C L I N I C A L C H E M I S T R Y 2
LEC 1: ENZYMES
MS. MARTINA DEANNE C. MENDOZA, RMT
2023 - 2024
Enzymes
• Enzymes are proteins that functions as biological catalysts and
are neither consumed nor permanently altered during a chemical
reaction
• They appear in the serum in increased amounts after cellular
injury or tissue damage
• Abnormal large amounts of enzymes in serum are used
clinically as evidence of organ damage
• Each enzyme catalyzes a single reaction or a limited number of
chemical reactions, and it is specific for a substrate that it
converts to a defined product
Factors Affecting Enzymatic Reaction
I. Enzymatic concentration
- the higher the enzyme concentration, the faster the reaction,
because more enzyme is present to bind with the substrate
II. Substrate concentration
- with the amount of enzyme exceeding the amount of substrate,
the reaction rate steadily increases as more substrate is added
- however, when the substrate concentration reaches a maximal
value, higher concentration of substrate no longer results in
increased rate of reaction (saturation kinetics)
III. Cofactors
- nonprotein compounds that must bind to particular enzymes
before a reaction occur
→ Coenzymes – is an organic compound (second
substrates)
- increasing its concentration will increase the velocity of an
enzymatic reaction
- it is essential to achieve absolute enzymatic activity
- example: NADP/NAD+ (nicotinamide adenine dinucleotide)
→ Activators – are inorganic ions which alters the
spatial configuration of the enzyme for proper substrate binding
- example: calcium, zinc, chloride, magnesium, and potassium
→ Metalloenzymes - are inorganic ion attached to a
molecule
- example: catalase and cytochrome oxidase
IV. Inhibitors
a) Competitive inhibitor
• It physically binds to the active site of an enzyme – both
the substrate and inhibitor compete for the same active site
of the enzyme.
• The inhibition is reversible when the substrate
concentration is significantly higher than the concentration
of the inhibitor.
• The effect of the inhibitor can be counteracted by adding
excess substrate to bind the enzyme.
• Dilution of serum results to reduction in the
concentration of this inhibitor, thus increasing the rate of
reaction.
• It has the ability to alter the apparent Michaelis-Menten
constant (Km).
b) Non-competitive inhibitor
• It does not compete with the substrate but look for areas
other than the active site (allosteric site).
• The substrate and inhibitor (commonly metallic ion) may
bind an enzyme simultaneously.
• Because the inhibitor binds the enzyme independently
from the substrate, increasing substrate concentrate does
not reverse the inhibition.
• The presence of the inhibitor when it is bound to the
enzyme, slows the rate of the reaction.
c) Uncompetitive inhibitor
• The inhibitor binds to the enzyme-substrate (ES) complex.
• Increasing the substrate concentration results in more ES
complexes to which the inhibitor binds and thereby
increases the inhibition.
• Increasing substrate concentration results to increase
inhibition.
V. Isoenzymes
- these are enzymes (polypeptide chains) having the same
catalytic reactions but slightly different molecular structures –
various forms occur because of differences in the amino acid
sequence of enzymes.
- the importance of the total enzyme activity is enhanced by
fractionating the isoenzymes.
- Isoenzymes may be differentiated based on electrophoretic
mobility and resistance to heat denaturation
VI. Temperature
- enzymes are active at 25°C, 30°C, or 37°C.
- 37°C is the optimum temperature for enzymatic activity.
- increasing temperature usually increases the rate of a chemical
reaction by increasing the movement of molecules.
- the rate of denaturation increases as the temperature increases,
and is usually significant at 40°C to 50°C.
- Denaturation: Causes change in enzyme structure that results
in loss of activity; may be caused by elevated temperature,
extreme change in pH, and certain chemicals.
- 60 – 65°C may result to inactivation of enzymes
- temperature coefficient (Q10) means for every 10°C increase
in temperature, there will be a two-fold increase in enzyme
activity.
VII. Hydrogen ion concentration (pH)
- most physiologic reactions occur in the pH range of 7 to 8
- extreme pH level may denature an enzyme or influence its ionic
state resulting in structural change or change in the charge of
amino acid residue in the active site.
VIII. Storage
- low temperatures (refrigeration/freezing) render enzymes
reversibly inactive.
- repeated freezing and thawing tends to denature proteins and
should be avoided. - -20°C is the ideal temperature for
preservation of enzymes (longer period of time).
- 2 to 8°C is the ideal storage temperature for substrates and
coenzymes.
- 22°C or room temperature is the ideal storage for LDH (LD4 and
LD5).
IX. Hemolysis - mostly increases enzyme concentration
X. Lactescense or milky specimen - decreases enzyme
concentration
Enzyme Nomenclature
• Enzymes are classified according to their biochemical functions,
indicating substrate and class of reaction catalyzed, and are
designated by individual identification numbers.
• The first digit places the enzyme in its classifications (six
classifications).
• The second and third digits, represents the subclass to which
the enzyme is assigned.
• The final and fourth number/s is a serial number that is specific
to each enzyme in a subclass.
Classifications of Enzymes
 Oxidoreductases catalyze an oxidation-reduction reaction
between two substrates
 Transferases catalyze the transfer of a group other than
hydrogen from one substrate to another.
 Hydrolases catalyze hydrolysis of various bonds.
 Lyases catalyze removal of groups from substrates without
hydrolysis; the product contains double bonds.
 Isomerases catalyze the interconversion of geometric,
optical, or positional isomers.
 Ligases catalyze the joining of two substrate molecules,
coupled with breaking of the pyrophosphate bond in
adenosine triphosphate (ATP) or a similar compound.
General Properties of Enzymes
Each enzyme contains:
 Active site = is water-free cavity, where the substrate
interacts with particular charged amino acid residues; is a
3-dimensional protein structure
 Allosteric site = is a cavity other than the active site; may
