ENZYMOLOGY

CARDIAC MARKERS
PANCREATIC MARKERS
PROSTATE MARKERS
CARDIAC MARKERS
• CK-MB

• AST

• LD (LDH) 1 & 2

• LD/HBD ratio

• MI Biomarkers
Creatinine Kinase
• EC 2.7.3.2
• Adenosine triphosphate: creatine N-
phosphotransferase (CK).

• Is a dimeric enzyme (82 kDa) that catalyzes the

reversible phosphorylation of creatine (Cr) by
adenosine triphosphate (ATP).
• Physiologically, when muscle contracts, ATP is
converted to adenosine diphosphate (ADP), and
CK catalyzes the rephosphorylation of ADP to
ATP using creatine phosphate (CrP) as the
phosphorylation reservoir.

• Mg2+ is an obligate activating ion that forms

complexes with ATP and ADP.

• Inhibited by excess ADP and by citrate, fluoride,

nitrate, acetate, iodide, bromide, malonate, and
l-thyroxine.
• CK is a dimer composed of two subunits, each
with a molecular weight of about 40,000 Da.
These subunits (B and M) are the products of
loci on chromosomes 14 and 19, respectively.
Because the active form of the enzyme is a
dimer, only three different pairs of subunits can
exist: BB (or CK-1), MB (or CK-2), and MM (or
CK-3).
• The enzyme in serum is relatively unstable,
activity being lost as a result of sulfhydryl group
oxidation at the active site of the enzyme.

• Activity can be partially restored by incubating

the enzyme preparation with sulfhydryl
compounds, such as N-acetylcysteine,
dithiothreitol (Cleland reagent), and
glutathione.
• CK activity is greatest in striated muscle and
heart tissue, which contain some 2500 and 550
U/g of protein, respectively.

• Other tissues, such as the brain, the

gastrointestinal tract, and the urinary bladder,
contain significantly less activity, and the liver
and erythrocytes are essentially devoid of
activity
Clinical Significance
• Serum CK is increased in nearly all patients
when injury, inflammation, or necrosis of
skeletal or heart muscle occurs.
• Elevation of serum CK activity may be the only
sign of subclinical neuromuscular disorders.
Increase Creatinine Kinase
• Duchenne’s muscular dystrophy
• Myocardial Infarction
• Hypothyroidism
• Pulmonary infarction
• Reye’s syndrome
• Strenuous exercise and intramuscular injections
• Cerebral vascular accident
• Rocky mountain spotted fever
• Carbon monoxidepoisoning
• Has 3 isoenzymes:
▫ CK-BB (CK1)= Brain
▫ CK-MB (CK2)= Heart, muscle
▫ CK-MM (CK3)= Muscle, heart

▫ Cardiac muscle – CK-MM and CK-MB.

▫ Skeletal muscle – CK-MM
▫ Brain, GI, colon, prostate, uterus = CK=BB
• Trauma to skeletal muscle causes increase in
total CK and MB isoenzyme, but % activity of
MB is <3% (>6% in MI)
 Catalyzes CK
phosphocreatine + ADP creatine + ATP

 Methods:
 Tanzer-Gilvarg: (forward)

 Phospho Kinase + LD Lactate + NAD

 Oliver- Rosalki: (reverse)

 Hexokinase + G6PD 6-P Gluconate + NADPH

Aspartate Aminotransferase
• It is commonly referred to as a transaminase
and is involved in the transfer of an amino group
between aspartate and a-keto acids.

• The older terminology, serum glutamic-

oxaloacetic transaminase (SGOT, or GOT), may
also be used.

• Pyridoxal phosphate functions as a coenzyme

Tissue Source
• AST is widely distributed in human tissue.
• The highest concentrations are found in cardiac
tissue, liver, and skeletal muscle.
• With smaller amounts found in the kidney,
pancreas, and erythrocytes.
Diagnostic Significance
• The clinical use of AST is limited mainly to the
evaluation of hepatocellular disorders and
skeletal muscle involvement.

• In AMI, AST levels begin to rise within 6 to 8

hours, peak at 24 hours, and generally return to
normal within 5 days. However, because of the
wide tissue distribution, AST levels are not
useful in the diagnosis of AMI.
AST Isoenzyme
• AST exists as two isoenzyme fractions located in
the cell cytoplasm and mitochondria.

• Cytoplasmic AST – predominant in serum.

• Mitochondria – cellular necrosis.
Assay for Enzyme Activity
• Karmen method - which incorporates a coupled
enzymatic reaction using malate dehydrogenase
(MD) as the indicator reaction and monitors the
change in absorbance at 340 nm continuously as
NADH is oxidized to NAD.

