2. BIOMARKER
Is a substance used as an indicator of a biologic state
It is characteristic and found only in tissue of interest
It is objectively measured
It is elevated as an indicator of normal biologic processes, pathogenic
processes, or pharmacologic responses to a therapeutic intervention
Detection of marker must enable intervention that prevent or minimize effects
of disease
3.
4.
5. CARDIAC MARKERS
Intracellular macromolecules (proteins) released from a heart
muscle when it is damaged as a result of myocardia infarction (MI).
Cardiac markers are biomarkers measured to evaluate heart
function.
They found in the blood.
They are normally present at all times, however, they are
significantly elevated during a damage of the heart muscle.
6. CRITERIA OF IDEAL CARDIAC MARKER
Good specificity: detects only cardiac damage
Optimal sensitivity and detectable while damage is reversible or preventable
Correlates with amount of injury and enables prognosis prediction
No single marker meets all needs
Cheap, rapidly measurable and easy to detect
7.
8. CARDIAC MARKERS
Aspartate aminotransferase (AST or GOT) 1954
Lactate dehydrogenase (LDH) 1955
Creatine kinase MB (CK-MB) 1960
CK Electrophoresis (1972); CK Immunoelectrophoresis (1975); CK mass (1985)
Myoglobin (Mb) 1975
Troponin I (TnI) 1989
Troponin T (TnT) 1992
B-type natriuretic peptide (BNP)
C-reactive protein (CRP)
Myeloperoxidase (MPO)
Ischemic modified albumin (IMA)
9. CLASSIFICATION ACCORDING TO USE
Obsolete markers:
• Aspartate transaminase
(AST or GOT)
• Lactate dehydrogenase
(LDH) and lactate
dehydrogenase
isoenzymes
Current markers and test
panels:
• Creatine kinase (CK)
and muscle-brain
creatine kinase (CK-
MB)
• Troponin T (TnT) and
Troponin I (TnI)
• Myoglobin (Mb)
Markers under
assessment with
potential for clinical use:
• CK-MB isoforms
• High sensitivity C-
reactive protein (hs-
CRP)
• B-type (formerly brain)
natriuretic peptide
(BNP)
10. CLASSIFICATION ACCORDING TO THE TYPE OF
CARDIAC PROBLEM
Myocardial injury markers:
Markers of myocardial necrosis: CK-MB, myoglobin and troponin
Markers of myocardial ischemia: IMA and H-FABP
Hemodynamic stress markers:
Natriuretic peptides
Inflammatory and prognostic markers:
hs-CRP
sCD40L and homocysteine
11.
12. WHAT IS THE AST TEST?
Aspartate transaminase (AST/ aspartate
aminotransferase / SGOT (serum glutamic
oxaloacetic transaminase), is a pyridoxal
phosphate (PLP)-
dependent transaminase enzyme.
AST catalyzes the reversible transfer of an α-
amino group between aspartate and glutamate
and, as such, is an important enzyme in amino
acid metabolism.
AST is found in the liver, heart, skeletal
muscle, kidneys, brain, and red blood cells, and
it is commonly measured clinically as a marker
for liver health.
13. Two isoenzymes are present in a wide variety of eukaryotes.
In humans:
GOT1/cAST, the cytosolic isoenzyme derives mainly
from red blood cells and heart.
GOT2/mAST, the mitochondrial isoenzyme is present
predominantly in liver.
These isoenzymes are thought to have evolved from a
common ancestral AST via gene duplication, and they share
a sequence homology of approximately 45%.
AST has also been found in a number of microorganisms,
including E. coli, H. mediterranei, and T. thermophilus.
In E. coli, the enzyme is encoded by the aspCgene and has
also been shown to exhibit the activity of an aromatic-
amino-acid transaminase
14. WHY IS THIS
TEST DONE?
The AST level is measured
to check the liver, kidneys,
heart, pancreas, muscles,
and red blood cells.
This test is also done to
check medical treatments
that may affect the liver.
