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CCHM 312
LECTURE / SECOND SEMESTER
Dianne Rose C. Mendoza, RMT, MPH

WEEK 2: ENZYMES

ENZYMES Examples:

BIOLOGIC PROTEINS that catalyze biochemical reactions without altering the (a) OXIDASE - cytochrome oxidase
equilibrium point of the reaction or being consumed or changed in composition
(b) DEHYDROGENASE
 Major structure is APOENZYME which protein in nature
 catalyze biochemical reactions = means enzyme molecule can readily speed o lactate dehydrogenase (LDH)
up hasten the rate of chemical reaction o malate dehydroenase (MDH)
 Enzymes are liberated as FREE ENZYMES at the end of the reaction o isocitrate dehydrogenase (ICD)

2. TRANSFERASE - catalyze the transfer of a chemical group from one substrate to

ENZYME NOMENCLATURE another

 Practical or Trivial Name Examples:

 Commonly used
(a) Aspartate Aminotransferase (AST)
 Also known as the RECOMMENDED NAME of an enzyme
molecule
(b) Alanine Aminotransferase (ALT)

 Systematic Name (c) Creatine Kinase (CN) or

 Standardized by the Enzyme Commission specifically by
International Union of Biochemistry o Creatine Phosphokinase (CPK)

(d) Gamma Glutamyl Transferase (GGI)

PRACTICAL/TRIVIAL NAME: (e) Ornithine Carbamyl Transferase (OCT)

According to the name of the substrate with the addition of the suffix “ASE” 3. HYDROLASE - hydrolyze the splitting of a bond by the addition of WATER
(hydrolysis reaction)
Examples:
Examples:
o Enzymes acting on lipids – LIPASE
o Enzymes acting on proteins – PROTEASE (a) ESTERASES

o Acid Phosphatase (ACP)

According to the type of reaction they catalyzed. o Alkaline Phosphatase (ALP)
o Cholinesterase (CLS)
Examples: o Lipase (LPS)

o Transfer of amino group from substrate to another - (b) PEPTIDASES

TRANSFERASE
o Transfer to phosphate group from a high energy phosphate o Trypsin (PTS)
compound to its substrate - KINASE o Pepsin (PPS)
o Effect of hydrolysis on phosphate esters – PHOSPHATASE o Leucine Aminopeptidase (LAP)
o Removal of hydrogen atoms from its substrate –
DEHYDROGENASE (c) GLYCOSIDASE

o Amylate, kAMS)
SYSTEMATIC NAME o Amylo 1,6 glycosidase
o Galactoxsdases
According to the numerical designation given by the Enzyme Commission (E.C.)

Examples:

o E. C. 1. 1. 1. 7 for Lactate Dehydrogenase

 Systematic name: L- lactate – NAD+ oxidoreductase
o E. C. 3. 2. 1 .1 for Amylase
o E. C. 2. 6.1. 2 for Alanine Aminotransferase
4. LYASES - remove groups from substrate WITHOUT hydrolysis, leaving only
double bonds in the molecular structure of the product.
Examples

The first number defines the CLASS to which the enzyme belongs, while the next two (a) aldolases
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. (b) glutamate decarboxylase

GENERAL CLASSIFICATION OF ENZYMES: (c) pyruvate decarboxylase

1. OXIDOREDUCTASES - removal or addition of electrons (reduction-oxidation (d) tryptophan decarboxylase.

["redox"] reaction.)

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5. ISOMERASES - catalyzes the intramolecular rearrangement of the substrate In such, the metal ion may serve as:
compound.
o a BRIDGE to hold the substrate and enzyme together
Examples: o the primary catalytic center
o stabilizing agent in the conformation for catalytic activity.
(a) glucose phosphate isomerase
Examples:
(b) ribose phosphate lsomerase
Amylase Cl, Br-
6. LIGASES (Synthetases) - joins two substrate molecules together using the energy
released from hydrolyzing a pyrophosphate bond to a high energy phosphate compound. LDH Zn2

 Synthase or Synthetase such as Glycogen Synthase Lipase Ca++

TERMS ASSOCIATED WITH ENZYMES: ENZYME KINETICS:

 An enzyme molecule can either be released as an ACTIVE or INACTIVE 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
HOLOENZYME - an ACTIVE substance formed by combination of a coenzyme Michaelis and Menten can either dissociate back to E + S or breakdown to product (P)
(cofactor) and apoenzyme. and free enzyme (provided that the product has a low affinity for the enzyme).