• The optimal pH is 7.3 to 7.8.

Source of Error
• Hemolysis should be avoided because it can
dramatically increase serum AST concentration.

• AST activity is stable in serum for 3 to 4 days at

refrigerated temperatures.

• Reference Range AST, 5 to 30 U/L (37°C)

Lactate DeHydrogenase 1 & 2
• LD 1 (Anodic), while LD 5 is (cathodic)

• LD 1 and LD2 (HEAT STABLE)

• LD5 (COLD LABILE)

• Used in evaluating Cardiac Disorders

• Normally LD1< LD2
• Normal Ratio is 0.5-0.75 or <1
• If LD1>LD2 it is called a flipped ratio
▫ Flipped ratio is seen in MI (provided sample
is not hemolyzed)
▫ 50% MI: flipped ratio in 48hours
▫ 80% MI: flipped ratio in 72 hours
 • Catalyzes:
reversible lactate pyruvate
using NAD+ as a cofactor.

Forward method: Wacker (pH 8.8)

Lactate → Pyruvate

Reverse method: Wrobleuski La Due (pH 7.2)

Pyruvate → Lactate
•
CLINICAL SIGNIFICANCE
• Increase in:
▫ Anemia (pernicious, hemolytic, megaloblastic)
▫ Myocardial infarction (MI)
▫ Muscle trauma
▫ Renal infarct
 • 4 SUBUNITS in each Isoenzyme
HIGH
SHORTHAND
ISOENZYME SUBUNITS CONCENTRATI
SUBUNITS
ON
Heart, RBC,
LD1 HHHH LD H4
Brain

Heart, RBC,
LD2 HHHM LD H3M
Brain

Brain, Kidney,
LD3 HHMM LD H2M2
Lung

Liver, Skeletal
LD4 HMMM LD HM3
muscle, kidney

Liver, Skeletal
LD5 MMMM LD M4
muscle, ileum
NON-ENZYMATIC PROTEIN MARKERS
• MYOGLOBIN
▫ Major protein responsible for oxygen supply of
striated muscle

• TROPONIN
▫ the troponin complex is a component of the thin
filament of striated muscle linked to actin
▫ Three Subunits:
 Troponin I: an inhibitory subunit
 Troponin T: tropomyosin-binding subunit
 Troponin C: Calcium-binding subunit
 MYOGLO TROP T TROP I CK-MB AST LD
BIN

Elevation 2-4 hours 3-4 hours 3-12 4-8 hours 6-8 hours 8-10 hours
after hours
chest
pain (MI)
Peak 6-10 hours 48 hours 12-24 12 - 24 24-48 72 hours
activity hours hours hours
Duration 2-5 days 2-5 days 5-10 days 2-3 days 4 days 10 days
of
elevation
Sensitivit Sensitive More More Not Not Insensitive
y but not sensitive sensitive entirely sensitive, ,
specific and and specific for not Nonspecifi
specific specific AMI specific c
than CK- than CK-
MB MB
Usefulne Negative Eliminates Eliminates Gold Detect Detect
ss predictabl need for need for standard infarction infarction
e marker LD LD > 3 days > 5 days
isoenzyme isoenzyme prior to prior to
PANCREATIC MARKERS
Are use for the investigation of pancreatic
disease, more specifically acute pancreatitis.

Most commonly used serum biomarkers:

Amylase
Lipase
Trypsin
AMYLASE

• EC 3.2.1.1
• 1,4-α-d glucan glucanohydrolase (AMY)
• is an enzyme of the hydrolase class
that catalyzes the hydrolysis of 1,4-α-
glucosidic linkages in polysaccharides.
• AMYs are calcium metalloenzymes, with the
calcium essential for functional integrity.

• AMY in human serum has a moderately sharp

pH optimum at 6.9 to 7.0.

• AMY is present in a number of organs and

tissues the greatest concentration is noted in the
salivary glands, which secrete a potent AMY (S-
type) to initiate hydrolysis of starches while the
food is still in the mouth and esophagus.
• AMYs normally occurring in human plasma are
small molecules with molecular weights varying
from 54,000 to 62,000 Da.

