myocardial infarction (when Macro-CK2 is present,
it is usually associated with poor prognosis)
¤ MI (less than 5% of the causes)
Gamma- Glutamyl Transferase (EC
2.3.2.1) or GGT
¨ GGT catalyzes the transfer of glutamyl moiety
from peptides to amino acids, other peptides, or
water molecules.
¨ GGT are plasma membrane bound on cells that
has high secretory or absorptive properties (such
as liver, canaliculi cells proximal renal tubules,
intestinal epithelium, and prostate gland).
Gamma- Glutamyl Transferase (EC
2.3.2.1) or GGT
¨ Half life of GGT is about 7 to 10 days. In
alcoholic liver disease, half-life increases to 28
days.
¨ Measurement
¤ Szasz Assay
¤ GGT activity is measured by cleavage of chromogen
o-carboxyl p-nitroaniline from a glutamate modified
form of the compound.
GGT elevations
¨ Liver damage is the major source of GGT release.
¨ Smoking à Moderate smoking raises GGT levels
by 10%, while heavy smoking by 20%.
¨ Medications increase GGT levels up to five
times normal, these drugs include ethanol,
phenytoin, barbiturates, carbamazepine, and
valproic acid.
GGT decrease
¨ Pregnancy à First trimester of pregnancy causes
25% decrease in GGT levels
¨ Oral contraceptives reduce GGT by 20%.
Uses of GGT
¨ Evaluation of liver injury (primary use)
¨ Test for alcoholic abuse (It is abnormal in only
30%, 50% of those consuming excessive amounts
of alcohol. More often elevated in maintenance
drinkers rather than alcohol drinkers)
Lactate Dehydogenase (EG 1.1.1.27)
or LD
¨ LD is a zinc-containing enzyme and its activity is
part of the glycolytic pathway.
¨ LD is a tertramer of two active subunits, H (for
heart) and M (muscle). Combinations of the subunits
produce five isoenzymes.
¤ LD1 (HHHH; H4)
¤ LD2 (HHHM; H3M)
¤ LD3 (HHMM; H2M2)
¤ LD4 (HMMM; HM3)
¤ LD5 (MMMM; M4)
LD
¨ Normal electrophoretic pattern
¤ All bands of isoenzymes are low
¤ LD2 is always higher than LD1

¨ Tissue Sources: heart, RBC, Kidney (LD1 and LD2);

lungs, pancreas, spleen (LD3); skeletal muscles, liver,
intestine (LD4 and LD5)
¨ Reference Values: 100-225 U/L (forward reaction)

80-280 U/L (reverse reaction)

Diagnostic Significance
¨ Highest level are seen in pernicious anemia and
hemolytic disorders
¨ Hepatic carcinoma and toxic hepatitis – 10 fold
increase
¨ Viral hepatitis and cirrhosis – 2-3x increased
¨ LD 1 and 2 flipped pattern – MI, hemolytic anemia
¨ LD5 – moderately increased in acute viral hepatitis,
and cirrhosis; markedly increased in hepatic
carcinoma and toxic hepatitis
Method of Determination
¨ Wacker Method
LD
(forward/direct reaction)
Lactate + NAD Pyruvate + NADH @ 340 nm
¨ Wroblewski La Due (Reverse/indirect reaction)
LD
Pyruvate + NADH Lactate + NAD
Amylase/Diastase E.C. 3.2.1.1
¨ Catalyzes the breakdown of starch
¨ Smallest enzyme
¨ Earliest pancreatic marker
¨ Isoenzymes: S-type (ptyalin); P-type (amylopsin)
¨ Reference Values: 60-180 SU/dL (Somogyi units)
95-290 U/L
¨ Optimum pH: 6.9-7.0
Diagnostic Significance
¨ Acute pancreatitis
¨ Rise at 2-12 hrs, peak at 24 hrs and normalize
within 3-5 days
¨ AMS in urine remains elevated up to 7 days
Amylase Determination
¨ Saccharogenic – measures the amount of reducing
sugars produced by the hydrolysis of starch
¨ Amyloclastic - amylase activity is evaluated by
following the decrease in substrate concentration
¨ Chronometric – measures the time required for
amylase to be completely hydrolyzed
¨ Amylometric – measures the amount of starch
hydrolyzed in a fixed period of time
Lipase
¨ An enzyme that hydrolyzes the ester linkages of
fats to produce alcohol and fatty acids
¨ Most specific pancreatic marker: secreted
exclusively in the pancreas, not affected by renal
disorders
¨ Reference Values: 0-1.0 U/mL
¨ Optimum pH: 7.8-8.0
Lipase Determination
¨ Cherry Crandall Method
¨ Principle:Hydrolysis of Olive Oil after incubation for
24 hrs @ 370C and titration of fatty acids using
NaOH
¨ Substrate: 50% Olive oil/Triolein
¨ End Product: Fatty Acid
Considerations in LPS Assays:
¨ Lipemic specimen à reduction lipase activity
¨ Opiates and morphines à increases LPS activity
due to its spastic effect on the duodenal musculature
and Sphincter of Oddi
Diagnostic Significance
¨ In acute pancreatitis, lipase levels rise 6 hours after
onset of attack, peak at 24 hours, remains elevated
for 7 days, and normalize in 8-14 days
¨ In chronic acute pancreatitis, acinar cell
degradation occurs resulting in loss of amylase and
lipase production
¨ LPS is also elevated à pancreatic duct obstruction
and tumors of the pancreas
Other Enzymes
Glucose-6-Phosphate Dehydrogenase
¨ Oxidoreductase
¨ Catalyzes the oxidation of glucose-6-phosphate à
6-phosphogluconate
¨ Important as the 1st step in the pentose phosphate
shunt
Leucine Aminopeptidase (LAP)
¨ Exhibits napthylamidase activity à enzyme attacks
the free amino end of the peptide chain
¨ Substrate: Acyl β napthylamide
¨ Rich in pancreas
Increased LAP:
¨ Hepatobiliary disease
¨ Pancreatic Cancer
¨ Last trimester of pregnancy
¨ Obstructive biliary disease
Measurement:
¨ Goldbarg-Rutenburg method à β napthylamide
reacts with ethylene diamine dihydrochloride à
blue end product
5’ Nucleotidase (5’ N)
¨ Used to differentiate obstructive from
hepatocellular jaundice and hepatobiliary from
osseous disease
¨ Increased à post-hepatic jaundice, intrahepatic
cholestasis and infiltrative lesions of liver
¨ Slightly increased à hepatocellular jaundice
¨ Normal à bone disease
Ornithine carbamoyl transferase (OCT)
¨ Found most exclusively in the liver
¨ Excellent marker for liver disease but it is rarely
used
Cholinesterase (EC 3.1.1.7) and
Pseudochollnesterase (EC 3.1.1.8)

