Int.
J. Life. Sci. Scienti. Res., 3(3):
1039-1046, May 2017
A Correlation of
Adenosine Deaminase (ADA) Activity and Lipid Peroxidant (MDA) in Serum and Pleural Fluid for Diagnosis
of Pulmonary Tuberculosis
Ghanshyam Gahlot1*, Gajanand Joshi1, Yogita Soni2, Surendra Jeengar3
1Biochemist, Department of Biochemistry, S.P.
Medical College, Bikaner, Rajasthan University of Health and Science, India
2Professor, Department of Biochemistry, S.P.
Medical College, Bikaner, Rajasthan University of Health and Science, India
3Assistant Professor, Department of Biochemistry, S.P.
Medical College, Bikaner, Rajasthan University of Health and Science, India
Corresponding Author: Dr.Ghanshyam Gahlot, Biochemist,
Department of Biochemistry, S.P. Medical College, Bikaner, Under
Chabili Ghati, Near Neem Ka
Gatta, Bikaner, Rajasthan .Pin code -334001
Abstract- Background: Tuberculosis
is one of the commonest chronic infectious diseases; highly endemic in India
kills five lakh patients every year. Oxidative Stress
plays important role in inflammatory and degenerative diseases including
pulmonary tuberculosis. There is hardly any one study available in literature
correlating oxidative stress, lipid profile values and antioxidant status
together with the pulmonary tuberculosis; so we decided to conduct this study.
Methods: Study
group included newly diagnosed 50 cases of pulmonary tuberculosis and control
group included 50, age and sex matched healthy volunteers and employees. All
the cases were subjected to complete physical and
systemic examinations, routine investigations including Sputum for AFB by Ziehl-Neelsen staining, AFB culture and Chest X-ray and
special tests like Erythrocyte sedimentation rate (ESR), Malondialdehyde (MDA) and Adenosine deaminase
(ADA) and findings recorded and statistically analysed.
Results: In
the study group with 33 males and 17 females, we were found Serum
MDA mean ± SD 2.91±0.99; Serum
ADA 38.15±13.47,
while the mean levels of pleural fluid MDA and ADA in tubercular patients were
found to be 1.65+0.53 nmoles/ml and 56.88+22.1
U/L respectively. While in controls with 61 males and 39 females,
these values were 1.72±0.45 nmoles/ml
(MDA), 20.15± 6.70 U/L (ADA) respectively.
Conclusion: Malondialdehyde
(MDA) and Adenosine deaminase (ADA) were
found statistically significantly higher in study group when compared with
control, (p <0.001). Antioxidant plays important role for
prevention of pulmonary Tuberculosis.
Key
words: Tuberculosis, Malondialdehyde,
Adenosine deaminase, Lipid peroxidation,
Oxidative stress
INTRODUCTION-
Tuberculosis is one of the commonest chronic
infectious diseases and highly endemic in India and five lakh
patients die every year [1]. It usually affects lungs but
cases of extra-pulmonary tuberculosis are not rare. Delay in diagnosis and in
initiating treatment results in poor prognosis and squeal in up to 25% of cases
[1-2]. Pulmonary
Tuberculosis (PTB) can be confirmed by sputum examination and diagnosed easily
but diagnosing extra-Pulmonary TB becomes frequently difficult, since the
specificity and sensitivity of non-invasive methods is very low. Several
workers have estimated the specificity and sensitivity of Adenosine Deaminase (ADA) and found out its reliability [3-6].
Several biochemical reactions occur in human body during health and disease; as
a result of these essential reactions, there is formation of highly reactive
oxygen species (ROS) which consist of free radicals (FR). In reactions with FR,
bio-molecules undergo oxidation and through donation of their own electrons,
they themselves become new secondary radicals that continue radical chain
reactions and support spatial and time-dependent oxidative stress (OS)
propagation and consequently lead to the cell/ tissue damage [7]. In healthy conditions at the cellular
level, there is a critical balance that exists between the FR generation and
the various antioxidant defence mechanisms. But during certain disease
processes there is a huge imbalance between these two mechanisms resulting in
OS, hence this condition is characterized by disturbance in the pro-oxidant –
antioxidant balance in favour of the former, which leads to a potential harm to
the cell [8]. ROS can damage proteins, lipids, nucleic acids and
other cellular components under oxidative stress conditions [9]. OS
plays an important role in inflammatory & degenerative diseases like
pulmonary tuberculosis [10].
