Background: Reactive oxygen species have been implicated in the pathogenesis of ischemic and reperfusion injury. In the current work we have measured malondialdehyde (MDA), total thiols, total CK, CK-MB and AST in ECG proven acute myocardial infarction (AMI) patients immediately after admission and 24 hours after administration of thrombolytic agent streptokinase, and in healthy controls. Methods: Blood samples from 44 AMI patients and 25 age and sex matched healthy controls were obtained and analyzed for MDA, total thiols using spectrophotometric methods and cardiac enzymes CK, CK-MB and AST using automated analyzer. Results: We have found significant increase  in MDA, CPK, CK-MB, AST (p< 0.001) and significant decrease in total thiols (p<0.001) in AMI patients after thrombolytic therapy compared to values at admission, and healthy controls. MDA correlated negatively with total thiols (r = - 0.333, p<0.05) and positively with CK-MB (r = 0.491, p<0.01) in AMI patients after thrombolytic therapy. Conclusions: Reperfusion following thrombolytic therapy increases reactive oxygen species with concomitant decrease in antioxidant total thiols

Babu, Suresh

Mungli, Prakash

Shetty, Jeevan K

English

CogPrints Cognitive Sciences Eprint Archive

Total Thiols and MDA Levels in Patients with Acute Myocardial Infarction Before and After Reperfusion Therapy

