Vol.2, No.1, 70-77 (2010)
doi:10.4236/health.2010.21012
SciRes
Copyright © 2010 Openly accessible at http://www.scirp.org/journal/HEALTH/
Health
Troponin based studies in search of a biomarker for
cardiac arrest
Pasha Ghazal1,2, Kaneez Fatima Shad1, Nikhat Sidduiqui2
1Dr Panjwani centre for molecular medicine and drug design, ICCBS, University of Karachi, Karachi, Pakistan
2Neurochemistry Research Unit, Department of Biochemistry, University of Karachi, Karachi, Pakistan;
ftmshad@yahoo.com, ftmshad@gmail.com
Received 3 November, 2009; revised 13 December, 2009; accepted 14 December, 2009.
ABSTRACT
Cardiac arrest is shown to be a cause of a large
number of deaths not only in Pakistan but
around the globe. The prevalence of this dis-
ease demands identification of its etiology. The
science of proteomics can be used to identify
cardiac specific proteins. The subsequent over
expression or under expression of these pro-
teins can be utilized as targets not only for
therapeutical interventions but also for identi-
fying molecular signatures for Cardiac diseases.
In context of a number of studies which have
shown that the specificity of serum biomarkers
like troponin (cTnI and cTnT) are questionable
as they may also appear in serum in pathologi-
cal conditions other than cardiac dysfunction,
the search of a specific marker for cardiac arrest
becomes imperative. In this study protein pro-
filing of cardiac arrest patients was performed
after its quantification through Bradford assay.
SDS-PAGE and 2 DE techniques were used as to
characterize proteins. The samples of the pa-
tients prior to characterizing of proteins were
subjected to lipid and cardiac enzymes profiling.
The results of these investigations have shown
an increase in almost all of these parameters by
many folds from that of normal values. In addi-
tion to this the samples were found out to be
positive for troponin T which strongly confirms
the incidence of the cardiac arrest. The results
of SDS-PAGE exhibited the induction of three
proteins of 100 kDa, 97 kDa and of 66 kDa with
100 kDa as the most highly expressed protein. In
addition to that SDS-PAGE gels have shown the
down regulation of 45 kDa protein, again indi-
cating the changes as a result of cardiac arrest.
2DE gel patterns of cardiac arrest samples
demonstrated higher number of protein spots as
compare to control in the alkaline range, which
might suggest their role in cardiac dysfunction.
Therefore it can be concluded that this study
may pave the grounds for identification of such
proteins which can serve not only as potential
therapeutical targets but also as candidate
markers for accurate diagnosis of the disease.
Keywords: Cardiac arrest; Troponins; Proteomics,
SDS-PAGE; 2DE; Therapeutical Targets.
1. INTRODUCTION
Cardiovascular diseases(CVD) have been identified as
being the single most significant cause of morbidity [1]
and global mortality, accounting for almost 17 million
deaths annually i.e. 30% of global mortality, moreover
survival rates from cardiac arrest is less than 1% [2]. This
situation is prevalent not only in the developing countries
but in the western industrialized part of the world as well.
The statistical analysis is in evidence to this, in United
States of the 2 400 000 US deaths in 1999, 720 000 (30%)
were directly attributed to cardiac diseases. Of this
number, the US Centers for Disease Control and Preven-
tion estimated that 462 000, or 64% of the subtotal, were
sudden cardiac deaths (SCDs) [3]. In the context of
phenomenal advances in the physical and biological
sciences, the limited number of viable therapeutic targets
and effective cardiovascular therapies are a source of
sheer surprise [4]. In addition to that, molecular causes
underlying cardiac dysfunction in most heart diseases are
unknown and expected to result from causal alterations in
gene and protein expression [5]. Increasing body of evi-
dence suggest that examining changes in the protein ex-
pressions arising due to intrinsic and extrinsic perturba-
tions [4], offer insight into the understanding of cellular
and molecular mechanisms that cannot be obtained from
genomic analysis[6]. Proteins are considered to be the
central executors of all life programs [7]. Up till now only
150 proteins have been identified from human heart tissue
using 2D-gel electrophoresis and sequencing [8]. There-
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71
fore in context of this situation, the emerging global
trends of CVD demands identification of upstream and
downstream effectors forming the basis of the disease
which could also serve as targets for therapeutical inter-
vention. The myofilament proteins, including Troponin T
are responsible for the contractile nature of the cardio-
myocytes. These proteins are highly regulated by a
number of specific post translational modifications
(PTMs), some of which have been discovered through
proteomic studies [9]. These altered PTMs lead to heart
failure and ischemia. Proteomic analysis facilitates in
comprehending the pathophysiology of the diseases in
non-biased manner and also provides oppourtunity for the
development of a suite of candidate biomarkers for the
diagnosis, staging and tracking of disease .Still, the
number of useful cardiovascular biomarkers are limited
[5]. Although, Troponin as proteins have proved to be
ideal biomarkers because of being low molecular weight
and due to complete specificity of the cardiac isoforms of
TnT and TnI. These protein markers arise from damaged
cardiomyocytes which release their cellular components
into blood after necrosis [1].