bind regulator molecules
• As a protein, each enzyme is composed of a specific amino acid
(primary structure), forming polypeptide chains (secondary
structure), which then folds (tertiary structure) and results in
structural cavities.
• The catalytic activity of an enzyme molecule depends generally
on the integrity of its structure.
• When all sites are filled, no further binding can occur until an
active site discharges its contents.
• When bound tightly to the enzyme, the coenzyme is called a
prosthetic group.
• Apoenzyme (enzyme portion) and coenzyme forms a complete
and active system known as holoenzyme (apoenzyme +
prosthetic group = holoenzyme).
• Prosthetic group is an organic cofactor tightly bound to the
enzyme.
• Digestive enzymes in its inactive form originally secreted from
the organ of production is called a proenzyme or zymogen.
Enzyme Theory
 Emil Fisher’s / Lock and Key Theory
• A German scientist, Emil Fischer postulated the lock and key
model in 1894 to explain the enzyme’s mode of action.
• It is based on the premise that the shape of the key (substrate)
must fit into the lock (enzyme).
• There is no change in the shape of the active site when the
substrate binds.
 Kochland’s / Included Fit Theory
• Proposes that the binding of a substrate or some other
molecule to an enzyme causes a change in the shape of the
enzyme so as to enhance or inhibit its activity.
• It retains the key-lock idea of a fit of the substrate at the active
site but postulates in addition that the substrate must do more
than simply fit into the already preformed shape of an active
site—it must cause a change in the shape of the enzyme that
results in the proper alignment of the catalytic groups on its
surface.
• This concept has been likened to the fit of a hand in a glove, the
hand (substrate) inducing a change in the shape of the glove
(enzyme).
• It is based on the substrate binding to the active site of the
enzyme.
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Enzyme Kinetics
• A chemical reaction may occur spontaneously if the free energy
or available kinetic energy is higher for the substrate than the
product.
• Enzymes catalyze physiologic reactions by lowering the
activation energy level that the substrate must reach for the
reaction to occur.
• An enzyme combines with only one substrate and catalyzes
only one reaction is known as absolute specificity.
• Enzymes combine with all the substrates in a chemical group is
called group specificity.
• Enzymes reacting with specific chemical bonds is known as
bond specificity.
Enzymatic Reaction
1. Zero-order reaction – the reaction rate depends only on
enzyme concentration.
2. First-order reaction – the reaction rate is directly
proportional to substrate concentration.
To measure the extent of enzymatic reactions, two general
methods may be used:
• Fixed-time – the reactants are combined; the reaction
proceeds for a designated time; the reaction is stopped, and
measurement is made.
• Continuous monitoring/kinetic assay – multiple
measurements of changed in absorbance are made during the
reaction; it is preferred than fixed-time.
Michaelis - Menten Equation
- represents the relationship between reaction velocity and
substrate concentration
P+E
Units for Expressing Enzymatic Activity
1. International Unit (IU or U) – 1 micromole of
substrate/minute
2. Katal Unit (KU) – 1 mole of substrate/second
• Enzymes are quantified based on their activity rather than
absolute values.
• The units used to report enzyme levels are activity units.
• The definition for activity unit must consider change in pH,
temperature, substrate, etc.
Enzymatic Activity
- enzymes are measured in terms of:
 Change in the substrate concentration
 Change in the product concentration
 Change in coenzyme concentration
Causes of Elevated Plasma Enzyme Levels
1. Tissue necrosis and degeneration (death of enzyme-containing
cells)
2. Impaired removal of enzyme from plasma
3. Normal cell turnover
4. Increased permeability of cell membrane
5. Increase in the production of enzymes by cells
6. Decreased clearance of enzymes from the circulation
Enzyme Measurement
• Serum is the preferred specimen for the measurement of
enzymes.
• Heparinized plasma is the alternate specimen; however, some
enzymes (AST and CK) are inhibited.
• Citrate, EDTA, and fluoride plasma specimens may cause false
decreased enzymatic activity.
• The substrate (reagent) is specific to the enzyme whose activity
will be measured.
• The presence of inhibitors can be diminished if the sample will
be diluted.
• During measurement, proteases may cause interferences by
dissolving enzymes and substrate.
Major Clinical Enzymes
Alkaline Phosphatase (ALP)
• It functions to liberate inorganic phosphate from an organic
phosphate ester with the concomitant production of an alcohol.
• It is predominantly found in the cell membranes.
• Major tissue cells: liver, bone, placenta, and intestinal
• Reference range: 30 – 90 U/L
• Major isoenzymes:
 Liver ALP
 Bone ALP
 Placental ALP
 Intestinal ALP
• The most abundant plasma ALP isoforms are coded by a single
gene on chromosome 1, producing liver, bone, and kidney
isoenzymes.
• Gene on chromosome 2 code for placental and intestinal ALP.
• In healthy sera, ALP levels are derived from liver (hepatic
sinusoid and canalicular surface) and bone (osteoblasts).
• It is normally elevated (bone isoenzyme) in children during
periods of growth and in adults older than age 50 years
(geriatric).
• During period of growth and muscle development, serum ALP
and creatine levels increase.
• In normal pregnancy, increased ALP activity can be detected
between 16 and 20 weeks of pregnancy.
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• In blood groups O and B, intestinal ALP is increased after
consumption of a fatty meal. ALP is also higher than in
individuals of groups A and AB.
• Placental ALP is lower in pregnant women of blood groups A
and AB.
• When total ALP activity is elevated, mostly it is contributed by
the liver isoenzyme.
 Carcinoplacental ALP
• CSF levels of PLAP are of diagnostic value in differentiating
whether tumor in the pineal body is pinealoma or a germ cell
tumor.
Methods
• ALP is inactivated by EDTA; hence, serum is the preferred
sample.
• ALP requires magnesium ions for activation.