Cognitive Sciences ePrint Archive

Original Article:
Total  Thiols  and  MDA  Levels  in  Patients  with  Acute  Myocardial  Infarction  Before  and  After 
Reperfusion Therapy
Suresh Babu,
Department of Biochemistry, JJM Medical College, Davangere, Karnataka, India, 
Jeevan K Shetty,
Department of Biochemistry, Kasturba Medical College, Manipal University, Karnataka, India,
Mungli Prakash,
Department of Biochemistry and Genetics, St. Mathews University, School of Medicine, Grand Cayman, Cayman Island, BWI.
Address For Correspondence:
Dr. Jeevan K Shetty,
Assistant Professor,
Department of Biochemistry,
Kasturba Medical College, 
Manipal- 576104, INDIA.
E-mail: drjkshetty1978@yahoo.com
Citation: Babu S, Shetty JK, Prakash M. Total thiols and MDA levels in Patients with Acute Myocardial Infarction Before and After 
reperfusion therapy. Online J Health Allied Scs. 2010;9(3):6
URL: http://www.ojhas.org/issue35/2010-3-6.htm
Open Access Archives: http://cogprints.org/view/subjects/OJHAS.html and http://openmed.nic.in/view/subjects/ojhas.html
Submitted: Jul 19, 2010; Accepted: Sep 18, 2010; Published: Oct 15, 2010
Abstract:
Background: Reactive oxygen species have been implicated 
in the pathogenesis of ischemic and reperfusion injury. In the 
current  work  we  have  measured  malondialdehyde  (MDA), 
total thiols, total CK, CK-MB and AST in ECG proven acute 
myocardial infarction (AMI) patients immediately after admis-
sion and 24 hours after administration of thrombolytic  agent 
streptokinase,  and  in  healthy  controls.  Methods: Blood 
samples from 44 AMI patients and 25 age and sex matched 
healthy controls were obtained and analyzed for MDA, total 
thiols using spectrophotometric methods and cardiac enzymes 
CK, CK-MB and AST using automated analyzer. Results: We 
have found significant increase  in MDA, CPK, CK-MB, AST 
(p< 0.001) and significant decrease in total thiols (p<0.001) in 
AMI patients after thrombolytic therapy compared to values at 
admission,  and healthy controls.  MDA correlated negatively 
with total thiols (r = - 0.333, p<0.05) and positively with CK-
MB (r  = 0.491,  p<0.01) in  AMI patients  after  thrombolytic 
therapy.  Conclusions:  Reperfusion  following  thrombolytic 
therapy increases  reactive  oxygen  species  with  concomitant 
decrease in antioxidant total thiols.
Key  Words:  MDA;  Total  thiols;  Myocardial  infarction; 
Reperfusion injury   
Introduction:
In  acute  myocardial  infarction  (AMI),  two  distinct  types  of 
damage occur to the heart: ischemic injury and reperfusion in-
jury. The first results from the initial loss of blood flow and 
the second upon the restoration of oxygenated blood flow. The 
heart can tolerate a brief exposure to ischemia as the inherent 
mechanisms  to  preserve  energy  levels  prevent  injury.(1)  
Reperfusion  of  ischemic  myocardium  is,  therefore,  a  pre-
requisite for cellular survival.  Reperfusion followed by AMI 
has several benefits such as significant increase in recovery of 
left  ventricular  functions.(2)  Along  with  beneficial  effects, 
reperfusion  has  also  been  attributed  to  production  of  large 
amounts of reactive oxygen species (ROS).(3)
Increased generation of ROS may be responsible,  at least in 
part, for irreversible peroxidative damages to myocardial tis-
sue,  which  may  persist  for  months  following  thrombolytic 
therapy.(4) Among the ROS, malondialdehyde (MDA), a lipid 
peroxidation end product, is considered as one of the marker 
of  cell  membrane  damage.(5)  The  major  antioxidant  in  the 
body fluids is the cystein-SH bound to protein, majority of it 
found on albumin, and glutathione (GSH). These –SH groups 
(total thiols) play a major role along with other antioxidants in 
the body to ameliorate the lipid peroxidative effects of ROS.
(6,7)
In the current work, we have measured major antioxidant total 
thiols,  and  important  lipid  peroxidation  end  product  MDA, 
along with cardiac enzymes total CK, CK-MB, AST in AMI 
patients  immediately  after  admission  and  24  hours  after 
thrombolytic therapy to compare their levels with age matched 
healthy controls. We have also tried to establish relationship 
between oxidative stress markers with cardiac enzymes.
Materials and Methods:
Subjects and Samples
The study was carried out in the department of biochemistry, 
JJM Medical College, Davangere, India. The study group con-
sisted of forty four AMI patients and twenty five age and sex 
matched healthy controls.  Mean age and sex of patients was 
55 ± 11 years, and 33 males/11females, and that of controls 47 
± 15 years,  and 16 males/09 females,  respectively.  Patients 
were recruited from Bapuji and Chigateri government hospit-
als,  who  were  brought  to  emergency  room  with  history  of 
chest pain within last six hours of onset.  They were diagnosed 
to have AMI according to clinical criteria, chest pain which 
lasted for up to 3 hours, ECG changes (ST elevation of 2mm 
or more in at least two leads). All AMI patients were treated 
with streptokinase 1,500,000 IU for 1 hour within six hours of 
onset of pain. 
Informed consent was obtained from all the subjects involved 
and ethical clearance was obtained from institutional ethical 
clearance  committee.  Blood  samples  were  drawn  into  plain 
1
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Volume 9, Issue 3; Jul-Sep 2010
vacutainers from the antecubital veins of AMI patients imme-
diately after admission and 24 hours after initiation of throm-
bolytic  therapy.  Similarly,  samples  were also obtained from 
age  and  sex  matched  healthy  controls.  Total  CK,  CK-MB, 
AST,  MDA, total  thiol  levels  were  measured in all  the ob-
tained samples after proper processing. 
Reagents
Special chemicals  like  5’  5’  dithio-bis  (2-nitrobenzoic  acid) 