Although the value of Troponins cannot be negated, yet
there is a room for identification of more specific bio-
markers as, elevated serum troponins can be seen not only
with acute cardiac injury but also with other non-cardiac
disorders [10]. This risk factor creates possibility for
pursuit of new biomarkers which decreases the risk of
elevation of these in diseases secondary to myocardial
infarction and may lead to wrong diagnosis of disease.
The present study which employed the technology of
proteomics is an effort in this regard, as with the advent of
proteomics, it is possible to examine global alterations in
the expression of diseased heart and hence has potential
to provide insights into the molecular and cellular mecha-
nisms of cardiac arrest. Ultimately, a better understanding
of the role of these proteins should increase the likelihood
of new effective therapies developed through rationale
design [11].
2. MATERIALS AND METHODS
2.1. Study Group
Collection of serum samples was made from 50 appar-
ently healthy Pakistani male from University of Karachi.
All of the subjects belonged to the same age group
(35-40), were non smokers and were not on any type of
medication. An equal number of samples were collected
from patients with cardiac arrest pathology who pre-
sented themselves at Karachi University clinic. Prior to
sampling, written consent was obtained from both groups
of subjects and studies were carried out in accordance
with human ethical approval from PCMD, University
of Karachi.
2.2. Sampling and Biochemical Analysis
The samples taken were stored at-70oc until used for
protein analysis. Prior to protein profiling serum from
cardiac arrest patients was also evaluated for lipid profile
and cardiac enzymes assay followed by Troponin T
quantification.
Assessment of different parameters of lipid profile was
made, which included test for cholesterol Triglycerides,
HDL and LDL and total lipids. HDL and LDL were
measured using Cholestrol precipitant method.
Cholesterol levels were estimated using kit-method
specifically, CHOD-POD enzymatic colourimetric me-
thod by spectrophotometer. The absorbance of the sample
and the standards was read at 550 nm against the blank.
TG levels were determined by using Bioscience kit spe-
cifically CPO-POD enzymatic colourimetric method. The
absorbance of the samples and standards was read at
505nm against the blank.
HDL and LDL were measured using Cholestrol pre-
cipitant method.
Precipitation of HDL and LDL cholesterol was made
by using precipitating reagent, according to the kit
method and the absorbance of the samples and standards
was read at 500 nm against the blank.
Cardiac enzyme profiling was carried out which in-
cluded tests for SGOT, CPK, LDH, and CK-MB.
The SGOT levels in Cardiac arrest patients were esti-
mated spectrophotometrically by kit method at 340nm
wavelength. The CPK levels in cardiac arrest patients
were estimated spectrophotometrically by kit method at
480 nm wavelength. The LDH levels in cardiac arrest
patients were estimated spectrophotometrically by Breuer
and Breuer kit method at 340,334 and 365 nm wave-
lengths.
The CK-MB levels in cardiac arrest patients were es-
timated spectrophotometrically by Breuer and Breuer kit
method at 340 nm and 334 nm wavelengths.
2.3. Troponin T Quantification
Troponin T quantification was made through Roche
Cardiac T reader.
2.4. Protein Quantification
After biochemical analysis serum samples were subjected
to protein quantification by using Bradford assay.
2.5. Protein Characterization
Separation of serum proteins was carried out using So-
dium Dodecyl Sulphate Polyacrylamide gel electropho-
resis (SDS-PAGE) of cardiac arrest samples.