15. Principle of the test
Since enzymes are present in very small quantity in
biological fluids and it is difficult to isolate them,
So, a convenient way to quantitate them is to
measure their catalytic activities and relate this
activity to concentration.
Serum is allowed to react with substrate and
product is OAA.
OAA reacts with 2, 3-dinitro-phenyl hydrazine
(DNPH) and gives brown colour and intensity of
this colour is measured at 550 nm.
The intensity of colour is directly proportional to
activity of SGPT, NaOH is added to stop the
reaction.
16. Principle 1
SGOT converts L-Aspartate and a
Ketoglutarate to Oxaloacetate and
Glutamate.
The Oxaloacetate formed reacts with
2,4, Dinitrophenyl hydrazine to
produce a hydrazone derivative,
which in an alkaline medium produces
a brown coloured complex whose
intensity is measured.
The reaction does not obey Beer's law
and hence a calibration curve is
plotted using a Pyruvate standard.
The activity of SGOT (AST) is read off
this calibration curve.
17.
18. OAA
• Mix well and allow to stand at R.T for 10 min. Measure the absorbances of the tubes 2-5
against tube 1(Blank).
• Plot a graph of the absorbances of tubes 2-5 on the 'Y' axis versus the corresponding
Enzyme activity on the 'X' axis.
21. WHAT DOES THE TEST RESULT MEAN?
Liver damage caused by:
Infection, such as viral hepatitis or mononucleosis
Gallbladder disease
Poisons, such as too much alcohol
Cancer
Muscle damage caused by:
A muscle disease
An injury, such as after a fall, auto accident, or seizure
Kidney, pancreas, heart, or liver disease or damage.
Taking a medicine that affects the test result.
No medical problems are known to cause an AST level that is lower than normal.
Sometimes the test result may be lower than normal but it does not mean there is a problem.
22. Clinical Significance
AST is associated with liver parenchymal cells.
The difference is that ALT is found predominantly in the liver, with clinically negligible quantities found in
the kidneys, heart, and skeletal muscle, while AST is found in the liver, heart, skeletal muscle, kidneys, brain,
RBC.
As a result, ALT is a more specific indicator of liver inflammation than AST, as AST may be elevated also in
diseases affecting other organs (myocardial infarction, acute pancreatitis, acute hemolytic anemia, severe
burns, acute renal disease, musculoskeletal diseases, and trauma).
AST was defined as a biochemical marker for the diagnosis of acute myocardial infarction in 1954. However,
the use of AST for such a diagnosis is now redundant and has been superseded by the cardiac troponins.
AST (SGOT) is commonly measured clinically as a part of diagnostic liver function tests, to determine liver
health.
Cant identify etiology of damage
Patients undergoing extended hemodialysis without supplemental vitamin B6 therapy may show low GPT in
serum.
23. Creatine Kinase
Found in high concentration in skeletal muscles,
myocardium and brain. Small amount is present in lung,
thyroid and adrenal glands
But not found at all in liver and kidney
Not present in RBC, so not affected by hemolysis
Normal serum level is 40-500 IU/L at body temperature
(55 to 170 units/L for men; 30 to 135 units/L for women;
68 to 580 units/L for newborns)
Cells that consume ATP rapidly (skeletal muscle,
brain, photoreceptor cells of the retina, hair cells of
the inner ear, spermatozoa and smooth muscle) PCr serves
as an energy reservoir
24.
25. Creatine Kinase
Clinically, creatine kinase is assayed in blood tests as a marker of
Myocardial infarction (heart attack),
Rhabdomyolysis (severe muscle breakdown),
Muscular dystrophy,
The autoimmune myositides and
Acute renal failure.
26. Creatine Kinase
A creatine kinase (CK) test may be used to detect inflammation of muscles
(myositis) or serious muscle damage and/or to diagnose rhabdomyolysis in case
of signs and symptoms (as muscle weakness, muscle aches, and dark urine).