(DTNB), reduced glutathione (GSH), and standard MDA were 
obtained from sigma  cheAMIcals,  St  Louis,  MO, USA. All 
other reagents were of analytical grade. 
Biochemical Determinations
Measurement of cardiac enzymes:
Cardiac enzymes CPK, CK-MB and AST were measured us-
ing enzymatic assay using auto analyzer.(8-10)
Total thiol assay:
Reaction mixture contained 900 µL 2 mM Na2 EDTA in 0.2 M 
Na2HPO4, 20 µL 10 mM DTNB in 0.2 M Na2HPO4  and 100 
µL of serum. Reaction mixture was incubated at room temper-
ature for 5 minutes;  absorbance read at 412nm. Appropriate 
sample and reagent blanks were prepared simultaneously and 
the  respective  absorbance  was  noted.  Corrected  absorbance 
values were used to calculate serum total thiols using the mol-
ar extinction coefficient 1600 M-1 cm-1 and values expressed as 
µM. The calibration curve was produced using GSH dissolved 
in phosphate buffered saline.(11)
MDA assay:
Reaction  mixture  contained 1 mL 0.67% thiobarbituric  acid 
(TBA), 500 µL 20% Tri carboxylic acid (TCA) and 100 µL 
serum.  Incubated  at  100oC  for  20  minutes;  centrifuge  at 
12,000rpm for 5 minutes. Absorbance of supernatant read at 
532  nm.  MDA  was  determined  by  using  molar  extinction 
coefficient 1.56 x 105 M-1 cm-1 and values expressed as nM.
(12)
Statistical Analysis
The results were expressed as mean ± standard error of mean 
(SEM). A p<0.05 was considered statistically significant. Stat-
istical analysis was performed using the statistical package for 
social sciences (SPSS-16, Chicago, USA). One way analysis 
of variance (ANNOVA) was used to compare the mean values 
in  three  groups,  followed  by multiple  comparison  post  hoc 
tests. Pearson correlation was applied to correlate between the 
parameters.
Results:
As shown in Table 1 we have found significant increase  in 
MDA, total CK, CK-MB, AST (p< 0.001) and significant de-
crease in total thiols (p<0.001) in AMI patients after throm-
bolytic therapy compared to values at admission and healthy 
controls.  There  is  significant  increase  in  levels  of  MDA 
(p<0.001) and decrease in total thiols (p<0.001) in AMI pa-
tients immediately after admission compared to controls.  On 
applying Pearson’s correlation total thiol levels in serum cor-
related negatively with MDA (r = - 0.333, p<0.05) (Figure 1). 
As depicted in Figure 2, there was significant positive correla-
tion between CK-MB and MDA (r = 0.491, p<0.001).
Table 1: Total thiols, MDA, Creatinine kinase, CK-MB and AST levels in healthy controls and myocardial infarction patients 
at admission and 24 hours after streptokinase therapy (Values expressed in mean ± SD).
Tests Controls (n = 25) AMI Cases (n = 44)On admission 24 hours after Streptokinase
Creatinine Kinase (U/L) 108 ± 27 188 ± 125** 608 ± 216#
CK-MB  (U/L) 17 ± 2 38±22** 151 ± 64#
AST(U/L) 24 ± 5 40±38** 80 ± 30#
MDA (nmoles/L) 215 ± 51 362±53** 1542 ± 353#
Total thiols (µmoles/L) 384 ± 70 306± 24** 196± 29#
**p< 0.001 compared to healthy controls; #p<0.001 compared to AMI on admission
Figure 1: Correlation between MDA and Total thiols in AMI patients 24 hours after thrombolytic therapy
2
Figure 2: Correlation between MDA and CK-MB in AMI patients 24 hours after thrombolytic therapy
Discussion:
We have found increase in the MDA levels immediately after 
myocardial infarction compared to healthy controls. This may 
be due to  oxidative  stress induced by acute ischemic  injury 
and similar observation was published by many previous au-
thors.(6,13)  We  have  also  observed  significant  increase  in 
MDA levels  24 hours  after  reperfusion,  which  possibly  ex-
plain increased oxygen availability to ischemic site generating 
increased amount  of  ROS, thereby causing further injury to 
cell membrane. This may possibly explain the reperfusion in-
jury observed by previous authors.(14,15)
We have also observed that increased MDA levels after reper-
fusion correlated positively with cardiac marker enzyme CK-
MB, 24 hours after thrombolytic therapy, which may again ex-
plain  the  ROS  mediated  damage  to  myocyte  membrane 
thereby increasing the release of  cardiospecific  marker  CK-
MB  fraction.(1)  Available  literature  explains  that  oxidative 
stress followed by reperfusion therapy may be due to variety 
of factors such as (1) Enhanced generation of ROS due to sud-
den massive increase in oxygen supply; (2) Reduced levels of 
antioxidants available, (3) Enhanced consumption, leakage or 
destruction of antioxidants, (4) Leakage of electrons from the 
disrupted mitochondrial electron transport chain and (5) Pha-
gocytic recruitment and activation.(16) All of above effects to-
gether  initiate  lipid  peroxidation  in  cell  membrane,  damage 
membrane proteins or cause DNA fragmentation. These pro-
cesses may result in a loss of contractile function of cardiac 
myocyte  and lead to severe myocardial  cell  damage collect-
ively termed as reperfusion injury.(17) 
We have found decrease in total thiols following reperfusion, 
which correlated negatively with MDA.  The major part of thi-
ols in plasma is derived from proteins, especially albumin, and 
they are susceptible to oxidation during reperfusion. Signific-
ant low level of total thiols during myocardial infarction and 
after reperfusion may indicate increased consumption of these 
thiol  groups  to  neutralize  increase  levels  of  ROS  in  these 
states.(18) Decreased availability of thiols may also affect oth-
er biochemical function of the cell where thiols play an im-
portant role for the catalysis process.(19)
In conclusion the cardiac ischemia  induces ROS production 
and subsequent reperfusion can result in toxic ROS overpro-
duction with concomitant decrease in antioxidants.
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Approved Recommendation on IFCC Methods for the Measurement of Catalytic Concentration of Enzymes.