 Apoenzyme – is the protein component, since it is protein in nature this

tends to be prone to denaturation so we consider this to be the HEAT
LABILE portion of enzyme.
o Enzymes are at around 56 and above degree C temperature
they do tend to undergo denaturation
 Cofactor – 2 kinds:
o ORGANIC MOLECULE – such as NAD and NADP and The MICHAELIS-MENTEN EQUATION gives the means to determine TOTAL
called as “Coenzyme”. Coenzyme bound to an apoenzyme we ENZYME CONCENTRATION in serum and other body fluids
call it as a “Prosthetic group”
o INORGANIC MOLECULE – like Fe, Mg, Mn –called as ¨ Accurately describes virtually all single-substrate enzyme-catalyzed reactions and many
ACTIVATORS which are made up of metal ion. HEAT- bisubstrate reactions in which the concentration of one substrate is constant throughout
STABLE the course of the reaction.
 Apoenzyme bind to metal ion =
METALLOENZYME
MICHAELIS-MENTEN CONSTANT

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

 Ex: ALP

METALLOENZYME - enzyme whose metal ions are intrinsically part the molecule
such as catalases and cytochrome oxidase.

PROENZYME - INACTIVE precursor of enzymes, also referred to as ZYMOGENS

 Ex: Pepsinogen in the stomach which will require hydrochloric acid to be
converted into Pepsin which is already an active molecule
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.
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
COFACTOR

 ORGANIC MOLECULE - COENZYME.

ENZYME SPECIFICITY
It hastens enzymatic reaction but undergoes a change or is consumed to another
product. Emil Fisher's LOCK and KEY THEORY

Examples: 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).
o NAD – nicotinamide Adenine dinucleotide
o NADP - nicotinamide Adenine dinucleotide phosphate

 METAL ION - ACTIVATOR

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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

NON- COMPETITIVE INHIBITOR. - These are substances that DO NOT

RESEMBLE the substrate and bind to the enzyme in areas other than the active site

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

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 ENZYME ASSAYS
ENDPOINT ANALYSIS
o Reaction is initiated by ADDITION of substrate
o Reaction is allowed to proceed for a period of time
o Measurement is done at the END of the reaction
o DISADVANTAGE: underestimation of the “true” enzyme activity and
linearity of reaction CANNOT BE OBSERVED

MULTI-POINT AND KINETIC ASSAY

o Change in concentration of the indicator substance at several intervals

o CONTINUOUS MEASUREMENT of change in concentration as function
of time

USE OF COUPLED REACTIONS:

o Enzymatic activity is measured by coupling the activity with

COLORIMETRIC REACTION
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. UNITS FOR EXPRESSING ENZYME ACTIVITY

INTERNATIONAL UNIT (I.U. or U)

o Equivalent to the amount of enzyme that catalyzes the conversion of 1

micromole of substrate per minute (1 μmol/min) under controlled
FACTORS AFFECTING ENZYME REACTIONS:
conditions.
ENZYME CONCENTRATION - An increase in the concentration of enzyme
KATAL UNIT (K.U.)
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.
o Equivalent to the amount of enzyme that catalyzes the conversion of 1 mole
Meaning, if the amount of enzyme is doubled, the reaction proceeds twice as fast.
of substrate per second (1kat = 1 mol/s) under controlled conditions.
SUBSTRATE CONCENTRATION - An increase in the concentration of substrate
produces also an increase in the rate of reaction, provided all other conditions are kept
MEANS OF MEASURING ENZYME ACTIVITY
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
 Change in Coenzyme Concentration
in increased rate of reaction (SATURATION KINETICS).
 Increase in Product Concentration
TEMPERATURE - The rate of any chemical reaction is usually increased 2-3 times for  Decrease in Substrate Concentration
every 10 degrees Celsius rise in temperature.