The urine may be dark because of the presence of myoglobin, substance
released by damaged muscles that can be harmful to the kidneys.
CK may be checked alone or along with other blood chemistry tests such as
electrolytes, BUN or creatinine (KFT). A urine myoglobin may also be ordered.
A health practitioner may use a CK test to help detect muscle damage in these
cases, especially if someone is taking a drug such as a statin, using ethanol or
cocaine, or exposed to toxin that has been linked with potential muscle damage.
In those who have experienced physical trauma, a CK test may sometimes be
used to evaluate and monitor muscle damage.
27. Creatine Kinase
In the cells, the "cytosolic" CK enzymes consist of two
subunits, which can be either B (brain type) or M (muscle
type) or combination.
There are three different isoenzymes: CK-MM, CK-BB and
CK-MB.
MM-CK is expressed in sarcomeric muscle (skeletal and
cardiac muscle), MB-CK is expressed in cardiac muscle,
and BB-CK is expressed in smooth muscle and in most
non-muscle tissues
The genes for these subunits are located on
different chromosomes: B on 14q32 and M on 19q13.
In addition to those three cytosolic CK isoforms, there are
two mitochondrial creatine kinase isoenzymes,
the ubiquitous and sarcomeric form.
The functional entity of the latter two mitochondrial CK
isoforms is an octamer consisting of four dimers each
28. Creatine Kinase
Skeletal muscle expresses CK-MM (98%) and
low levels of CK-MB (2%).
Myocardium (heart muscle) expresses CK-MM
at 70-80% and CK-MB at 20–30%.
gene protein
CKB brain, BB-CK
CKBE
ectopic expression, BM-CK,
Cardiac
CKM muscle, MM-CK
CKMT1A, CKMT1B ubiquitous mtCK; or umtCK
CKMT2 sarcomeric mtCK; or smtCK
29.
30. PCr is not only an energy buffer
but also a cellular transport form
of energy between subcellular
sites of energy (ATP) production
(mitochondria and glycolysis)
and those of energy utilization
(ATPases)
31.
32. Isoenzymes
Higher levels of the three isoenzymes mean different things:
CK-MM generally rises if you have muscle damage in your heart, brain, or skeleton
after a crush injury, seizures, muscular dystrophy, muscle inflammation, or another
skeletal muscle disorder.
CK-MB generally rises after a heart attack, inflammation of the heart muscle, muscular
dystrophy, and other problems related to the heart.
CK-BB tends to rise if you have a brain injury, meningitis, abnormal cell growth, severe
shock, stroke, hypothermia, or restricted blood flow to the bowel.
The normal levels for the three isoenzymes of creatine kinase are:
CK-MM: 100% ; CK-MB: 0% ; CK-BB: 0%
34. Measurement
Creatine Kinase activity is determined by a coupled enzyme reaction resulting in the production
of NAD(P)H, measured at 340 nm, proportionate to the CK activity present in the sample.
In this reaction, phosphocreatine and ADP are converted to creatine and ATP.
The generated ATP is used by hexokinase to phosphorylate glucose resulting in glucose-6-
phosphate,
G-6-P is oxidized by NADP in the presence of glucose-6-phosphate dehydrogenase to produce
NADPH and 6-phospho-D-gluconate.
One unit of CK is the amount of enzyme that will transfer 1.0 μmole of phosphate from
phosphocreatine to ADP per minute at pH 6.0.
The results vary widely because of differing analytical methodology and also differences due to
age, sex, race, and level of physical activity
35.
36.
37.
38. Creatine
Kinase
Measurement
Elevation of CK is an indication of muscle damage (injury,
rhabdomyolysis, myocardial infarction, myositis and
myocarditis).
There is an inverse relationship in the serum levels of T3 and
CK in thyroid disease. In hypothyroid patients, with decrease
in serum T3 there is a significant increase in CK.
Isoenzyme determination has been used extensively as an
indication for myocardial damage in heart attacks.