Mycobacterium tuberculosis (M.
tuberculosis) is an intracellular pathogen which grows and replicates in the
host macrophages. The pathogen activates the invaded macrophages and results in
free radical burst [11-12]. These FR induce lipid peroxidation (LP), a chain process which affects
polyunsaturated fatty acids (PUFA) mainly localized in cell membranes, in which
end products such as malondialdehyde (MDA) is
generated [13]. MDA is itself responsible for some of the damaging
effects of free radicals on Deoxyribonucleic acid (DNA) and on cell membranes [14].
High levels of lipid peroxidation products like MDA is
seen in advanced tuberculosis and can be measured in the blood as a parameter
of oxidative stress [15]. There are number of studies
available in the literature where different researchers have tried to find out
the level of oxidative stress, lipid profile values and antioxidant status
separately in pulmonary tuberculosis (PTB) patients, there is hardly any one
study available in literature correlating these three parameters together with
the disease; so we decided to conduct the study [15].
Lipid Peroxidation
converts poly unsaturated fatty acids present in cell membrane to the primary
product of Lipid peroxides and to secondary metabolites such as malondialdehyde and thus, causing cell injury and death via
DNA strand breakage and membrane damage [16]. Malondialdehyde (MDA) is the important marker of lipid peroxidation. Adenosine deaminase
is an enzyme involved in purine metabolism [17].
ADA catalyses irreversible hydrolytic deamination
of adenosine to produce inosine and ammonia [18].
Adenosine deaminase is secreted by T-lymphocytes and
macrophages during infection, so ADA is marker in chronic inflammatory
conditions such as tuberculous pleural effusions [19].
Normal serum and pleural fluid level of ADA is < 30 U/L. Increased in
various forms of Tuberculosis making it a marker for tuberculosis of lungs with
pleural effusion. In the present study we measured levels of MDA, ADA in normal
control and subject groups.
MATERIAL
AND METHODS- The study was carried out in the
Department of Biochemistry in collaboration with the Department of Respiratory
Medicine at S.P. Medical College and Associated group of P.B.M. Hospitals,
Bikaner, Rajasthan, India. The study was approved by
ethics committee and informed consent was taken from all the patients. The
study was undertaken between Aug, 2014 to Sep, 2015. A
total number of 100 subjects of both sexes, aged between 18-60 years were
included in this study.
Sample
Selection Criteria:-The class which was clinically
suspicious of other infection diseases besides pulmonary tuberculosis like
infectious mononucleosis, typhoid, viral hepatitis, HIV infections and
malignant tumor were not included in this study as this disease can also affect
serum and pleural fluid ADA levels. Patients suffering from diseases of heart,
liver, kidney, skeletal muscles and RBCs, which tend to alter MDA levels were also excluded from the study.
Subjects
were divided into two groups:
Group A =
50 Healthy subjects as controls
Group B =
50 Pulmonary Tuberculosis subjects
ESR,
MDA, ADA determination were done in pulmonary tuberculosis subjects as well as
in healthy subjects. Confirmation of Pulmonary Tuberculosis patients by sputum
smear, Mauntox test determined the disease status.
Collection of Samples
(a) Blood sample: -
Venous blood (5ml) was withdrawn and transferred to clean dry centrifuge tube.
Blood was allowed to clot at room temperature and centrifuged.
(b) Pleural fluid –
0.9ml of pleural fluid was taken in a test tube containing 10ml of mixture of
0.05 ml of glycerol and 0.05 ml of ethylene glycol.
Analytical
grade chemicals, standard were used and following estimation were done.
(1)
Erythrocyte sedimentation Rate (ESR)
(2)
Determination of Malondialdehyde (MDA)
(3)
Determination of Adenosine deaminase activity (ADA)
ESR
estimation:- By using of Westergen’s
method [20], 1 part of anticoagulant (3.8% tri sodium citrate
solution) + 4 parts of Blood, filled in to pipette with blood by sucking till
the O mark and clamped it vertically in the tube Read the upper level of red
cells exactly after one hour. It is expressed as the fall of RBC’s in mm at the
end of first hour (mm/hr).