I. Regan ALP  Electrophoresis

- It is found in lung, breast, ovarian, and gynecological cancers. • Liver and bone ALPs are the most anodal isoenzymes; intestinal
- It is the most heat-stable ALP (65°C for 30 minutes). ALP is the least anodal (LiBoPlaIn).
- It is inhibited by phenylalanine reagent, a bone ALP co-migrator. • Use of neuraminidase and wheat germ lectin improves the
- It is coded by the gene on chromosome 2. separation of bone and liver ALPs.

II. Nagao ALP  Heat Fractionation / Stability Test

- It is found in adenocarcinoma of the pancreas and bile duct, and • It is performed at 56°C for 10 – 15 minutes.
in pleural cancer. • Placental ALP is the most heat-stable; bone ALP is the most
- It is a variant of Regan ALP. heat labile.
- It is inhibited by L-leucine and phenylalanine. • Decreasing order of ALP heat stability: Placental, intestinal,
liver, and bone
Diagnostic Significance • (MNEMONICS: Promise Ikaw Lang Beh)

• ALP is increases in obstructive jaundice due to greater rate of
secretion.
• When there is obstruction in the flow of conjugated bilirubin
into the canaliculus, it is accompanied by elevated plasma B2,
ALP, and gamma-glutamyltransferase (GGT).
 Chemical Inhibition Test
• This method uses different concentrations of phenylalanine,
synthetic urea, and levamisole solutions.
• Inhibited by Phenylalanine: Placental, Intestinal, and Regan
• Inhibited by 3M urea: Bone ALP
• Inhibited by Levamisole: Liver and Bone ALP

• For bone disorders, highest elevation occurs in Paget’s disease
(osteitis deformans).
• B1x isoform is used to study low bone mineral disease (BMD)
in patients with chronic kidney disease.
 Bowers and Mc Comb (Szasz Modification)
• Reference method; most specific; routine method
• Substrate: p-nitro phenyl phosphate (PNPP)
• Same substrate with Bessy, Lowry & Brock

• Serum ALP is elevated in cases of abortion and may be

increased when there is difficulty in pregnancy and birthing.

• Transient low serum ALP may occur after blood transfusion or

cardiopulmonary bypass.

• Prolonged low levels of ALP occur in hypophosphatasia

(defective bone and teeth mineralization).

• Bone ALP isoform known as B1x is detected in the serum of Notes to Remember!
dialysis patients.
• Zinc is a component of ALP, and magnesium is the enzyme
• B1x isoform is used to study low bone mineral disease (BMD) activator.
in patients with chronic kidney disease.
• Hemolysis and diet (fatty meals) are sources of analytic errors
• Serum ALP is elevated in cases of abortion and may be and may cause elevated serum ALP.
increased when there is difficulty in pregnancy and birthing.
• ALP is sensitive if stored at low temperature (4°C), leads to
• Transient low serum ALP may occur after blood transfusion or increased serum level.
cardiopulmonary bypass.
• Decreased ALP is seen in zinc deficiency.
• Prolonged low levels of ALP occur in hypophosphatasia
(defective bone and teeth mineralization). Increased ALP
 Obstructive jaundice  Osteoblastic bone tumors
ALP as a Tumor Marker  Osteitis deformans (osteosarcoma)
 Osteomalacia  Sprue
• Placental alkaline phosphatase (PLAP) is utilized as a tumor  Rickets  Hyperparathyroidism
marker in serum and cerebrospinal fluid (CSF) for most germ cell  Hepatitis and cirrhosis
tumors. (slight increased)