Troponin measurement has largely replaced this in many
hospitals, although some centers still rely on CK-MB.
There is 50 per cent loss of serum CK activity after 6 hours at
RT and 24 hours at refrigerated temperature.
Hence, all determinations of serum CK activity should be
done on fresh blood samples.
39. Trends of CK during MI
Studies suggested that serum CK activity is a more sensitive
indicator in early stage of myocardial Ischaemia
After myocardial infarction, serum value is found to increase after
about 6 hours, reaches a peak level in 24 to 30 hours, and returns
to normal level in 2 to 4 days (usually in 72 hours)
40. Trends of SGOT during MI
Concentration of the enzyme is very high, in myocardium
In acute myocardial infarction, serum activity rises sharply
within the first 12 hours, with a peak level at 24 hours or over
and returns to normal within 3 to 5 days
41. Lactate
Dehydrogenase
(LDH)
Present in a wide variety of organisms, including plants and animals (sk. Muscle, liver, kidney, RBC).
Lactate dehydrogenases exist in four distinct enzyme classes.
Two of them are cytochrome c-dependent enzymes, each acting on either D-lactate or L-lactate. The other two are NAD(P)-
dependent enzymes, each acting on either D-lactate or L-lactate.
LDH catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of NADH and NAD+. It converts
pyruvate, the final product of glycolysis, to lactate when oxygen is absent or in short supply
LDH has five distinct isoenzymes LDH1, LDH2, LDH3, LDH4 and LDH5.
They can be separated by electrophoresis.
LDHI has more positive charge and fastest in electrophoretic mobility while LDH5 is the slowest
42. LDH is an oligomeric
(tetrameric) enzyme made up of
four polypeptide subunits.
Two types of subunits namely M
(for muscle) and H (for heart)
are produced by different
genes. M-subunit is basic while
H subunit is, acidic.
43. LDHl (H4) is predominantly found in heart muscle and is
inhibited by pyruvate – the substrate.
Hence, pyruvate is not converted to lactate in cardiac muscle
but is converted to acetyl CoA which enters citric acid cycle.
LDH5 (M4) is mostly present in skeletal muscle and the
inhibition of this enzyme by pyruvate is minimal, hence
pyruvate is converted to lactate.
Further, LDH5 has low Km (high affinity) while LDHl has high
Km (low affinity) for pyruvate.
The differential analytical activities of LDHl and LDH5 in heart
and skeletal muscle, respectively, are well suited for the
aerobic (presence of oxygen) and anaerobic (absence of
oxygen) conditions, prevailing in these tissues.
44. Measurement
Either colorimetric estimation by measuring
color of pyruvate-DNPH or by rate of reaction
assay for the change in the absorbance of
NADH
Heparinised plasma or serum can be used
All the keto acid react with DNPH to form
corresponding colored compound (hydrazine)
Serum + Substrate buffer (sodium pyruvate)
+NADH Lactate + NAD+
45. Tissue breakdown releases LDH, and therefore LDH can be
measured as a surrogate for tissue breakdown,
e.g. hemolysis.
Other disorders indicated by elevated LDH include cancer,
meningitis, encephalitis, acute pancreatitis, and HIV.
It can also be used as a marker of myocardial infarction.
Following a myocardial infarction, levels of LDH peak at 3–4
days and remain elevated for up to 10 days.
False positive because of hemolysis
Red blood cells are rich in LDH, hence avoid haemolysis.
Haemolysed samples should not be assayed.
46. Interpretation
70-240 U/L
MI – starts increasing at 6-12 hr and
reaches peak at 48 hour, decline to
baseline in 10 days (LDH1 and/or
LDH2)
Increases in muscular dystrophy and
in myoglobinuria
Hepatitis and tumors of liver
Pulmonary infarction increases LDH
but not CK
47. Trends of LDH during MI
Normal serum LDH activity ranges from 60 to 250 IU/L
S-LDH level > 1500 IU/L in acute myocardial infarction
In acute myocardial infarction, serum activity rises within 12 to
24 hours, attains peak at 48 hours (2 to 4 days) reaching about
1000 IU/L and then return gradually to normal from 8th to 14th
day
48. γ-GLUTAMYL TRANSPEPTIDASE (G-GTP)
Also called γ-Glutamyl transferase (γ-GT).