Estimation of Malondialdehyde
(MDA): MDA concentration was estimated as reactive
substances by a thiobarbituric acid assay method [21].
Reagent used in TCA-TBA-Hcl-Prepared by dissolving
15% w/v Tri chloro acetic acid and 0.375/w/v thio barbituric acid in 0.25 N-Hcl and to make 100 ml. The 0.4ml of serum 0.6ml TCS-TBA-Hcl reagent was mixed well and kept in boiling on water
bath for 10 min. After cooling, add 1.0 ml freshly prepared IN NaOH so as to eliminate centrifugation absorbance of pink
color was measured at 535nm and against blank calculated by
16.0 X O.D. 535 nmoles/ml.
Estimation
of ADA –Kit method:-
(a)
ADA – MTB reagent L1= phosphate buffer
(b)
ADA-MTB reagent (l2) =Adenosine reagent
(c)
ADA – MTB reagent (L3) = Phenol reagent
(d)
ADA-MTB reagent (L4) = Hypochlorite
reagent.
(e)
ADA – MTB standard(S) = ADA standard
Statistical Analysis- The
data obtained for various parameters was subjected to statistical analysis. Arithmetic
mean and standard deviation were calculated of all the parameters studied, to compute ‘t Values’ (student’s t-test). On the basis of t values ‘p values’ (probability) were
determined to make out the significance of variance between the mean values of
individual parameters among the two groups of the subjects studied.
RESULT AND DISCUSSIONS-
It was observed, the mean ESR level was found to be 4.10 + 2.07mm in
first hour with a range of 1.0 to 9.0 mm in control group. The mean ESR level
was significantly raised to 20.86+ 7.01 with the range of 9.0 to 36.0mm
in first hour in study group of pulmonary Tuberculosis. The rise was
statistically significant as evident by P-value (P<0.001) showed in Fig. 1.
The mean serum malondialdehyde
(MDA) concentration was found to be 1.72±0.45 with a range of
0.64 to 2.56 nmoles/ml in healthy control subjects.
These results resembled with the observation made by Madhav
et al. [22] (Table 1; Fig.
1). The serum MDA level was increased to 2.91±0.99 nmoles/ml
with a range of 1.44 to 4.8 nmoles/ml
in pulmonary tuberculosis group. The increase was statistically
significant as compared to that of control group as evident by p-value
(p<0.005). The results of present study are in close collaboration with the
findings of Madhav et al. [22]. It might
be possible that increased oxidative stress and decreased antioxidant activity
in patients of pulmonary tuberculosis resulted increased lipid peroxidation leads to increased MDA concentration as
reported [23].
Table
1: Serum MDA concentration (nmoles/ml) in healthy
control and pulmonary tuberculosis patients (Study Group)
|
|
Male |
Female |
Total |
|||
|
Control
Group |
Study
Group |
Control
Group |
Study
Group |
Control
Group |
Study
Group |
|
|
Mean |
1.60 |
2.83 |
1.82 |
3.07 |
1.72 |
2.91 |
|
SD |
0.42 |
0.88 |
0.45 |
1.18 |
0.45 |
0.99 |
|
Range |
1.1 - 2.6 |
1.1 - 4.8 |
0.6 - 2.6 |
1.4 - 4.8 |
0.6 - 2.6 |
1.4 - 4.8 |
|
SE |
0.09 |
0.16 |
0.09 |
0.28 |
0.06 |
0.14 |
|
‘t’ |
6.98 |
4.31 |
7.76 |
|||
|
p-value |
< 0.001 |
< 0.005 |
||||
|
Statistical Significance |
HS** |
S* |
||||
*S = Significant
**HS
= Highly Significant
.jpg)
Fig
(1): Serum MDA concentration (nmole/ml) in healthy
control and pulmonary tuberculosis (PTB) patients
.jpg)
It
is revealed from Table 2, the mean pleural fluid MDA concentration was found to
be 1.65±0.53 nmoles/ml with a range of 0.64 to 3.04 nmoles/ml in the
present series of study. These results are in close agreement with the finding
of Gupta [24]. The increased concentration of MDA in pleural fluid
might be due to decrease in cellular immunity.