HYDROGEN ION CONCENTRATION or pH - Enzymatic reactions proceed at their

PITFALLS IN ENZYME ASSAYS
FASTEST RATE at an OPTIMUM pH and are considerably slowed or even stopped
at higher or lower pH values.
 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
ENZYME INHIBITION: effects of anticoagulants on enzyme activity.
 Lactescent or milky serum causes variable absorption by the
spectrophotometer.
 Storage
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NAGAO ALP

STORAGE OF SAMPLES - Adenocarcinoma of the pancreas and bile duct

- Pleural Cancer
 Most enzymes are stable at 6C for at least 24 hrs. - VARIANT OF REGAN
 Few enzymes are INACTIVATED at REFRIGERATOR TEMP (LD 4 and 5)
- inhibited by L-leucine and phenylalanine

KASAHARA ALP
QUALITY CONTROL PROGRAM FOR ENZYME ASSAYS
– hepatoma/hepatocellular carcinoma
 Strict adherence to ZERO-ORDER KINETICS
 Proportionality with increments of sample
 Use of pooled frozen serum or stable reference materials as controls METHODS OF DETERMINATION
 Replicate measurements to evaluate precision of assays
BOWERS and MC COMB (continuous-monitoring technique)

pH: 10.15
SPECIFIC ENZYMES Wavelength: 405 nm
p-nitrophenylphosphate ------------- p-nitrophenol + phosphate ion
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 (ALP)

- Alkaline Orthophosphoric Monoester Phosphohydrolase

REFERENCE VALUE: 30 - 90 U/L
pH: 8 - 10

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: MEASUREMENT

o Placental isoenzyme Assays to measure ALP activity use p-nitrophenylphosphate substrate at an alkaline
o Intestinal isoenzyme pH.
o Liver isoenzyme
Activators for this enzyme are Zinc, Magnesium. and other Cations,
o Bone isoenzymes.
o Isoenzymes CHELATORS (such as EDTA, citrate, oxalate) can FALSELY LOWER activity.

Activity of enzyme INCREASES SLIGHTLY on STORAGE due to loss of

inhibitors.
ISOENZYME
ALP is relatively stable at 4C for up to ONE (1) WEEK.

OPTIMUM pH: 8.6 - 10

INCREASED ALP is seen on the ff. conditions:

o Osteitis deformans/Paget’s disease

o Osteomalacia
o Rickets
o Osteoblastic bone tumors
o Bone Cancer
o Sprue
o Hyperparathyroidism
o Obstructive jaundice
o Hepatitis and cirrhosis
CARCINOPLACENTAL ALP

REGAN ALP ALP ELEVATIONS

- lung, breast, ovarian and gynecological cancers LIVER and BONE DISEASES (most common causes)
- Bone ALP co-migrator
- MOST HEAT STABLE Hepatic causes including CHOLESTASIS.
- inhibited by PHENYLALANINE REAGENT
Osteosarcoma, tumor metastates to bone (increases osteoblastic activity; ii turn, increases
bone ALP activity)
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DIABETES, Renal Failure - GAUCHER'S CELLS
- Hairy cell leukemia
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.
CHEMICAL INHIBITION TECHNIQUE
DECREASED ALP

- After blood transfusions or cardiopulmonary bypass (transiently decrease)

- MALNUTRITION
- HYPOPHOSPHATEMIA (prolonged, SEVERELY low levels)
- Zinc deficiency (prolonged, severely low levels.
o ZINC is a necessary cofactor in ALP activity.

