MYOCARDIAL MARKERS
Key words: enzyme activity; cytoplasmic and mitochondrial enzymes; alanine
aminotransferase (ALT-EC 2.6.1.2.); aspartate aminotransferase (AST-EC 2.6.1.1.);
creatine kinase (CK-EC 2.7.3.2.); creatine, lactate dehydrogenase (LD – EC
1.1.1.27); myoglobin; troponin; isoenzymes, electrophoresis
Reagents:
1. Sample of human serum
2. Reagent set AST, BioSystems
a. Reagent A: Tris 121 mmol/l, L-aspartate 362 mmol/l, lactate dehydrogenase
>660U/l, sodium hydroxide 255 mmol/l, pH 7,8
b. Reagent B: NADH 1,3 mmol/l, 2-oxoglutarate 75 mmol/l, sodium
hydroxide
148 mmol/l
c. Working solution: mixture of reagent A and reagent B
3.
d.
e.
f.
Reagent set LD, BioSystems
Reagent A: N-methyl-D-glucamine 0,406 mmol/l, lactate 62,5 mmol/l, pH 9,4
+
Reagent B: NAD 50 mmol/l
Working solution: mixture of reagent A and reagent B
4. Reagent set CK 50, BioSystems
g. Reagent A:imidazol 125 mmol/l, EDTA 2 mmol/l, magnesium acetate 12,5
mmol/l, N-acetyl cysteine 25 mmol/l, hexokinase 6 000 U/l, NADP 2,4 mmol/l,
pH 6,
h. Reagent B: creatine phosphate 250 mmol/l, ADP 15 mmol/l, AMP 25 mmol/l,
P1,P5di(adenosine-5’-)pentaphosphate 102 mmol/l, glucose -6-phosphate
dehydrogenase
8 000U/l
c. Working solution: mixture of reagent A and reagent B
Diagnosis of muscular disorders (cardiac or skeletal muscle) is based on the clinical
examination, series of diagnostic procedures (electrocardiography for heart
disorders; electromyography, muscle biopsy or molecular genetic testing for skeletal
muscular disorders) and on the laboratory testing of several biochemical markers.
The diagnostic tests available to detect heart muscle damage are: myoglobin, lactate
dehydrogenase, creatin kinase (mainly isoenzyme CK-MB), aspartate
aminotransferase, troponin T and I.
Laboratory tests available to recognized acute myocardial infarction:
Acute chest pain could by associated with acute myocardial ischemia, angina
pectoris or pulmonary emboli. The biochemical diagnosis is based on the detection
of a panel of intracellular cytoplasmatic or mitochondrial proteins, which reached the
blood circulation after the cell injury. At the begining of the ischemia, the permeability
of cell surface membrane is increased, thus cytoplasmatic proteins/enzymes are
able to enter the blood circulation. If the arterial occlusion is not released, the
mitochondrial proteins/enzymes will escape from the necrotic cells.
The determination of enzymatic activity of aspartate transaminase belongs
to the standard blood tests. It has a cytoplasmatic as well as a mitochondrial
isoforme. In the serum, the activity of AST starts to increase within 4 to 6 hours after
the onset of the myocardial ischemia, a maximum reaches in 1-2 days, and it usually
persists for 5 days. The increased serum AST activity is not specific for the
myocardial injury; the higher AST activity is also associated with the liver disorders,
an injury of skeletal muscles or a hemolysis. Occasionaly we observe the high serum
alanine transaminase (ALT) activity. ALT is found in the highest concentration in
the cytoplasm of liver cells The highest concentration of ALT is found in the
cytoplasm of hepatocytes; to a lesser extent, it can be found in skeletal and cardiac
muscle. The presence of ALT in serum gives evidence about the severe damage of
hepatocytes as a result of the congestive heart failure during myocardial ischemia.
Creatine kinase (CK) is a cytoplasmic and mitochondrial enzyme. However
the course of CK is similar to AST, the CK rises earlier. CK exists as a dimmer
consisting of two subunits: B (brain) and M (muscle). These subunits are combined
to form three isoenzyme forms: CK-BB - predominantly in the brain, CK-MM –
predominantly in skeletal and cardiac muscles, CK-MB – highly specific for cardiac
muscle. Elevated CK level, especially CK-MB level, is of the most clinical
significance of acute myocardial infarct. After an acute infarction, total CK rises in 4
to 18 hours, maximum activity occurs in 24 hours. However we used to determine
the enzymatic activity of CK, CK-MB, but recently we can identify the CK-MB antigen
(so called CK-MBmass) by immunodetection. This test is much more sensitive, but
more expensive.