SDS-PAGE was performed in a Bio-Rad Mini-PROT-
EAN III apparatus using Discontinuous gel system ac-
cording to Laemmli, 1970 protocol. The experiment was
conducted in the electrode tank buffer. Tris/Glycine
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72
(pH8.3) using 4% stacking gels and 10% separating gels.
100g of protein was loaded on to the wells of the
stacking gel. Glass plates were assembled with electrodes
and electrode tank buffer was poured between the slabs.
The comb was removed from stacking gel. 100g protein
samples were loaded into each well along with 5 l of
protein marker. Electrophoresis was carried out at con-
stant voltage of 90 volts. After completion of electro-
phoresis, the glass plates were taken out and gel was
removed. The gel was left overnight in staining solution.
The staining solution was discarded and destaining solu-
tion was added until a clear background was obtained and
bands were visible. The gel was then photographed and
analyzed through Quantity One software.
Protein Profiling was carried out by Two Dimensional
Gel Electrophoresis.The 2D electrophoresis was carried
out by the modified procedure of O Farrell (1975). De-
naturing Acrylamide tube gels were used to separate
proteins (isoelectric focusing, IEF) and then run on SDS-
Polyacrylamide gel (10%).0.688g of urea was dissolved
in 0.5 ml of double distilled water at 40oC in a water bath.
0.16 ml monomer solution, 0.06 ml ampholytes and 0.025
ml NP–40 were added. The solution was then de-
gassed.0.83l of TEMED and 5.83 l APS were added to
solution and poured into the capillary tubes. Gels were
allowed to polymerize for 1hr. Tubes and upper reservoir
was filled with NaOH (2mM), while lower reservoir was
filled was phosphoric acid. Gels were then pre-run at
200V for 10 minutes, then at 300V for 15 minutes fol-
lowed by 400V for 15 minutes. After the pre-run, 100 g
of protein samples was then loaded on the gel surface.
9.0M urea (10l) was used to overlay the samples. The
tubes and upper reservoir were filled again with NaOH
(2mM). Gels were run at 750V for 5hrs. After isoelectric
focusing, the gels were protruded from the tubes and
equilibrated with SDB for 15 minutes. Gels were then
loaded on the prepared SDS polyacrylamide gel (10%
separating, 4% stacking). 0.5% Agarose was layered on
the IEF gel and left to solidify for 15min. Electrophoresis
was performed as for SDS-PAGE. The gel was then
stained with Coomassie brilliant blue overnight. This was
followed by destaining of the gels until a clear back-
ground was obtained and bands were visible.
2.6. Silver Staining of Polyacrylamide Gels
Silver staining protocol was used as described by Swan et
al., (1995) for the visualization of proteins specifically
low abundance protein. The gel was placed in fixative for
10 min and then rinsed with double distilled water for
another 10 min. The double distilled water was replaced
with fixer/sensitizer for 15 min. Gel was rinsed first with
ethanol (40%) and then with double distilled water for 20
min each. For 1 min the gel was immersed in sensitizer,
and then rinsed twice with double distilled water (1
min/wash). Gel was then placed in staining solution for
20 min and rinsed again in double distilled water for 1
min. The bands were developed in the developer until the
desire level of staining was obtained after which the de-
veloper was discarded and the gel was immersed in stop
solution for 5 min. The stop solution was replaced with
storage solution and the photograph of the gel was taken.
Throughout the procedure the gel was agitated on an
orbital shaker.
2.7. Statistical Analysis
Statistical analysis using student’s t test was carried out in
order to establish the degree of significance for lipid profile
and for assessment of cardiac enzymatic level for both the
control and cardiac arrest samples. The p-value was found
to be less than 0.001 with 95% confidence level which
clearly depicts the statistical significance of the results.
3. RESULTS
3.1. Protein Yield
The protein yield of cardiac arrest patients and normal
control were expressed as µg of protein per ml. As shown
in Figure 1 the yield of serum protein samples was ±72.
The protein yield of the diseased samples was found out
to be much less than that of the controls which is in direct
relation to the cardiac arrest pathology.
3.2. Lipid Profile
The lipid profile test was performed on samples of car-
diac arrest and on its respective control before protein
profiling was carried out (see Table 1).