Table 2: Comparison of Serum MDA (nmoles/ml) and PF-MDA (nmoles/ml)
levels in pulmonary tuberculosis patients (Study Group)
|
|
Male |
Female |
Total |
|||
|
Serum MDA |
PF-MDA |
Serum MDA |
PF-MDA |
Serum MDA |
PF-MDA |
|
|
Mean |
2.83 |
1.61 |
3.07 |
1.72 |
2.91 |
1.65 |
|
SD |
0.88 |
0.55 |
1.18 |
0.50 |
0.99 |
0.53 |
|
Range |
1.44-4.8 |
0.64-3.0 |
1.4-4.8 |
0.64-2.5 |
1.4-4.8 |
0.64-3.0 |
|
SE |
0.15 |
0.09 |
0.28 |
0.11 |
0.14 |
0.07 |
|
‘t’ |
6.63 |
4.45 |
7.93 |
|||
|
p-value |
< 0.001 |
|||||
|
Statistical Significance |
HS** |
|||||
PF=
Pleural Fluid
**HS
= Highly Significant
It
has been observed from Table 3, Fig. 2 that the mean serum Adenosine Deaminase (ADA) level was found to be 20.15±6.70 U/L with a
range of 13.3 to 47.1 U/L in normal control subjects.
The results of present study resembled with the findings of Kelbel
et al. [25].
Table
3: Serum ADA (U/L) concentration in healthy control and pulmonary tuberculosis
patients (Study Group)
|
|
Male |
Female |
Total |
|||
|
Control
Group |
Study
Group |
Control
Group |
Study
Group |
Control
Group |
Study
Group |
|
|
Mean |
21.55 |
38.42 |
18.96 |
37.67 |
20.15 |
38.15 |
|
SD |
8.45 |
12.17 |
4.58 |
15.90 |
6.70 |
13.47 |
|
Range |
13.3- 47.1 |
17.8 - 68.7 |
13.3 - 31.2 |
14.0 - 80.0 |
13.3 - 47.1 |
14.0 - 80.0 |
|
SE |
1.76 |
2.15 |
0.88 |
3.72 |
0.94 |
1.90 |
|
‘t’ |
6.09 |
4.89 |
8.53 |
|||
|
p-value |
< 0.001 |
|||||
|
Statistical Significance |
HS** |
|||||
**HS = Highly Significant
.jpg)
Fig
(2): Serum ADA concentration (U/L) in healthy control and pulmonary
tuberculosis (PTB) patients
The serum ADA level was found to be 38.15±13.47
U/L with a range of 14.0 to 80.0 U/L in the pulmonary
tuberculosis patients. The results of present study are close agreement
with the finding of Blake and Berman [26]. The increase
concentration of serum ADA in tubercular patients as compared to that of
control was statistically significant as evident by p-value (p<0.001) (Table
3; Fig. 2). It might be due to decreased cellular immunity in pulmonary
tuberculosis [27], also
reported that the plasma ADA activity is higher in disease where cellular
immunity is impaired.
Table
4 indicates the mean pleural fluid ADA level was found to be 56.58±22.21 U/L
with as range of 16.25 to 94.32 U/L in the present
series of study. These results are in collaboration with the finding of Mathur et al. [28]. The decreased cellular
immunity in tuberculosis resulting increased pleural fluid ADA concentration as
observed in the present series of study.
Table 4: Comparison of
Serum ADA (U/L) and PF-ADA (U/L) levels in pulmonary tuberculosis patients
(Study Group)
|
|
Male |
Female |
Total |
|||
|
Serum
ADA |
PF-ADA |
Serum
ADA |
PF-ADA |
Serum
ADA |
PF-ADA |
|
|
Mean |
38.42 |
56.08 |
37.67 |
57.48 |
38.15 |
56.58 |
|
SD |
12.17 |
21.95 |
15.90 |
23.27 |
13.47 |
22.21 |
|
Range |
17.8 - 68.7 |
16.2 - 94.3 |
14.0 - 80.0 |
16.7 - 92.6 |
14.0 - 80.0 |
16.2 - 94.3 |
|
SE |
2.15 |
3.88 |
3.81 |
5.48 |
1.92 |
3.14 |
|
‘t’ |
4.18 |
3.58 |
5.22 |
|||
|
p-value |
< 0.001 |
|||||
|
Statistical Significance |
HS** |
|||||
PF=Pleural Fluid
**HS
= Highly Significant
It
is evident from Table 5 that mean serum MDA and ADA levels were found to be
2.91±0.99 nmoles/ml and 38.15±13.47 U/L respectively.