Copper Resistant: Prostatic ACP

Tartrate Resistant: RBC ACP

B. ACID PHOSPHATASE
Catalyzes same reaction made by ALP
Active at pH 3 - 5.0
Diagnostic Significance: detection of PROSTATIC CARCINOMA ACP ELEVATIONS
Tissue sources: PROSTATE (major source), RBC, platelets and bone
Moderate elevation of Total ACP

REFERENCE VALUES: o Female Breast CA

o Paget’s disease
Total ACP male - 2.5 - 11.7 o Hyperparathyrodism
Prostatic ACP – 0 - 3.5 ng/mL
Non-prostatic ACP elevations

o Neimann-Pick disease
ACID PHOSPHATASE (EC 3.1.2.3) or ACP o Gaucher’s disease
o Myelocytic leukemia
- 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 (5) BANDS AMINOTRANSFERASES

Catalyzes the transfer of an amino group of one amino acid to a hydrocarbon to form a
different amino acid
ACP ISOENZYMES
Aminotransferases are of two types:
BAND 1, the MAJOR SOURCE is PROSTATE GLAND. Prostatic ACP activity is
inhibited by TARTRATE. o Aspartate Aminotransferase (EC 2.6.1.1) or AST
o Alanine Aminotransferase (EC 2.6.1.2) or ALT
BAND 2 and 4 isoenzymes are from GRANULOCYTES.

BAND 3 is the major form present in PLASMA. This isoenzyme (band 3) is derived COFACTOR  pyridoxal-S'-phosphate or P-5’-P
from platelets, erythrocytes, and monocytes.
ALT
BAND 5 is found mainly in OSTEOCLASTS. This isoenzyme, (band 5) is
RESISTANT to TARTRATE inhibition. ¨ L-alanine + alpha ketoglutarate « pyruvate + glutamate

AST

METHODS OF DETERMINATION ¨ 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.

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 (4) DAYS from the incident.
- Specimen
o AST is stable in serum at refrigerator temperature for up to THREE (3)
WEEKS, indefinitely if frozen. ALT has the same stability but markedly
decreases with freezing.
o 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.
o 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

TARTRATE-RESISTANT ACP (BONE ISOENZYME) Has 2 isoenzymes fractions: CYTOPLASM and MITOCHONDRIAL

- Active osteoclast-mediated bone resorption MAJOR tissue source: CARDIAC TISSUE, LIVER and SKELETAL MUSCLES

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Other sources: kidney, pancreas and RBC o Diagnosis of ACUTE or CHRONIC VIRAL HEPATITIS  ALT
increases to a greater degree than AST
REFERENCE VALUES: 5 - 37 U/L
Monitors the course of hepatitis treatment and the effect of drug therapy

Aminotransferase levels are altered in:

METHOD OF DETERMINATION
o Hepatocyte injury (INCREASE in AST, and ALT but to a lesser degree)
- KARMEN METHOD – pH 7.5; 340 nm o Muscle injury (INCREASE in both enzymes)
- Uses malate dehydrogenase and monitors the change in absorbance o Kidney infarcts (INCREASE in both enzymes)
o 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.

CLINICAL SIGNIFICANCE (INCREASED AST activity)
In the evaluation of myocardial infarction, hepatocellular disorders and skeletal muscle
involvement
MI  AST level is usually 4-10 times the upper limit of normal
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.

Diagnosis of CHRONIC ALCOHOL ABUSE and DRUG HEPATOTOXICITY

CK ISOENZYMES
Pulmonary infarction, pericarditis, acute hepatitis
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,
CLINICAL SIGNIFICANCE (DECREASED AST activity) CK-MB comprises 0% to 1% of total CK in type 1 fibers, and 2% to 6% of total CK
in type 2 fibers).
¨ DECREASED level is seen during PREGNANCY
CK3 or CK-MM. (also found in SKELETAL MUSCLES)

Macro-CK (an OLIGOMER present in mitochondria and is seldom released into

ALT (Alanine aminotransferase) circulation)

 Has enzymatic activity similar to AST REFERENCE VALUE: 15-160 U/L Male
 Highest concentration is in the LIVER
15-130 U/L Female
 Other sources: kidney, pancreas, RBC, heart, skeletal muscles, lungs
 REFERENCE VALUES: 6 - 37 U/L 6% of total CK  CK-MB