Comparison between normal serum and control of
different plasma lipid parameters was performed in order
to assess the degree of severity of the pathology as shown
in Figure 2. It was found out that there is a 1.44796 fold
increase in cholesterol levels followed by a 0.775 fold
decrease in TG levels, 1.415 decrease in HDL levels,
1.266 fold increase in LDL levels and 1.322fold increase
in total lipids when comparison was made between car-
diac arrest sample and its respective control.
Figure 1. Quantification of proteins in cardiac ar-
rest sample by ELISA using Bradford Assay.
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73
Table 1. Comparison and contrast between control and cardiac
arrest sample of different plasma lipid parameters.
Parameters Normal serum
(mg/dl)
Cardiac arrest
sample (mg/dl)
Cholesterol 200±14.61 288.9592±14.59
Triglycerides(TG) 274±14.14 212.3878±16.86
HDL 59±14.152 41.69388±4.77
LDL 154±14.16 195.0612±16.22
Total. lipids 624±14.15 825±14.710
HDL: High density lipoproteins
LDL: Low density lipoproteins
0
100
200
300
400
500
600
700
cholestrol TGHDLLDLT.lipids
mg/d
l
(a)
0
100
200
300
400
500
600
700
800
900
cholestrol TGHDLLDLT.lipids
mg/d
l
(b)
Figure 2. (a) Standard error bars for different parameters
of lipid profiling of controls. (n=50, p>0.001). (b) Sta n-
dard error bars for different parameters of lipid profiling
of cardiac arrest sample (n= 50, p>0.001).
3.3. Cardiac Enzyme Test
Tests were performed on serum samples of cardiac arrest
patients and their respective control for assessment of
cardiac enzyme levels. When comparative studies were
made it was found out that SGOT level demonstrated an
increase by 2.988 fold, CPK by 1.246 fold LDH by
1.31935 fold and CK-MB by 2.34fold between serum of
diseased patients and control (see Table 2).
3.4. Troponins T Test
The cardiac arrest samples used were found out to be
troponin T positive. The tests for troponins are carried out
in situations when there is a many fold increase in HDL,
LDL and cholesterol levels of the patient. The troponin T
values of all the healthy subjects were assumed to be
within the reference range of (0-<0.05 ng /ml) while that
0
50
100
150
200
250
300
350
400
SGOT CPKLDH CKMB
μl/dl
(a)
0
50
100
150
200
250
300
350
400
450
500
SGOTCPKLDH CKMB
μl/dl
(b)
Figure 3. (a) Standard error bars for cardiac enzymatic
levels of control. (n=50, p>0.001). (b) Standard error bars
for cardiac enzymatic levels of cardiac arrest sample.
(n=50, p>0.001).
Table 2. Comparisons and contrast of different cardiac enzymes
in normal serum and cardiac arrest sample.
Cardiac enzymesNormal serum (µ/l) Cardiac arrest sample
SGOT 24.5±1.414 73.2449±15.78
CPK 173.35±7.77 216.1633±14.08
LDH 324±14.142 427.4694 ±22.87
CK-MB 22.4±1.414 52.4898±13.73
SGOT: Serum glutamic oxaloacetic transaminase
CPK: Creatine phosphokinase
LDH: Lactate dehydrogenase
CK-MB: Creatinine Kinase
of patients were found out to be within (0.8-1.04±0.1413)
which is in absolute concordance with the established
ranges for the determination of myocardial damage. (0.1-
2.0 ng/ml).
3.5. Protein Components Analyzed Using
SDS-PAGE (10%)
Serum protein (100g) of control and cardiac arrest pa-
tients (n=50 for each group) were subjected to SDS Page
(10%). The proteins were visualized by Coomassie bril-
liant blue.
As shown in Figure 4 the electrophoretic patterns of
the components of control (normal serum) and of cardiac
arrest sample demonstrated approximately 16 and 9
bands respectively in the silver stained gel. The molecular
weight of the protein components ranged from 200-
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74
25kDa. The silver stained electrophoretic patterns were
used for the molecular weight determination and semi-
quantitative analysis was made using Quantity one soft-
ware. Protein components (P1, P7, P11, P14, and P16)
with molecular weights (200kDa, 100kDa, 97kDa, 66kDa,
45kDa and 25 kDa) respectively were found to be both
common in control and cardiac arrest sample as shown in
Table 3. The difference in expression of protein compo-
nents between control and cardiac arrest patient samples
was obtained through R.Q values (see Table 4 The
135kDa (P4), 116kDa (P6), 87kDa (P9) protein compo-
nents were present in the cardiac arrest sample but were
absent in normal control. The results showed that the
45kDa (P14) protein present in control was under ex-
pressed or down regulated in cardiac arrest sample, where
as the 100kDa (P7), 97kDa (P8) and 66kDa (P11) were
over expressed in cardiac arrest samples (see Table 5).