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 Acid phosphatase (ACP)
• It is active at pH 5.0.
• The major isoenzymes are coded for by different genes, with
varied molecular weights, structures, and inhibition properties.
• ACP activity in the bones is associated with the osteoclasts.
• Tissue sources: Prostate (major source), RBCs, platelets, liver,
and bone
• Prostatic and bone ACPs are the clinically significant
isoenzymes.
• Reference range:
Male = 2.5 – 11.7 U/L (Total ACP)
0 – 3.5 ng/mL (Prostatic ACP)
Based on the differences at the structural level of the gene, ACP
can be divided into 5 isoenzymes:
Prostatic ACP AcPP, human chromosomal location 3q21-
q23
Erythrocytic ACP AcP1, human chromosomal location 2p25
Lysosomal ACP AcP2, human chromosomal location 11p12-
p11
Testicular ACP AcPT, human chromosomal location 19q13
Macrophagic AcP5, human chromosomal location
ACP 19p13.3-p13.1
Diagnostic Significance
• The major cause of elevated plasma ACP is prostatic disease.
• ACP is measure in the detection of prostatic adenocarcinoma.
• ACP assay aids in the detection of metastatic prostatic cancer,
yet total ACP is not a sensitive marker, and prostate specific
antigen (PSA) is a more useful screening and diagnostic tools.
• ACP is also useful in the forensic chemistry, in the investigation
of rape cases – vaginal washings are examined for seminal fluid-
ACP activity, which can persist for up to 4 days.
• ACP activity > 50 IU/L indicates the presence of seminal fluid in
the sample, in the case of forensic investigation.
• TRAP-5b is a marker for bone remodeling and for bone mineral
disease in patients with chronic kidney disease.
• TRAP-5b is associated with bone resorption while the bone ALP
isoforms (B/I, B1, and B2) are related with deposition.
• TRAP-5b is considered to be a marker of the osteoclasts.
• Increased ACP is observed in thrombocytopenia.
ACP as a Tumor Marker
• Prostatic acid phosphatase (PAP) is used together with prostate
specific antigen (PSA) to monitor recurrence of prostate cancer.
• PSA is more sensitive than PAP in detecting stages A and B
prostatic cancer.
• After surgical treatment of prostate cancer, ACP levels fall
faster than PSA, and plasma levels are expected to be
undetectable following complete removal of tumor.
• Tartrate-resistant acid phosphatase (TRAP/TRACP) is present
in some forms of cancer such as chronic leukemia, lymphomas,
and hairy cell leukemia.
• TRACP-5a is found in Gaucher cells, or in the leukocytes of
patients with hairy cell leukemia.
Methods
• Reference method: Roy and Hillman
• Substrate: Thymolphthalein Monophosphate
Notes to Remember!
• Total ACP is measured by its ability to cleave groups at an
acidic pH.
• Serum sample must be free from hemolysis.
• Icteric serum causes falsely decreased TRAP activity, but not
for total ACP.
• Thymolphthalein monophosphate is the specific substrate and
choice for quantitative endpoint reaction.
• α-naphthyl phosphate substrate is preferred for continuous
monitoring methods.
• If not assayed immediately, serum should be frozen or acidified
to a pH lower than 6.5.
• With acidification, ACP is stable for 2 days at room temperature.
• Serum ACP decreases within 1 to 2 hours if left at room
temperature.
• Prostatic ACP is inhibited by 20=mM L-tartrate ions while 1-
mM cupric sulfate and 2% formaldehyde ions inhibit red cell ACP.
Increased ACP (w/ Metastatic Bone Involvement)
 Prostatic carcinoma  Gaucher’s disease
 Breast, lung, and thyroid  Niemann-pick disease
carcinoma
Aspartate Aminotransferase (AST)
• Previously known as serum glutamic oxaloacetic transaminase
(SGOT).
• It is involved in the transfer of an amino group between
aspartate and α-keto acids with the formation of oxaloacetate
and glutamate.
• Isoenzyme fraction: Cytoplasmic AST (ASTc) and Mitochondrial
AST (ASTm)
• Major tissue source: Cardiac tissue, liver, and skeletal muscle
• Other sources: Kidney, pancreas, and RBC
• ASTc is the abundant fraction in healthy serum.
• In case of tissue necrosis, ASTm is the predominant isoenzyme.
Page 5 of 12
• AST and LD have higher activity in the liver than ALT, but both
are non-specific enzymes.
• AST and LD have equal concentrations in the hepatocytes.
• The kidney has higher AST activity compared to ALT and LD.
• Reference range: 5 – 37 U/L
Diagnostic Significance
• It is vital in the evaluation of myocardial infarction,
hepatocellular disorders, and skeletal muscle involvement.
• In acute myocardial infarction (AMI), AST levels begin to rise at
6 – 8 hours, peak at 24 hours, and normalize within 5 days.
• AST is used for monitoring therapy with potentially hepatotoxic
drugs; a result more than 3x the upper border of normal should
signal cessation of therapy.
• AST is slight to moderately increased in muscular dystrophy or
disorders compared to ALT which has minimal elevation or even
normal.
Methods
 Colorimetric method: Karmen Method
• It uses malate dehydrogenase (MD) and monitors the change in
the absorbance oat 340 nm continuously as NADH is oxidized to
NAD+ .
• Use of heparin may inhibit the activity of AST.
• AST activity is stable in serum for 3 to 4 days at refrigerated
temperature.
• Hemolysis should be avoided because it increases AST 10x the
upper reference limit.
• Interferences: Hemolyzed and icteric samples (false increased)
and presence of heavy metals (false decreased)
• Reference method: Reitman and Frankel
• Substrate: Aspartic Alpha & Ketoglutaric Acid
Alanine Aminotransferase (ALT)
• Previously known as serum glutamic pyruvic transaminase
(SGPT).
• It catalyzes the transfer of an amino group from alanine to α-
ketoglutarate with the formation of glutamate and pyruvate.
• Its highest concentration is in the liver, hence elevated plasma
concentration denotes hepatic injury.
• It is more liver-tissue-specific compared to AST.
• Major tissue source: Liver
• Other sources: Kidney, pancreas, heart, skeletal muscles, lungs,
and RBCs
• Reference range: 6 – 37 U/L
Diagnostic Significance
• Significant in the evaluation of hepatic disorders – markedly
increase concentration in acute inflammatory conditions
involving the liver.
• Higher elevations are found in hepatocellular disease more
than the extra-hepatic and intra-hepatic disorders.
• ALT is slightly increased in obstructive jaundice but markedly
increased in necrotic jaundice.
• The highest elevation of transferase is seen in acute hepatitis.
• It monitors the course of liver treatment and the effects of drug
therapy.
• ALT levels are used to screen blood donors.
• ALT measurement is a more sensitive and specific screening
test for post-transfusion hepatitis or occupational toxic exposure
compared to AST.
Methods
• Both ALT and AST require pyridoxal phosphate, an essential
cofactor that should always be added in any measurement.
• Absence of pyridoxal phosphate (vitamin B6) will result to
diminished activity of both transferases.
• The use of icteric and lipemic samples may cause a significant
interference.
• Use of hemolyzed sample cause false increase of AST activity
while a slight elevation or none at all may be observed in ALT.
 Coupled Enzymatic Reaction
• This reaction takes place at pH 7.5 and monitors the change in
the absorbance at 34 nm continuously as NADH is oxidized to
NAD+ .
• Using hemolyzed sample may cause slight elevations in ALT
activity.
Notes to Remember!
• The highest elevations of transferases are seen in acute
hepatitis.
• Viral hepatitis (A, B, and C) is one of the most common causes
of acute liver injury resulting in increased plasma levels of ALT
and AST.
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• High serum levels of ALT and AST occur most commonly in Increased Serum Amylase
acute hepatitis and/or mechanical damage to the liver.  Acute pancreatitis  Alcoholism
 Ectopic pregnancy  Mumps
• Severe viral or toxic hepatitis may produce elevations of  Peptic ulcers
transferee up to 20x the normal limits.
Macroamylasemia
• Moderate elevation of transferases is observed in chronic
hepatitis, hepatic cancer, and infectious mononucleosis. • It is characterized by elevated macroamylase in serum.