METHOD OF DETERMINATION
o CK-BB  MOST RAPIDLY moving isoenzyme
- Coupled Enzymatic reaction: pH 7.5; 340 nm o CK-MB  HYBRID
- REITMAN-FRANKEL METHOD
o CK-MM  SLOWEST and MOST COMMON form

DIAGNOSTIC SIGNIFICANCE

o AMI

o DUCHENNE’S MUSCULAR DYSTROPHY  HIGHEST

MEASUREMENT
ELEVATION of total CK
Aminotransferase activity measurement is done by coupled enzymatic reactions, using
NADH as the final reaction product.
MEASUREMENT
Reagents with NH4 will give FALSELY INCREASED ALT and AST owing to the
conversion of NADH to NAD by the ammonium ion.
ELECTROPHORESIS is the method of choice. All isoenzymes can be measured at
one time because of technical difficulties, it has been seldom used.
International Federation of Clinical Chemistry (IFCC) recommended that methods
should include P-5'-P in the reagents.
Immuno inhibition 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

DIAGNOSTIC SIGNIFICANCE
CK-MB. It may use two different antibodies or by using the "Conan" monoclonal
antibody, specific for CK-MB.
Significant in the evaluation of hepatic disorders

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METHODS OF DETERMINATION o MI (less than 5% of the causes)

TANZER-GILVARG ASSAY (FORWARD / Direct method)

pH 9.0 @ 340 nm 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).

Half-life of GGT is about 7 TO 10 DAYS. In alcoholic liver disease, half-life increases

to 28 DAYS.

MEASUREMENT
OLIVER-ROSALKI METHOD (REVERSE / indirect method)
o SZASZ ASSAY
o GGT activity is measured by cleavage of chromogeno-carboxyl p-
pH 6.8 @ 340 nm
“MOST COMMONLY USED” nitroaniline from a glutamate modified form of the compound.
Faster Reaction

GGT ELEVATIONS

LIVER DAMAGE is the major source of GGT release.

SMOKING  Moderate smoking raises GGT levels by 10%, while heavy smoking
by 20%.

Medications increase GGT levels up to five (5) times normal, these drugs include
ethanol, phenytoin, barbiturates, carbamazepine, and valproic acid.
CONSIDERATIONS IN CK ASSAYS:
GGT DECREASE
- CK is LIGHT SENSITIVE

- Anticoagulants (Oxalates and Fluoride)  inhibits CK action Pregnancy  First trimester of pregnancy causes 25% DECREASE in GGT levels
- CK in serum is VERY UNSTABLE and is rapidly lost during storage 
Oral contraceptives reduce GGT by 20%.
activity can be regenerated by adding substances with –SH groups (CYSTEINE,
DITHIOTHREITOL, MERCAPTOETHANOL)
USES OF GGT
- Exercise and IM injections causes CK ELEVATIONS
- Evaluation of liver injury (PRIMARY USE)
CK levels are increased in:
- Test for alcoholic abuse (It is abnormal in only 30%, 50% of those consuming
o Progressive muscular dystrophy excessive amounts of alcohol. More often elevated in maintenance drinkers rather
o Poliomyelitis than alcohol drinkers)
o Acute psychosis
o Alcoholic myopathy
o Delirium tremens LACTATE DEHYDOGENASE (EG 1.1.1.27) or LD
o Hypothyroidism
o Malignant hyperthermia LD is a ZINC-CONTAINING ENZYME and its activity is part of the GLYCOLYTIC
o Acute cerebrovascular disease PATHWAY.
o Trichinosis and dermatomyositis
LD is a TERTRAMER of two active subunits, H (for heart) and M (muscle).
Combinations of the subunits produce five isoenzymes.