Lactate dehydrogenase (LD) is the less specific test for the damage
of cardial muscle. It is is a ubiquitous cytoplasmic enzyme found in nearly all cells of
the body. LD exists as a tetramer arisen as a combination of two subunits: H (heart)
and M (muscle). Combinations of these subunits can result in any one of five
isoenzymes. These are LD-1 (H4), LD-2 (H3M), LD-3 (H2M2), LD-4 (HM3), LD-5
(M4). The LD isoenzymes can be characterized on the basis of their electrophoretic
mobility. Separation of LD isoenzymes may help to localize the tissue of origin
associated with an increased serum total LD activity. Isoenzymes that migrate the
fastest (LD-1) and the next fastest (LD-2) toward the anode are predominante in the
heart and erythrocytes. For the AIM is characteristic the increase of isoform H4
(LD1), while the increase of isoform M4 (LD5) is connected with the liver or skeletal
muscle disorders. The LD activity increases very slowly, and it usually backs to
normal in 10–14 days.
Myoglobin is the primary oxygen-carrying pigment of muscle tissue. It has the
advantage of responding very rapidly (maximum in 0,5-2 hours) to myocardial
ischemia, increasing and decreasing earlier than CK-MB or troponins (see below).
The big disadvantage is, that it lacks specificity; the elevated concentration of
Myoglobin is also associated with the muscle tissue, crush syndrome, compartment
syndrome, and an impairment of the glomerular filtration.
The most specific and sensitive markers of acute myocardial damage are
troponins: Troponin T (TnT) and Troponin I (TnI). They are comprised in the
troponin complex of both skeletal as well as in cardiac muscle tissue, although the
isoforms of troponins are different. Troponins are release in 2–4 hours (the cytosolic
pool of the myocytes) and persist for up to 7-10 days (due to prolonged degradation
of actin and myosin filaments).
According the WHO recommendation, the common cardiac markers are AST,
CK, CK-MB, CK-MB mass, troponins and myoglobin. Among the newer promising
markers the fatty acid-binding proteins, isoenzyme glycogenphosphorylase BB and
ischemia-modified albumin belong.
Fatty acid-binding proteins (FABP) are tissue specific intracellular molecules
of about 15 kD. They are a class of cytoplasmic proteins that bind long chain fatty
acid and play an important role in the intracellular utilization of fatty acids. Different
types of FABP have been detected and we recognize Heart FABP, Liver FABP and
Intestinal FABP, etc. Human cardiac muscle has high content of FABP (10-20 mol %
of cytoplasmic proteins). Heart FABP (HFABP) is a sensitive biomarker of
myocardial necrosis.
Laboratory tests available to recognized skeletal muscle injury
(rhabdomyolysis):
Rhambomyolysis is a grave, life threaten disorder. The skeletal muscle tissue
is breaks down rapidly. It is associated with increased plasmatic concentration of
myoglobin and with higher enzymatic activity of CK, AST, LD and aldolase in serum.
Warburg´s optical test
To determine the activity of enzymes (LD,ALT,AST etc.) is used Wartburg optical
test. The principle of this assay is based on the ability of reduced forms of
coenzymes NADH and NADPH to absorb light at wavelength 340nm whereas their
oxidized forms not. The rate of change in optical density at 340nm is proportionate to
studied enzyme activity. Enzymes that use different coenzymes (not NADH or
NADPH) could be analyzed in systems of coupled reactions where the final reaction
has NADH or NADPH as coenzyme.
(http://www.bmglabtech.com/images/apps/170-2.jpg)
1. Determination of creatine kinase activity
Principle:
Creatine kinase (CK) is a cytoplasmic and mitochondrial enzyme that
catalyzes both the formation of ATP and the reversible phosphorylation of creatine:
creatine
+
ATP

creatine phosphate
+
ADP
The catalytic concentration is determined from the rate of NADPH formation,
measured at 340 nm, by means of the hexokinase (HK) and glucose-6-phosphate
dehydrogenase (G6P-DH) coupled reactions:
Creatine phosphate + ADP→ Creatine + ATP
ATP + Glucose → ADP + Glucose -6-phosphate
Glucose-6-phosphate + NADP
+
→ 6-Phosphogluconate + NADPH + H
+
Procedure:
- Pipette reagents into a cuvette as described in table 3:
Table 3
Working reagent
Sample
1 ml
50µl
- Mix and insert the cuvette into photometr. Start the stopwatch.