¨ These enzymes cleave one of the body's major

neurotransmitter known as acetylcholine.
¨ True Cholinesterase
n It has high activity in CNS, red blood cells, lung, and spleen.
¨ Pseudocholinesterase or acylcholine acylhydrolase
n It is important in cleavage of succinylcholine. (muscle relaxant
used during surgery)
n It is primarily produced in the liver, but is also synthesized by
myocardium and pancreas.
Measurement
¨ True Cholinesterase uses acetylcholine while
pseudocholinesterase uses butyrylthiocholin as a
substrate.
¨ The released thiocholine reacts with Ellman's
reagent (dithiobisnitobenzoic acid DTNB releasing
6-mercapto-2nitro benzoic acid, which is
measured photometrically.
¨ Kalow and Genest à uses benzoylcholine as
substrate
Clinical Significance
¨ Enzyme measurement is done:
¤ To monitor those exposed to cholinesterase inhibitors
(pesticides such as organophosphate insecticides),
Pseudocholinesterase activity falls before cholinesterase
in RBCs with poisoning.
¤ As a liver function test. Pseudocholinesterase production
reflects synthetic function of the liver. Activity is low in
malnutrition.
¤ For diagnosis of genetic variants à prolonged apnea
after using succinylcholine during anesthesia.
Angiotensin-Converting Enzyme (EC:
3.4.15.1) or ACE
¨ It is also known as kininase 11 or peptidyl
dipeptidase A.
¨ ACE is responsible for conversion of angiotensin I to
angiotensin H, and inactivation of bradykinin,
enkephalin, tachykinins.
¨ Enzyme structure is described as a single
polypeptide chain with zinc at the active center.
(Activity lost with chelating agents).
¨ Most activity is present in the lungs but it is found in
endothelial cells throughout the body.
Angiotensin-Converting Enzyme (EC:
3.4.15.1) or ACE
¨ Measurement
¤ Activity is measured by ACE's ability to cleave synthetic
peptides, releasing hippuric acid, of other Indicator
molecules.
¨ Disease Correlation
¤ Most common reason for ordering ACE levels is for
diagnosing and monitoring sarcoidosis.
¤ As the disease progresses to fibrosis, ACE levels decline.
¤ ACE elevations are also seen pulmonary involvement.
Myeloperoxidase
¨ Hydrogen peroxide oxidoreductase
¨ Stored in azurophilic granules of PMNs and
monocytes
¨ Catalyzes the conversion of chloride anion and
hydrogen peroxide to hypochlorite
¨ Clinical significance: MPO is released into the
extracellular fluid and general circulation during
inflammatory conditions
¤ Active mediator in the atherosclerotic CV disease
Aldolase
¨ Enzyme involved in the conversion of fructose 1,6-
bisphosphate into dihydroxyacetone phosphate and
glyceraldehyde-3-phosphate
¨ Aldolase A – muscle, RBC and brain
¨ Aldolase B – liver, kidney, enterocytes
¨ Aldolase C – brain
¨ Elevated aldolase level can be seen in skeletal
muscle damage, IM, muscular dystrophy
Enzymes as Markers of Hepatic Disorders
Hepatic Disorders
¨ Acute injury (Acute hepatitis) and Necrosis
¤ ALT, AST, ALP, Bilirubin (B1 and B2), LD4 and LD5
¤ Normal Total Protein and Albumin
Hepatic Disorders
¨ Cirrhosis
¤ Bilirubin (B1 and B2), NH3
¤ Slightly increased/Normal – ALT, AST, ALP and LD

¤ Decreased/Low total protein and albumin

¤ Increased Globulin
Hepatic Disorders
¨ Biliary Tract Obstruction
¤ Increased ALP, Bilirubin (B2), GGT, 5’-nucleotidase, LAP
Enzymes as Cardiac Markers
Enzymes for Myocardial Infarction

Appear in Disappearanc
Peak
Serum e
CK 4-6 hrs after 12-24 hrs 1-2 days
AST 6-8 hrs after 48 hrs 4-5 days
8-10 hrs
LD 72 hrs 7-12 days
after