• Slightly increased in hepatic cirrhosis, alcoholic hepatitis, and
obstructive jaundice.
• The De Ritis ratio (ALT:AST) in acute hepatitis is > 1.0, whereas
in alcohol-induced hepatic injury, the AST:ALT ratio is 3:1.
• The De Ritis ratio reflects the time course of acute viral
hepatitis and is generally a vital clue to the patient’s prognosis.
• It is an acquired benign condition, more frequent in men and
usually discovered incidentally in the fifth through seventh
decades.
• Macroamylase is a combination of an amylase attached to a
protein (either IgG or IgA); this enzyme-protein complex cannot
be excreted in the urine due to its “macro” size, hence, it remains
in the blood circulation.

• Severe viral or toxic hepatitis, De Ritis ratio (AST:ALT) is < 1.0. • Clinical findings: Persistent increased serum amylase w/o
symptoms; serum lipase is normal
Amylase / Alpha-1-4Glucohydrolase
Methods
• It catalyzes the breakdown of starch and glycogen.
• Samples with high activity of AMS should be diluted with NaCl
• It is an important enzyme in the physiologic digestion of starch. to prevent inactivation.

• It is a calcium-containing enzyme. • Elevated triglyceride may inhibit serum AMS activity.

• It is the smallest enzyme in size; thus, it is filtered by the renal
glomeruli and normally appears in the urine.
• It is the earliest pancreatic marker.
• The major isoenzymes are both present in normal, healthy sera.
• The administration of morphine and other opiates for pain
relief before blood sampling will lead to falsely elevated serum
AMS levels.
• Samples with citrate, oxalate, and heparin should be avoided as
it can inhibit AMS.

• Major isoenzymes: S-type (ptyalin) and P-type (amylopsin)
• Isoforms of salivary amylase: S1, S2, and S3
• Isoforms of pancreatic amylase: P1, P2, and P3 (predominant
isoform in acute pancreatitis)
• Major sources: Acinar cells of the pancreas and the salivary
glands
• Other sources: Fallopian tubes, small intestine, skeletal muscles,
and adipose tissue
• Reference value: 60 – 80 Somogyi unit (SU)/dL or 95 – 290
U/L
• Amylase is stable in serum and urine specimens for 7 days at
room temperature.
• Specimen precaution: Avoid contamination with saliva (false
increase of serum AMS)
• Substrate: Starch
 Saccharogenic
• It is the classic reference method expressed in Somogy unit
(SU).
• It measures the amount of reducing sugars produced by the
hydrolysis of starch by the usual glucose methods. Amyloclastic
• It measures amylase activity by following the decreases in
substrate concentration (degradation of starch).

• Conversion factor: 1.85 (Somogyi units to IU)  Chromogenic

• It determines amylase activity by the increase in color intensity
Diagnostic Significance of the soluble dye-substrate solution produced in the reaction.

• In acute pancreatitis (AP), AMS levels rise 2-12 hours after  Coupled-enzyme
onset of attack, peak at 24 hours, and normalize within 3-5 days. • It quantifies amylase activity by a continuous-monitoring
technique.
• Increased AMS blood levels are accompanied by increased
urinary excretion.

• AMS in urine remains elevated for up to 7 days.

• Normal Amylase/Creatinine ratio: 1%-4% (0.01-0.04)

• A:C ratio (AP) = > 4% (up to 15%)