There was a 5.6 fold up regulation of 100kDa protein,
4.5 fold up regulation of 97 kDa protein, while 2.13 fold
up regulation of 66 kDa protein, and in contrast there was
a 0.75 fold, down regulation of 45kDa protein in the
serum of cardiac arrest patients. 100kDa molecular wei-
ght protein was the most highly expressed followed by
97kDa and 45kDa. Differential expression of above
mentioned proteins in cardiac arrest patients illustrate
their possible role in the pathogenesis of cardiac arrest.
100kDa, 97kDa and 45kDa proteins can be identified as
potential markers of cardiac arrest .Up regulation of these
proteins are involved in the pathogenesis of this disease.
Low level of 45 kDa protein depicts that the high level of
this protein should be maintained to avoid the patho-
genesis.
3.6. Protein Components Resolved Using
Isoelectric Focusing (IEF)
2 DE performed on cardiac arrest sample had shown large
number of proteins in acidic range of pI range of 4.75-7.1
(see Figure 6), however those proteins which were ex-
clusively expressed in cardiac arrest sample were alkaline
in nature with pI range 7.6-10. The 2DE gel patterns of
control showed large number of proteins towards acidic
side with in the pI range of 4.3-6.4(see Figure 5).While
there are only 8 protein spots present in control as well as
in the diseased samples and were in the pI range 4.6 to 8.2.
This is an important observation suggesting a possible
role of alkaline proteins in the progression of cardiac
arrest and can be confirmed in future after further char-
acterization.
These findings suggest the potential role of these pro-
teins in disease progression and pathology. The altered
expression of several proteins observed in the present
study strengthened the hypothesis of the involvement of
these proteins in disease etiology. The proteins identified
here could be used in future as diagnostic and therapeutic
biomarker.
Figure 4. SDS-PAGE analysis of cardiac arrest sample, with normal serum and marker.
Openly accessible at
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75
Figure 5. 2D gel electrophoresis serum proteins from controls resolved through IEF and visualized by silver staining. The
spots show differentially expressed proteins. Protein marker used was with in the range of Mr 66-24kDa.
Figure 6. 2DE electrophoretic pattern of cardiac arrest proteins resolved through IEF and visualized by silver staining. The spots
show differentially expressed proteins Protein marker use was with in the range of Mr 200-29 kDa.
P. Ghazal et al. / HEALTH 2 (2010) 72-79
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76
Table 3. Difference in expression of protein components be-
tween control and cardiac arrest patient samples as obtained
through R.Q values using quantity one software.
Protein
components
Molecular
weight(kDa)
Relative
quantity%
(control)
Relative
quantity%
(cardiac arrest
samples)
P1 200 3.8 3.7
P2 165 2.3 -
P3 155 3.3 -
P4 135 - 4.8
P5 120 2.6 -
P6 116 - 5.1
P7 100 2.4 13.6
P8 97 2.3 10.3
P9 87 - 4.4
P10 76 4.6 -
P11 66 4.5 9.6
P12 62 4.9 -
P13 52 2.7 -
P14 45 5.4 4.1
P15 35 3.0 -
P16 25 0.9 0.2
Table 4. Protein components differentially expressed as in car-
diac arrest sample and in control. (n=50) as visualized by 10%
SDS.
Protein
components
Molecular
weight(kDa) Control Cardiac arrest sample
P1 200 + +
P2 189 - +
P3 165 + -
P4 155 + -
P5 120 + -
P6 116 - +
P7 100 + +
P8 97 + +
P9 87 - +
P10 76 + -
P11 66 + +
P12 62 + -
P13 52 + -
P14 45 + +
P15 35 + -
P16 25 + +
Table 5. Protein components of patient in control and cardiac
arrest sample which have been up regulated or down regulated.