The serum MDA and ADA levels were increased significantly as shown by p-value
(p<0.001).
Table
5: Comparison of Serum MDA (nmoles/ml) and Serum ADA
(U/L) levels in pulmonary tuberculosis patients (Study Group)
|
|
Male |
Female |
Total |
|||
|
Serum
MDA |
Serum
ADA |
Serum
MDA |
Serum
ADA |
Serum
MDA |
Serum
ADA |
|
|
Mean |
2.83 |
38.42 |
3.07 |
37.67 |
2.91 |
38.15 |
|
SD |
0.88 |
12.17 |
1.18 |
15.90 |
0.99 |
13.47 |
|
Range |
1.4 - 4.8 |
17.8 - 68.7 |
1.4 - 4.8 |
14.0 -80.0 |
1.4 - 4.8 |
14.0 -80.0 |
|
SE |
0.15 |
2.15 |
0.28 |
3.81 |
0.14 |
1.92 |
|
‘t’ |
16.55 |
9.05 |
18.25 |
|||
|
p-value |
< 0.001 |
|||||
|
Statistical Significance |
HS** |
|||||
**HS = Highly Significant
The
mean levels of pleural fluid MDA and ADA in tubercular patients were found to
be 1.65±0.53 nmoles/ml and 56.88±22.21 U/L
respectively (Table 6). The PF-MDA and PF-ADA levels were raised significantly
in tubercular patients; might be due to reduced immunity in these patients.
Table
6: Comparison of PF-MDA (nmoles/ml) and PF-ADA (U/L)
levels in pulmonary tuberculosis patients (Study Group)
|
|
Male |
Female |
Total |
|||
|
PF-MDA |
PF-ADA |
PF-MDA |
PF-ADA |
PF-MDA |
PF-ADA |
|
|
Mean |
1.61 |
56.08 |
1.72 |
57.48 |
1.65 |
56.58 |
|
SD |
0.55 |
21.95 |
0.50 |
23.27 |
0.53 |
22.21 |
|
Range |
0.6 - 3.0 |
16.2 - 94.3 |
0.6 - 2.6 |
16.7 - 92.6 |
0.6 - 3.0 |
16.2 - 94.3 |
|
SE |
0.09 |
3.88 |
0.11 |
5.48 |
0.07 |
3.14 |
|
‘t’ |
14.45 |
10.17 |
17.49 |
|||
|
p-value |
< 0.001 |
|||||
|
Statistical Significance |
HS** |
|||||
PF= Pleural Fluid
**HS
= Highly Significant
CONCLUSIONS-
The
serum MDA and ADA concentration was found to be increased significantly in
tubercular patients as compared to that of control group; might be due to
increased oxidative stress associated with reduced cellular activity. A
positive correlation was recorded between the increase of serum MDA and ADA
concentration in tubercular patients because tubercular patients possessed
oxidative stress along with decrease cellular immunity. When serum MDA and ADA
concentration were correlated with control group, a negative correlation was
recorded; this might be due to the fact that serum MDA and ADA concentrations
are independent to that of normal control level but dependent on the severity
of the disease. The pleural fluid MDA and ADA concentration was found to be
raised in tubercular patients; might be due to reduced immunity level in
disease state. A positive correlation was observed between pleural fluid MDA
and pleural fluid ADA concentration in pulmonary tuberculosis. This might be
due to increased oxidative stress resulting decreased cellular immunity in
tubercular patients.
FUNDING-
Institution
Sardar Patel Medical College, Bikaner grant from the
Department of Biochemistry, Government of Rajasthan.
ACKNOWLEDGEMENTS-
The research was supported by Principal, S.P. Medical College and Controller of
attached hospitals, Bikaner and all indoor and outdoor patients making it
possible for us to conduct this work in this institution. I am extremely grateful to Principal and Controller
and our department.
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