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 Lipase / Triacylglycerol Acylhydrolase
• It is an enzyme that hydrolyzes the ester linkages of fats to
produce alcohol and fatty acid.
• It catalyzes partial hydrolysis of dietary TAG in the intestine to
the 2- monoglyceride intermediate, with the production of long
chain fatty acids.
• It is the most specific pancreatic marker. • Its primary and
major source is the pancreas and is not affected by renal
disorders.
• Isoenzymes: Pancreatic LPS (predominant in serum), intestinal
LPS, lipoprotein LPS, and gastric LPS
• Isoforms of LPS: L1, L2 (sensitive and specific marker), and L3
• Major tissue source: Pancreas
• Other sources: Stomach, liver, intestine, adipose tissue, breast
milk, and WBCs
• Reference range: 0 – 1.0 U/mL
Diagnostic Significance
• In AP, LPS levels rise 6 hours after onset of attack, peak at 24
hours, remains elevated for 7 days, and normalize in 8-14 days.
• Serum LPS may be elevated for 2 weeks.
• Persistent and prolonged elevations of serum lipase more than
2 weeks may indicate the presence of pancreatic cyst.
• In chronic pancreatitis, acinar cell degradation occurs, resulting
in loss of amylase and lipase production.
Methods
• In the traditional method, olive oil is a substrate because other
esterases can hydrolyze TAG and synthetic diglycerides.
• Addition of colipase (protein secreted by the pancreas) and bile
salts will make assay more sensitive and specific for the
detection of acute pancreatitis.
• Calcium is an important activator for lipase.
• The enzymatic reaction is at optimum pH 8.8.
• Icterus, lipemia, and hemolysis do not interfere with
turbidimetric lipase assays.
• Reference method: Cherry-Crandall
• It involves hydrolysis of olive oil after incubation for 24 hours
at 37°C and titration of fatty acids using NaOH.
• Substrate: 50% Olive oil/Triolein
• End product: Fatty acid
• Reference range: 0 – 1.5 U/mL (0 – 417 U/L)
• Conversion factor: 278, from U/mL to U/L)
Other Methods
 Tietz and Fiereck
• Substrate: Olive oil (Triglyceride)
• pH of the buffer: 8.0
• Incubation: 3 hours
• End product: Fatty acid
 Peroxidase coupling
• It is currently the most commonly used method.
• Substrate: Triolein (pure form of TAG)
• End product: Fatty acid
Lactate Dehydrogenase (LD)
• It catalyzes the interconversion of lactic and pyruvic acids.
• It is a zinc-containing enzyme that is part of the glycolytic
pathway and Krebs cycle.
• It is a hydrogen-transfer enzyme that uses the coenzyme
nicotinamide dinucleotide (NAD).
• It is a tetrameric molecule containing four subunits of two
possible forms (H and M).
• It is present in almost all cells in the body.
• In plasma, the majority of LD activity originates from the
breakdown of erythrocytes and platelets, with varying
contributions from other organ sources.
• The heart and red blood cells are the predominant sources of
LD.
• Tissue sources: Heart, erythrocytes, kidneys, lungs, pancreas,
spleen (LD-3); skeletal muscles, liver, intestine (LD-4 and LD-5).
• Reference range: 100 – 225 U/L (Forward reaction)
80 – 280 U/L (Reverse reaction)
LD Tissue Sources
• LD-1 is relatively abundant in cardiac muscles, whereas LD-5 is
more abundant in skeletal muscle.
• LD-1 is not found in the skeletal muscles and liver.
• LD-2 is the major isoenzyme in the sera of healthy persons.
• RBCs and cardiac tissues contain high levels of LD-1 and LD-2.
• LD-3 and LD-4 are also present in in minimal activity in the
heart and erythrocytes.
• LD-4 is the most abundant isoenzyme in the skeletal muscle.
• LD-5 is most abundant isoenzyme in the liver but almost
undetectable in the heart, RBCs, and renal cortex.
• LD will be clinically significant if separated into isoenzyme
fractions.
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Diagnostic Significance  Direct immunoassay
• It is currently the most widely used method in the clinical
• Highest LD serum levels are seen in pernicious anemia and laboratory.
hemolytic disorders.

• In AMI, LD levels begin to rise within 12 to 24 hours, peak

levels within 48 to 72 hours, and remains elevated for 10 to 14
days.

• LD-1 > LD-2, also known as the “flipped pattern”, is seen in

myocardial infarction and hemolytic anemia.

• LD activity in pleural fluids is useful for differentiating

transudates (low LD) from exudates (high LD).

• Viral hepatitis and cirrhosis would give LD slightly increased

values (2 – 3x URL) – this elevation is temporary, usually the Notes to Remember!
plasma level will return to normal after onset of symptoms.
• Hemolyzed sample should be totally avoided due to very high
• Hepatic carcinoma and toxic hepatitis will have tenfold increase. levels of LD in the red blood cells.

• LD-5 is moderately increased in acute viral hepatitis and
cirrhosis and markedly increased in hepatic cancer and toxic
hepatitis.
• LD activity may be considered as a marker in occupational
hazards such as in silica-induced toxicity.
• Serum LD is higher than the plasma LD due to release of the
enzyme from platelets.
• LD activity in serum or plasma is temperature-dependent
(mostly LD-1 and LD-5), and it varies when LD is suspended at
room temperature or in cold storage.

• An elevated total LD is a non-specific result because of its • Decreased activity is observed when samples are frozen (LD-5
presence from several tissues. is cold-labile), therefore samples should be processed within 24
hours after collection and stored at 25°C.
LD as a Tumor Marker
• There is transient elevation of total LD after blood transfusion
• LD-2, LD-3, and LD-4 are cancer markers (predominantly LD-3) but plasma level returns to baseline within 24 hours.
for acute leukemia, germ cell tumors, and breast and lung
cancers. Increased LD
 Anemia (pernicious,  Hepatitis and hepatic
• Serum LD is increased in metastatic CA due to its multiple hemolytic, and cancer
organ sources. megaloblastic)  Muscular dystrophy
 Myocardial infarction  Delerium tremens
• Serum LD level is a prognostic CA marker as it may determine  Leukemia  Malignancy
response to therapy.  Renal infarction  Pneumocystis jerovecii
 pneumonia
• In patients who have undergone radiotherapy for prostate
cancer, elevated LD-5 levels have been found to be strongly Creatine Kinase (CK)
indicative of high tumor proliferation rates and relapse of
following radiotherapy. • It catalyzes the transfer of a phosphate group between creatine
phosphate and adenosine phosphate.
Other LD Isoforms
• It is involved in the storage of high-energy creatine phosphate
• LD-6 represents the alcohol dehydrogenase enzyme; the sixth in the muscles.
band in electrophoresis; responsible for the metabolic
conversion of methanol and ethylene glycol to toxic compounds; • It is a dimeric molecule with small molecular size, composed of
present in patients with arteriosclerotic failure; may be elevated a pair of two different monomers called M and B.
in drug hepatoxicity and obstructive jaundice.
• It is found in small amounts throughout the body, but high
• LD composed of four C subunits is detected in spermatozoa and concentrations are in the muscles and brain, although CK from
in semen; not found yet in human serum or even in individuals brain tissue never crosses the blood-brain barrier to reach
with seminoma (malignant germ cell tumor of testicle). normal plasma.