• The enzyme is thus small enough to pass

through the glomeruli of the kidneys, and AMY
is the only plasma enzyme normally found in
urine
Amylase
• found in the SALIVARY GLANDS and
PANCREAS
• Breaks down starch to simple sugars
• Substrate: starch

starch/ amylum AMS maltose Maltase glucose

• Substrates:
▫ Pancreatic AMS: diastase
▫ Salivary AMS: ptyalin

• Serum AMS is usually pancreatic in origin

▫ microAMS: unbound, free. 50,000 dal. AMS
found in urine
▫ macroAMS: bound to IgG, IgA. High MW.
Measured in serum
 • Methods

Measures amount of
Saccharogenic maltose produced
(glucose: Somogyi)
Measures starch
Iodometric/amyloclastic
remaining
Measures dye released
Chromogenic from breakdown of
polysaccharide
Measures change of NAD
Kinetic Method
to NADH at 340nm
• With the exception of heparin, all
common anticoagulants inhibit AMY
activity because they chelate Ca2+.

• AMY is quite stable; activity is fully

retained during storage for 4 days at
room temperature, for 2 weeks at −4
°C, for 1 year at −25 °C, and for 5
years at −75 °C.
• Reference Interval using the IFCC recommended
method at 37 °C, the serum reference interval
was 31 to 107 U/L.
 LIPASE
• Human pancreatic lipase

• EC3.1.1.3; triacylglycerol acylhydrolase (LPS)

• The LPS gene resides on chromosome 10.

• LPS concentration in the pancreas is about

5000-fold greater than in other tissues.
• For full catalytic activity and greatest specificity,
the presence of bile salts and a cofactor called
colipase.

• Lipases are defined as enzymes that hydrolyze

glycerol esters of long-chain fatty acids.
(Breaks down Triglyceride into fatty acids and
glycerol)
Clinical Significance
• LPS measurement of serum is used to diagnose
acute pancreatitis.
• The clinical sensitivity and specificity is 80 to
100%.
• After an attack of acute pancreatitis, serum LPS
activity increases within 4 to 8 hours, peaks at
about 24 hours, and decreases within 7 to 14
days.
• Elevations between 2 and 50 times the upper
reference limit have been reported.
LPS ASSAY

• Substrate: Olive Oil

• End Product: Fatty Acids
• Methods:
▫ Cherry-Crandall
▫ Sigma-Tietz
▫ Titration
 TRYPSIN
• EC 3.4.21.4 (TRY)
• Pancreas specific serine protease characterized
by the presence at the active site of serine and
histidine.
• TRY hydrolyzes peptide bonds formed by the
carboxyl groups of lysine or arginine with other
amino acids, although esters and amides
involving these amino acids are actually split
more rapidly than peptide bonds.
• The acinar cells of the pancreas synthesize 2
major trypsins (1 and 2) in the form of the
inactive proenzymes (or zymogens) trypsinogens
-1 and -2.

• The two trypsinogens represent approximately

19% of the total protein in pancreatic juice;
normally, the pancreas secretes trypsinogen-1 at
about two fold to fourfold the concentration of
trypsinogen-2, but in pancreatic disease, the
ratio of trypsinogen-1 and -2 is reversed.
• TRY-1 is also described as cationic and TRY-2 as
anionic because of their differing electrophoretic
mobility.
• In healthy individuals, free trypsinogen-1
is the major form found in serum.
After an attack of acute pancreatitis,
serum TRY-1 rises in parallel with
serum AMY activity to peak values
ranging from 2 to 400 times the
upper reference limit.
• Serum trypsinogen-2 increases more than
trypsinogen-1 in acute pancreatitis, the
concentrations of the former being on average
about 10-fold those of the latter.

• Urinary trypsinogen-2 measurement has shown

high sensitivity and negative predictive value for
the diagnosis of acute pancreatitis.
PROSTATIC CANCER PROFILE
• ACP

• PSA
ACP (pACp)
• Optimum pH: 5

• Very Labile, Add 5M acetate buffer or citrate

tablet to preserve

• ACP are found in Prostate, RBC, bone, liver,

kidneys, platelets

• Substrate: organic Phosphate such as

β-glyceroPO4 and ρ-nitrophenylPO4
• Method
▫ Chemical Inhibition Test

 If Total ACP is normal: Stop test

 If elevated: suggestive of prostatic CA
do p-ACP by Chemical
Inhibition Test

Cu++ Tartrate C4H4O6-2

RBC-ACP Inactivated (+) Unaffected (-)
p-ACP Unaffected (-) Inactivated (+)
PSA (Prostate-Specific Antigen)
• Member of the kallikrein family of serine
proteases uniquely produced form the epithelial
cells of the prostate gland
• Most useful TM for Prostate Cancer
• Drawback: weak in distinguishing prostate CA
from nonmalignant prostate lesions
• Reference range: 0-4ng/mL