CLINICAL SIGNIFICANCE: o LD1 (HHHH; H4)

o LD2 (HHHM; H3M)
Increases in CK MB may be due to: o LD3 (HHMM; H2M2)
o LD4 (HMMM; HM3)
o Cardiac or skeletal muscle damage o LD5 (MMMM; M4)
o Chronic myopathies
o Chronic renal failure LD
o Acute respiratory exertion
Normal electrophoretic pattern
CK-BB is increased in:
o All bands of isoenzymes are LOW
o Smooth muscle injury (intestinal ischemia) o LD2 is always higher than LD1
o Malignancies (prostate cancer, small cell carcinoma of the lung, and
intestinal malignancies). Tissue Sources:

Macro-CK2 is present in heart, RBC, Kidney (LD1 and LD2)

o Malignancies lungs, pancreas, spleen (LD3)

o Myocardial infarction (when Macro-CK2 is present, it is usually associated
skeletal muscles, liver, intestine (LD4 and LD5)
with poor prognosis)

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REFERENCE VALUES: MOST SPECIFIC PANCREATIC MARKER: secreted exclusively in the pancreas,
not affected by renal disorders
FORWARD Reaction - 100-225 U/L
REVERSE reaction - 80-280 U/L

DIAGNOSTIC SIGNIFICANCE REFERENCE VALUES:

¨ HIGHEST LEVEL are seen in Pernicious Anemia and Hemolytic Disorders 0 - 1.0 U/mL
Optimum pH: 7.8 - 8.0
¨ Hepatic carcinoma and toxic hepatitis – 10 fold increase

¨ Viral hepatitis and cirrhosis – 2 - 3x increased LIPASE DETERMINATION

¨ LD 1 and 2 FLIPPED PATTERN – MI, hemolytic anemia - CHERRY CRANDALL METHOD

- Principle: Hydrolysis of Olive Oil after incubation for 24 hrs @ 37C and titration
¨ LD5 – MODERATELY INCREASED in ACUTE VIRAL HEPATITIS, and
CIRRHOSIS; markedly increased in hepatic carcinoma and toxic hepatitis of fatty acids using NaOH
- SUBSTRATE: 50% Olive oil/Triolein
METHOD OF DETERMINATION - End Product: FATTY ACID

WACKER METHOD (FORWARD/direct reaction) CONSIDERATIONS IN LPS ASSAYS:

Lipemic specimen  reduction lipase activity

Opiates and morphines  increases LPS activity due to its spastic effect on the
duodenal musculature and Sphincter of Oddi
WROBLEWSKI LA DUE (REVERSE/indirect reaction)
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
AMYLASE/DIASTASE E.C. 3.2.1.1
LPS is also ELEVATED  pancreatic duct obstruction and tumors of the pancreas
 Catalyzes the breakdown of STARCH
 SMALLEST ENZYME
 EARLIEST Pancreatic Marker OTHER ENZYMES
 Isoenzymes: S-type (PTYALIN); P-type (AMYLOPSIN)
GLUCOSE-6-PHOSPHATE DEHYDROGENASE
REFERENCE VALUES:
- OXIDOREDUCTASE

60-180 SU/dL (Somogyi units) - Catalyzes the oxidation of glucose-6-phosphate  6-phosphogluconate

95-290 U/L - Important as the 1st step in the pentose phosphate shunt

Optimum pH: 6.9 - 7.0

LEUCINE AMINOPEPTIDASE (LAP)

DIAGNOSTIC SIGNIFICANCE
- Exhibits NAPTHYLAMIDASE ACTIVITY  enzyme attacks the free amino
 Acute pancreatitis end of the peptide chain
 Rise at 2-12 hrs, peak at 24 hrs and normalize within 3-5 days - SUBSTRATE: Acyl β napthylamide
 AMS in urine remains elevated up to 7 days
- RICH IN PANCREAS
AMYLASE DETERMINATION
INCREASED LAP:

SACCHAROGENIC – measures the amount of reducing sugars produced by the

o Hepatobiliary disease
hydrolysis of starch
o Pancreatic Cancer
AMYLOCLASTIC - amylase activity is evaluated by following the decrease in o Last trimester of pregnancy
substrate concentration o Obstructive biliary disease