- After 30 seconds, record initial absorbance (340 nm) and at 1 minute intervals thereafter for 3
minutes.
- Calculate the difference between consecutive absorbances, and the average absorbance
difference per minute (∆A/min).
Calculation:
The CK concentration in the sample is calculated using the following formula:
The molar absorbance  of NADPH at 340 nm is 6 300, the lightpath l is 1 cm, the total reaction
volume Vt is 1.05, the sample volume Vs is 0.05 and 1 U/l are 16.67 nkat/l. The following formulas
are deduced for the calculation of the catalytic concentration:
Table
Time
CKA340nm
30 sec
1 min
2 min
3 min
∆A/min
2. Determination of aspartate aminotransferase activity
Principle:
Aspartate aminotransferase (AST) catalyzes the transfer of an aminogroup
between amino- and oxoacids:
L-aspartate
+
2-oxoglutarate

oxaloacetate
+
L-glutamate
Procedure:
- Pipette reagents into a cuvette as described in table:
Temperature
Working reagent
Sample
37°C
30°C
1 ml
50µl
1ml
100µ
- Mix and insert the cuvette into photometr. Start the stopwatch.
- After 30 seconds, record initial absorbance (340 nm) and at 1 minute intervals
thereafter for 3 minutes.
- Calculate the difference between consecutive absorbances, and the average
absorbance difference per minute (∆A/min).
- Write down the measured results in table 2:
Table
Time
ASTA340nm
30 sec
1 min
2 min
3 min
∆A/min
Calculation:
The ASTconcentration in the sample is calculated using the following general
formula:
The molar absorbance  of NADPH at 340 nm is 6 300, the lightpath l is 1 cm, the
total reaction volume Vt is 1.1, the sample volume Vs is 0.1 and 1 U/l are 16.67
nkat/l. The following formulas are deduced for the calculation of the catalytic
concentration:
3. Determination of lactate dehydrogenase activity
Lactate dehydrogenase is an oxidoreductase enzyme, whose activity is
+
necessary for the regeneration of NAD and continued glycolysis in contracting
musle. In liver, LD catalyzes the conversion of lactate to pyruvate with simultaneous
+
reduction of NAD to NADH. The basic reaction can proceed in the forward or
reverse direction:
lactate + NAD
+
 pyruvate + NADH + H
+
The catalytic concentration is determined from the rate of NADH formation,
measured at 340 nm.
Procedure:
-
Pipette reagents into a cuvette as described in table 3:
Table 4
Working reagent
Sample
1 ml
20µl
- Mix and insert the cuvette into photometr. Start the stopwatch.
- After 30 seconds, record initial absorbance (340 nm) and at 1 minute intervals
thereafter for 3 minutes.
- Calculate the difference between consecutive absorbances, and the average
absorbance difference per minute (∆A/min).
Calculation:
The LD concentration in the sample is calculated using the following general formula:
The molar absorbance  of NADPH at 340 nm is 6 300, the lightpath l is 1 cm, the total reaction
volume Vt is 1.025, the sample volume Vs is 0.025 and 1 U/l are 0,0166 nkat/l. The following
formulas are deduced for the calculation of the catalytic concentration:
Table 5
Time
LDA340nm
30 sec
1 min
2 min
3 min
∆A/min
References values:
Marker
ALT
AST
CK
LD
U/l
29
25
Men
10 - 65
Women
7 - 55
83 - 143
µ kat/l
0.48
0.42
Men
Women
0.167 – 1.084 0.117 – 0.917
1.38 – 2.38
4.. Determination of AST, ALT and CK by using Reflotron system (Roche)
For rapid determination of enzyme activity the reflectance photometry technique is
used. The Reflotron is a microprocessor-controlled reflectance photometer which
measures the intensity of the reflected light from homogenous inner surface. The
reactions are performed on the reagent strips. The particular test steps take place within
the reagent strip: plasma separation and the actual chemical reaction for the
determination of analyte. Every reagent strip has on its reverse side a magnetic tape with
all test- and lot-specific data.
For this test the whole blood can be used in addition to serum and plasma. The
blood sample passes through the plasma separation layer that separates the corpuscular
components of the blood. The plasma flows through a reaction zone where the chemical
reaction occurs. The intensity of colour is measured by the optics of the instrument.
- Perform the determination of these enzymes in your fresh capillary blood or in patient
serum sample (32l. Technical instructions are described in the chapter -Lipids III.
- The result of the enzyme activity is displayed in U/l for 37°C. Calculate the activity in
kat/l by using this equation:
1U= 0,0166kat/l