Methods
• Lactate is a more specific substrate compared to pyruvate.
• LD-1 prefers the forward reaction, whereas LD-5 prefers the
reverse reaction.
• LD is stable at room temperature for 48 hours and at least 6
days on storage at 4°C.
• Major tissue sources: Brain, smooth and skeletal muscles, and
cardiac muscles
• Reference range: Male = 15 – 160 U/L
Female = 15 – 130 U/L CK-MB = < 6% of total CK
Isoenzymes: CK-BB (brain type), CK-MB (hybrid type), and
CK-MM (muscle type)

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• CK-1 is the most anodal and labile isoenzyme; CK-3 is the least Methods
anodal.

• CK-BB is the dominant isoenzyme of CK found in brain,

intestine, and smooth muscle.

• Serum of adults rarely contains CK-BB of brain origin due to its

high molecular size; it may be normally present in neonatal sera.

• Cardiac tissues contain significant amounts of CK-MB (20%) –

myocardium is the only tissue from which CK-MB enters the
serum in significant quantities.

• CK-MB in serum of healthy person is

Atypical Isoforms of CK:

1. Intramitochondrial CK  CK Relative Index (CKI)

• It is also known as the MtCK or non-muscle/ubiquitous MtCK
(uMtCK).
• Elevated serum MtCK is seen in cirrhosis or hepatitis.
• It may be reliable independent predictor of development of
hepatocellular carcinoma.
• It is an expression of the percentage of the total CK that is
attributed to CK-MB.
• This is computed to know possible release of CK-MB from
noncardiac tissues when total CK is very high.

2. Sarcomeric Muscle CK
• It supports the myofibrillar structure and contractility.

Diagnostic Significance Aldolase (ALDO)

• Highest elevation of CK is seen in Duchenne’s muscular
dystrophy (50x URL).
• CK-BB is increased in cerebrovascular injury.
• CK-MB is found mainly in myocardial tissue – it is used as a
serodiagnostic test for AMI.
• Demonstration of elevated levels of CK-MB, ≥ 6% of the total CK,
is considered the most specific indicator of myocardial damage,
particularly AMI.
• Following AMI, the CK-MB levels begin to rise within 4 to 8
hours, peak at 12 to 24 hours, and normalize within 48 to 72
hours.
• Total CK is markedly elevated after trauma to skeletal muscle
from crush injury, convulsions, tetany, surgical incision, or
intramuscular injections.
• CK is a diagnostic test for neuroleptic malignant syndrome.
• Only CK is located in the smooth muscles; not ALT, AST, and LD.
• CK-MB is not elevated in angina, and it has been replaced by
cardiac troponins in the assessment of AMI.
• CK-MB though increased in AMI within 4 hours of chest pain,
had low sensitivity and specificity as compared to glycogen
phosphorylase isoenzyme BB (GPBB) and myoglobin.
• It is a glycolytic enzyme that splits fructose 1,6-diphosphate
into two triose phosphate molecules in the metabolism of
glucose.
• It is included in the panel of markers for skeletal muscle injury.
• Increased: Skeletal muscle disease, leukemia, hemolytic anemia,
and hepatic cancer
Other Clinically Significant Enzymes
Gamma-Glutamyl Transferase (GGT)
• It catalyzes the transfer of glutamyl groups between peptides
or amino acids through linkage at a gamma-carboxyl group.
• It is located in the canaliculi of the hepatic cells and particularly
in the epithelial cells lining the biliary duct, also in the kidney,
prostate, and pancreas.
• In the canalicular system in the liver, it is located on the hepatic
surface while the ALP is on the canalicular surface; hence, it is
useful in differentiating the source of an increased ALP level.
• It is elevated among individuals undergoing warfarin,
phenobarbital, and phenytoin therapies.
• It is elevated in all hepatobiliary disorders such as biliary tract
obstructions.

Increased Total CK • It is the most sensitive marker of acute alcoholic hepatitis.