CHRONOMETRIC – measures the time required for amylase to be completely

hydrolyzed
MEASUREMENT:
AMYLOMETRIC – measures the amount of starch hydrolyzed in a fixed period of
time
GOLDBARG-RUTENBURG METHOD  β napthylamidereacts with ethylene

diamine dihydrochloride  BLUE END PRODUCT

LIPASE

An enzyme that hydrolyzes the ester linkages of fats to produce ALCOHOL and
FATTY ACIDS
5’ NUCLEOTIDASE (5’ N)
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Used to differentiate obstructive from hepatocellular jaundice and hepatobiliary from Disease Correlation
osseous disease
o Most common reason for ordering ACE levels is for diagnosing and
monitoring SARCOIDOSIS.
Increased  post-hepatic jaundice, intrahepaticcholestasis and infiltrative lesions of
o As the disease progresses to FIBROSIS, ACE levels DECLINE.
liver
o ACE ELEVATIONS are also seen PULMONARY INVOLVEMENT.

Slightly increased  hepatocellular jaundice

MYELOPEROXIDASE
Normal  bone disease
Hydrogen peroxide oxidoreductase

Stored in azurophilic granules of PMNs and MONOCYTES

ORNITHINE CARBAMOYL TRANSFERASE (OCT)
Catalyzes the conversion of chloride anion and hydrogen peroxide to
Found most exclusively in the LIVER HYPOCHLORITE

EXCELLENT MARKER for LIVER DISEASE but it is RARELY USED CLINICAL SIGNIFICANCE: MPO is released into the extracellular fluid and
general circulation during INFLAMMATORY conditions

Active mediator in the atherosclerotic CV disease

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. ALDOLASE

TRUE CHOLINESTERASE Enzyme involved in the conversion of fructose 1,6-bisphosphate into

dihydroxyacetone phosphate and glyceraldehyde-3-phosphate
 It has high activity in CNS, red blood cells, lung, and spleen.
o ALDOLASE A – muscle, RBC and brain
PSEUDOCHOLINESTERASE or acylcholine acylhydrolase o ALDOLASE B – liver, kidney, enterocytes
o ALDOLASE C – brain
 It is important in cleavage of SUCCINYLCHOLINE. (muscle relaxant
used during surgery) liver, but is also synthesized by myocardium and ELEVATED ALDOLASE LEVEL can be seen in skeletal muscle damage, IM,
pancreas. muscular dystrophy

MEASUREMENT
ENZYMES AS MARKERS OF HEPATIC DISORDERS

True Cholinesterase uses ACETYLCHOLINE while pseudocholinesterase uses

HEPATIC DISORDERS
BUTYRYLTHIOCHOLIN as a substrate.
Acute injury (ACUTE HEPATITIS) and NECROSIS
¨ The released thiocholine reacts with ELLMAN'S REAGENT (dithiobisnitobenzoic
acid DTNB releasing 6-mercapto-2nitro benzoic acid, which is measured
o INCREASE ALT, AST, ALP, Bilirubin (B1 and B2), LD4 and LD5
photometrically.
o NORMAL Total Protein and Albumin

¨ KALOW and GENEST  uses BENZOYLCHOLINE as substrate CIRRHOSIS

CLINICAL SIGNIFICANCE o Bilirubin (B1 and B2), NH3

o SLIGHTLY INCREASED/NORMAL – ALT, AST, ALP and LD
Enzyme measurement is done: o DECREASED/LOW Total Protein and Albumin
o INCREASED Globulin
o To monitor those exposed to cholinesterase inhibitors (pesticides such as
organophosphate insecticides), Pseudocholinesterase activity falls before BILIARY TRACT OBSTRUCTION
cholinesterase in RBCs with poisoning.
o As a LIVER FUNCTION TEST. Pseudocholinesterase production reflects o INCREASED ALP, Bilirubin (B2), GGT, 5’-nucleotidase, LAP
synthetic function of the liver. Activity is low in malnutrition.
o For diagnosis of genetic variants  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 peptidyldipeptidase 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). ENZYMES AS CARDIAC MARKERS

Most activity is present in the LUNGS but it is found in ENDOTHELIAL CELLS ENZYMES FOR MYOCARDIAL INFARCTION
throughout the body.

MEASUREMENT

o Activity is measured by ACE's ability to cleave synthetic peptides, releasing

hippuric acid, of other Indicator molecules.

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