Protein
components
Molecular
weight(kDa) Control Cardiac arrest
sample
P7 100
normal up regulate
P8 97 normal
up regulate
P11 66 normal
up regulate
P14 45 normal
down regulate
4. DISCUSSION
Heart diseases resulting in heart failure are a leading
cause of morbidity and mortality in developing countries
[1]. In context of this situation, the importance of a spe-
cific biomarker which precisely marks the prognosis of
the disease like cardiac arrest, increases by many folds. In
terms of availability and ease of measurement , a protein
that is very abundant in the target cell, has a means of
reaching blood and ideally a specific form reflective only
of the target cell in the tissue can serve out to be a good
candidate marker. We focused on heart muscle contractile
proteins as their levels in blood reflect the amount of
damage to the heart due to the necrosis of cardiomyocytes
[12]. The quest for a specific biomarker for ischemic
injury and heart failure began in 1950 and in 1990’s
trponins, specifically cardiac TnT and TnI became ideal
because of their low molecular weight, the apparent
specificity of the cardiac isoforms of TnT and TnI for the
myocardium and in addition to this, the ease with which
they can be detected in serum by chemical laboratory
analysis, 4-6 hours of an acute event and may remain
elevated for 7-10 days after the event [10]. However,
elevated serum troponins can be seen not only with acute
cardiac injury but also with other non-cardiac disorders
including pulmonary embolus, sepsis and renal failures, a
number of studies [13-15], have confirmed that cTnI
along with cTnT were found to be elevated in renal failure
cases. This finding supported our search of a more spe-
cific protein as biomarker for cardiac arrest.
Nevertheless, the presence of troponins in the serum
are detected only after the occurrence of cardiac injury.i.e
when the ideal period for thereuptical intervention has
passed by a large extent.Consequently, the present study
was an effort in this regard. In this study, the serum
sample of only those cardiac arrest patients were included,
which were found out to be positive for troponins cTnT
and cTnI so in the presence of these established bio-
markers, some new proteins could be identified which
could serve the purpose of new diagnostic or prognostic
biomarkers. Apart, from that another move was made
towards the discovery of such proteins which could serve
as potential drug targets for the development of new
thereuptical approaches for combating heart diseases. As
a result of which in this study, protein components from
cardiac arrest sample and their respective controls were
differentially expressed on SDS-PAGE gel pattern and
2DE.Most of the diseases cause induction or suppression
of some proteins associated with them. Differentially
expressed three proteins P7(100kDa), P8(97kDa),and
P11(66kDa) on SDS-PAGE were found out to be over
expressed in the cardiac arrest sample ,suggesting their
role in progression of disease while it can be hypothe-
sized that normal expression of the P14(45kDa) which
was found out to be down regulated in this study can be
conducive in rectification of the disease. From 2DE gel
patterns of the cardiac arrest samples it can be concluded
that that there is a role of alkaline proteins in the etiology
of the disease. The overall study concludes that the above
mentioned three proteins with 100kDa protein being the
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77
Table 6. Difference in expression of protein components be-
tween control and cardiac arrest patient samples as obtained
through R.f values using graphical methods. The protein com-
ponents were obtained through 2D gel electrophoresis.
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P1 168 7.6 - +
P2 163 7.25 - +
P3 156 8.2 - +
P4 118 10 - +
P5 112 8.7 - +
P6 102 9.03 - +
P7 99 7.68 + +
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P9 85 4.9 - +
P10 80 5.02 + +
P11 76 7.1 + +
P12 71 6.4 + -
P13 67.4 5.715 + +
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P15 54 6.61 + +
P16 49 6.565 + +
P17 43 4.6 + +
P19 26 8.2 + +
P20 22 4.3 + -
P21 18 7.3 - +
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This study had been successful in detection of three pro-
teins P7(100kDa), P8(97kDa) and P11(66kDa) on SDS-
PAGE which were found out to be over expressed in the
cardiac arrest sample, with 100 kDa protein being the
most highly expressed and 45 kDa being down regulated
and can be identified as potential biomarkers and targets
for therapeutical intervention. In addition to that 2 DE
had been conducive in proposing that proteins of alkaline
nature play a major role in cardiac arrest pathology.
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(1998). Cardiac troponins in patients with renal dysfunc-
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[15] Apple, F.S, Murakami, M.M., Pearce, L.A. and Herzog,
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