γ-Glutamyl transpeptidase catalyses the transfer of the γ-glutamyl group
from one peptide to another peptide or to an amino acid.
Normal value: Normal serum activity has been shown to be:
Men: 10 to 47 IU/L
Women: 7 to 30 IU/L
Site: Highest tissue activity of this enzyme is found in kidneys, but activity
is relatively high in liver, lungs, pancreas and prostate.
Some activity is present in intestinal mucosa, thyroid gland and spleen.
Normal heart contains very little γ-GT.
49.
50. Trends of Gamma-GT during MI
Several investigators recently have demonstrated
increases in serum γ-GT in acute myocardial infarction.
Increase serum activity is found to be late, peak activity
between 7th and 11th day and lasts as long as a month
51. Troponin
Troponin is a type of protein found in the cardiac muscles.
Troponin isn't normally found in the blood.
When heart muscles become damaged, troponin is sent into the bloodstream. As heart damage
increases, greater amounts of troponin are released in the blood.
Troponin-C is a Ca-
binding component.
Troponin-I is an
inhibitory component.
Troponin-T is a
tropomyosin-binding
component
52.
53. Troponins help in the contraction of muscles, which is calcium-dependent, and there is the
interaction of myosin and actin.
Troponin-T has no cross-reactivity with skeletal muscle, so this assay is 100 % specific for cardiac
disease.
There are two cardiac troponins :
Troponin-T.
Troponin-I.
These are highly specific for cardiac muscle injury.
Their role is just like CK-MB, But Troponins are more specific than CK-MB. Troponins will be
normal or slightly raised in non-cardiac muscle injuries.
Troponins are measured by:
Monoclonal antibody immunoassay.
Enzyme-linked immunoassay.
Monoclonal sandwich antibody qualitative technique.
54. The Pattern Of Troponin-T In Acute Myocardial
Infaction:
Raised even in the first 3 hours.
The peak level is 12 to 48 hours.
Remain elevated up to 10 to 14 days
55. The Pattern Of Troponin-I In Acute Myocardial
Infaction:
Raised in the first 4 to 8 hours.
Peal level is around 12 hours.
Remains elevated 7 to 10 days.
56. Troponin and
CK-MB
Difference:
Causative reason Troponin T CK-MB
Skeletal muscle
injury
Normal Raised
Brain / Lung
injury
Normal Raised
Cardiac injury Raised Raised
Duration Raised for a
longer time
raised for a
shorter time
Sensitivity More sensitive Less sensitive
57. Advantage of
Troponin
over CK-MB:
Troponins
CK-MB
Specificity
More specifically, for
muscle injury.
Normal in noncardiac
muscle injury
Increased in
skeletal muscle
injury, brain,
lung, and renal
failure
Increased
level
Increased early and
elevated longer than CK-
Sensitivity
More sensitive to cardiac
muscle injury than CK-MB
Less sensitive to
cardiac muscle
injury
Importance
More important for the
evaluation of chest pain
Less important
for the
of chest pain
58. Cardiac
Markers:
Marker Detectable
Peak
level
Return to
normal
Troponin-T 4 to 8 hours
12 to 48
hours
7 to 10
days
Troponin- I 4 to 6 hours 12 hours
3 to 10
days
CK-MB 4 to 8 hours
12 to 24
hours
72 to 96
hours
LDH 2 to 4 days 2-4 days
10 days
(8
th
-14
th
days)
Y-Glutamyl
transpeptidase
7-11
Days
Lasts
month
Editor's Notes
Optimum pH when lactate as substrate -10; Optimum pH when pyruvate as substrate -7;