Cardio protective mechanisms of PKC isozyme-selective activators and inhibitors in the treatment of ischemia-reperfusion injury. Pharmacol Res.

Creatine phosphokinase-MB (CPK-MB) and the diagnosis of myocardial infarction.

IFCC Method for Creatine Kinase.

Lipid profile and non-enzymic antioxidant status in patients with acute coronary syndrome in South India. Heart Lung Circ.

Oxidative stress after acute myocardial infarction: effect of thrombolytic treatment.

Oxidative stress and ischemic myocardial syndromes. Med Sci Monit.

Oxidative stress and left ventricular remodelling after myocardial infarction. Cardiovasc Res.

Sudeshna Tripathy, Pannuri Vikram, Manish Verma. Serum paraoxonase activity and protein thiols in patients with hyperlipidemia.

The state of lipid peroxidation and antioxidants following thrombolytic therapy with rt-PA and streptokinase in acute myocardial infarction. Jpn Heart J.

Background: Reactive oxygen species have been implicated in the pathogenesis of ischemic and reperfusion injury. In the current work we have measured malondialdehyde (MDA), total thiols, total CK, CK-MB and AST in ECG proven acute myocardial infarction (AMI) patients immediately after admission and 24 hours after administration of thrombolytic agent streptokinase, and in healthy controls. Methods: Blood samples from 44 AMI patients and 25 age and sex matched healthy controls were obtained and analyzed for MDA, total thiols using spectrophotometric methods and cardiac enzymes CK, CK-MB and AST using automated analyzer. Results: We have found significant increase  in MDA, CPK, CK-MB, AST (p< 0.001) and significant decrease in total thiols (p<0.001) in AMI patients after thrombolytic therapy compared to values at admission, and healthy controls. MDA correlated negatively with total thiols (r = - 0.333, p<0.05) and positively with CK-MB (r = 0.491, p<0.01) in AMI patients after thrombolytic therapy. Conclusions: Reperfusion following thrombolytic therapy increases reactive oxygen species with concomitant decrease in antioxidant total thiol

Suresh Babu

Jeevan K Shetty

Mungli Prakash

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Total Thiols and MDA Levels in Patients with Acute Myocardial Infarction Before and After Reperfusion Therapy

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