 Duchenne’s muscular  Rhabdomyolysis
dystrophy  Carbon monoxide • It is a sensitive indicator of alcoholism (occult alcoholism);
 Myocardial infarction poisoning however, it is often elevated in alcoholics even without liver
 Hypothyroidism  Rocky mountain spotted disease.
 Pulmonary infarction fever
 Reye’s syndrome  Cerebral vascular • It is useful in monitoring the effects of abstention from alcohol.
acccident (occasional)
• It is also increased in pancreatitis and prostatic disorders.
Page 10 of 12
• Increased: Obstructive jaundice, alcoholism, and diabetes
mellitus
• Tests for obstructive jaundice: ALP, GGT, 5’NT, and LAP
• Substrate: Gamma-glutamyl-p-nitroanilide
• Methods: Szass, Rosalki, and Tarrow; Orlowski
• Other methods: Electrochemical
• Reference range: 5 – 40 U/L or 8 – 61 U/L
5’ Nucleotidase (5’ NT)
• It is a phosphoric monoester hydrolase; predominantly
secreted from the liver.
• It is a differential test for serum ALP, to detect the source of the
elevated ALP, if liver or bone.
• It is a marker for hepatobiliary disease and infiltrative lesions
of the liver.
• It is routinely increased in cholestatic disorder.
• Substrate: 5’-monophosphate (5’-IMP)
• Common method: Quantitative Enzymatic
• Other methods: Dixon and Purdon, Campbell, Belfield and
Goldberg
Pseudocholinesterase (PChE)
• It is also known as the butyrylcholinesterase (BuChe) or
acetycholine acylhydrolase.
• It is secreted by the liver, hence, measurement of this enzyme
reflects synthetic function rather than hepatocyte injury; a
secondary liver function test.
• It is a marker of insecticide/pesticide poisoning
(organophosphate poisoning).
• It also determines high exposure to nerve poisons such as sarin
(methyl isopropyl fluorophosphate).
• It is used to monitor the effect of muscle relaxants
(succinylcholine) after surgery; pre-operative screening for
patients with a history or family history of prolonged paralysis
and apnea after use of the muscle relaxant, succinylcholine, for
anesthesia.
• It catalyzes the removal of benzyl group from cocaine by acting
as an “antixenobiotic enzyme”.
• It is involved in the metabolism of anticholinergic drugs.
• It is readily available in plasma compared to
acetylcholinesterase.
• Tissue source: Liver, heart, pancreas, and CNS (white matter of
the brain)
• Serum level in poisoning: Low PChE
• Decreased: Malnutrition and liver disease (acute hepatitis,
cirrhosis, and carcinoma metastatic to the liver)
• Substrate: Butyrylcholine
• Methods: Ellman technique and potentiometric
Angiotensin - Converting Enzyme
• It is also known as peptidyl-dipeptidase A or kininase II.
• It is an aspartic acid protease.
• It is a hydrolase enzyme that requires zinc for activation.
• It is found primarily in the vascular endothelium of the lungs
and kidneys.
• It is a component of the renin-angiotensin system.
• It converts the inactive angiotensin I to its active form, the
angiotensin II, within the lungs.
• It is a diagnostic test for sarcoidosis.
• It is a possible indicator of neuronal dysfunction (Alzheimer’s
disease using CSF sample).
• It is a critical target for inhibitory drugs designed to lower
blood pressure.
• It normally has low concentration in plasma, since it is mostly
cellbound.
• ACE2 is the cellular receptor of SARS and SARS-CoV-2.
• Forms of ACE: somatic (sACE – present in many tissues) and
testes (tACE)
• Main function: To cleave histidine-leucine sequence from a
decapeptide called angiotensin I.
• Major tissue sources: Lungs and Kidneys
• Other sources: Heart, muscles, neurons, macrophages, and
epitheloid cells
• Diagnostic significance: Diagnosing and monitoring of
sarcoidosis
• Increased: Sarcoidosis, multiple sclerosis, pneumonia, acute
and chronic bronchitis, leprosy, HIV infection, and Addison’s
disease
• Sample: Serum
• Substrate: Furylacryloylphenylalanine-glycylglycine (FAPGG)
• Method: Spectrophotometric assay
Glucose-6-Phosphate Dehydrogenase
• It functions to maintain NADPH in the reduced form in the
erythrocytes.
• It is a newborn screening marker.
• Deficiency of this enzyme can lead to drug-induced hemolytic
anemia after taking primaquine, an antimalarial drug.
• Tissue sources: RBCs, spleen, lymph nodes, and adrenal cortex
• Increased: Myocardial infarction and megaloblastic anemia
Page 11 of 12
• Specimen: Red cell hemolysate and serum ACUTE MYOCARDIAL INFARCTION / CARDIAC PROFILE
CARDIAC ELEVATION PEAK NORMALIZE
Ceruloplasmin MARKER
Myoglobin 1 - 4 hours 6 - 9 hours 18 - 24 hours
• It is a copper-carrying protein with an enzymatic activity. Trop I 4 - 6 hours 12 - 18 hours 6 days
CK - MB 4 - 8 hours 12 - 24 hours 3 days
• It is a marker of Wilson’s disease (decreased ceruloplasmin Trop T 4 - 10 hours 2 days 7 - 10 days
level). AST 6 - 8 hours 24 hours 4 - 5 days
LD - Total 10 - 24 hours 48 - 72 hours 10 days
• Sample: Serum

• Methods: Immunoassay and Copper oxidase activity

Ornithine Carbamoyltransferase

• It is a marker for hepatobiliary diseases.

• Its serum concentration may provide a useful marker of disease

severity, and thus could be a useful marker for a high risk of
hepatitis C occurrence.

• Sample: Serum

• Serum level in hepatobiliary disease and Hepatitis C: Increased

Trypsin

• It is produced in secretory granules of the pancreatic acinar cell.

• It is a major enzyme that serves as an activator of digestive

enzymes.

• It is the inactive trypsinogen form in acinar cells.

• It is only secreted in the pancreas, hence a more specific

marker of acute pancreatitis compared to routine amylase test.

• Sample: Serum or Tissue

• Measurement: Immunoassay and Immunohistochemical

staining (tissue)

Enzyme Classification Example

oxidoreductase LDH
transferase AST, ALT, CK
hydrolase ACP, ALP, CHS, LPS, AMS
lyases aldolase
isomerase glucose PO4 - isomerase
ligases synthase

Tissue Specificity of Enzymes

Enzyme Principal
Tissue(s)
Acid phosphatase RBCs, prostate
Alanine Liver
High Specificity aminotransferase
Amylase Pancrease, salivary
gland
Lipase Pancreas
Aspartate Liver, heart,
Moderate aminotransferase skeletal muscle
Specificity Creatine kinase Heart, skeletal
muscle, brain
Alkaline phosphate Liver, bone, kidney
Low Specificity Lactate All tissues
dehydrogenase

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