CHAPTER – 2

REVIEW OF LITERATURE

This chapter presents the literature in the area of medicinal chemistry

wherein the relation of various bio-chemical parameters with the disease
Pulmonary Tuberculosis have been reviewed. Therefore the pulmonary
tuberculosis and its parameters have been separately dealt with.

Glycoproteins and Pulmonary Tuberculosis

Morishita1 (1965) conducted a prospective study on protein

bound polysaccharide in the dermatological field and concluded that
variations in the contents of serum polysaccharide which can be
regarded characteristic of each disease were hardly recognize, though
increase tendencies in the hexose wre observed in the exudative stage
of acute eczema, urticaria, lupus vulgaries and in certain cancers and
decrease tendencies in toxicolclermia, lupus erythematodes discoides,
melanosis and in some cases of xanthoma. High levels of hescosamine
wre observed in acute systemic lupus erythematodes and malignant
tumorsand decrease were seen in urticaria etc.
Mittae2 et al (1966) estimated that the average serum glycol
protein levels in 45 untreated new cases and 35 treated chronic cases of
proved pulmonary tuberculosis without any complication and of 24
normal healthy adults were estimated rise increases of pulmonary
tuberculosis.

Bradley P. William3 et al (1977) found out that the correlations

among serum protein bound carbohydrates ,serum glycoproteins
,lymphocyte reactivity and tumor burden in cancer patients ,their results
showed that serum protein bound carbohydrates or
glycoproteins may be of adjunctive value In assessing tumor burden and
immune reactivity in cancer patients .

Leif C. Anderson4 et al (1980) observed specific changes in the

surface glycoprotein patterns which correlate with the degree of
maturation and functional activation of T cells. Surface molecules
carrying Tcell specific antigens were identified by Immune precipitation
from lysates of surface labeled thymocytes and T-Lymphocytesusing
rabbit anti-human T cell antibodies.

Stefenelli. N5 et al (1984-85) determined the levels of sialic acid

inpatients suffering with various malignant tumors ,bacterial infections
,rheumatoid diseases and chronic liver diseases.Higher values are
found in the groups with neoplasms ,inflammatory diseases and active
rheumatoid diseases, and chronic liver diseases .Higher values are found
in the groups with neoplasms ,inflammatory diseases and active
rheumatoid diseases.

U Singh6 et al (1989) conducted a study on serum glycoprotein

in patients of pulmonary tuberculosis and found that the levels of
serum glycoprotein were found to increase in all the stages of
pulmonary tuberculosis with a marginal difference and the treatement
with anti tuberclosis drugs resulted in a decrease in ESR and the
various fractions of serum glycoprotein.

Fassbender Klaus7 et al (1995) used acute phase reactant xl-

acid glycoprotein AGP, orosomucoid as an additional tool in the
differential diagnosis of bacterial lung infections and concluded that
compared with those in healthy controls the serum concentrations AGP
were significantly increased in both tuberculosis and Non tuberculosis
bacterial pheumonia.

77
 Shinohara and co-workers8 (2008) studied the relationship
between the silalic acid concentration in serum and whole saliva in rats
with naturally occurring gingivitis. They suggested that the amount of
sialic acid in saliva can be a useful index of the severity of periodontal
disease.

Kalyan Goswami9 et al (2003) in their research came up with a

conclusion that there exists a significant negative correlation between
carbonylation and sialic acid content of serum proteins ill
hyperthyroidism and enhanced desialylation and carbonylation of
serumproteins by vitro H2O2 Treatment suggest that oxidative stress
can cause desialyation of serum glycoproteins.

Albillos A.10 et al (2004) illustrated that increased

lipopolysaccharide-binding protein was the only factor independently
associated with severe bacterial infection ill a multivariate
analysis.Monitoring of serum lipopolysaccharide –binding protein
could , therefore help to target cirrhotic patients with ascites for
antibiotic prophylaxis.

Kasahara J.11 et al (2004) illustrated that how serum

OXLOL/beta 2GPI compares, generated by oxidative stress and
associated with high dietary protein and salt intake, might be a novel risk
factor and a diagnostic marker for the development of chronic renal
diseases, especially IGA nephropathy.

Song –Ze Ding12 et al (2007) studied the effect of Helicobacton

pylori infection on oxidative stress levels and programmed cell death
in human gastric epithelial cells, they could deduce that infection of
DCFH2-DA treated gastric ehithellal well lines with H. Pylani was
associated with a rapid increase in flusorescence compared to levels of

78
fluorescence measured in uninfected control cells, indicicsating the
increased accumulation of interacellular ROS in in infected cell lines.

H.M. Erdogan13 et al (2008) designed a study to disclose some

indicators of oxidative stress and inflammation in natural cases of
bouine leptospirosis and the results obtained suggests that oxidation
damage along with other mechanisms might have taken part in the
pathogenesis of bouine leptospirosis should that increased total sialic
acid levels lipid bound sialic acid levels, MDA, NO, Uric acid, total
protein, glucose and decreased GHS and albumin concentrations were
suggestive of inflammation and oxidative stress in diseased bulbs.
Julien Brillaultl4 et al (2009) Moxifloxacin (MXF) is a
fluoroquinolone antibiotic that IS effective against respiratory
infections. However, the mechanisms of MXF lung diffusion are
unknown. Active transport in other tissues has been suggested for
several members of the fluoroquinolone family. In this study, transport
of MXF was systematically investigated across a Calu-3 lung epithelial
cell model. MXF showed polarized transport, with the secretory
permeability being twice as high as the absorptive permeability. The
secretary permeability was concentration dependent (apparent Pmax =
13.6 × 10-6 cm.s-1; apparent Km = 147 M), suggesting saturated
transport at concentrations higher than 350 g/ml. The P-glycoprotein
inhibitor PSC-833 inhibited MXF transport in both directions, whereas
probenecid, a multidrug resistance-related protein inhibitor, appeared
to have no effect in the Calu-3 model. Moreover, rifampin, a known
inducer of efflux transport proteins, upregulated the expression of P-
glycoprotein in Calu-3 cells and enhanced MXF active transport. In
conclusion, this study clearly indicates that MXF is subject to
Pglycoprotein- mediated active transport in the Calu-3 model. This P-

79
glycoprotein-dependent secretion may lead to higher MXF epithelial
lining fluid concentrations than those in plasma. Furthermore, drug- drug
interactions may be expected when MXF is combined with other P-
glycoprotein substrates or modulators.

Vitamin E and Pulmonary Tuberculosis

Fernandez Danaresf15 et al. (1989) studied the vitamin status in

patients with inflammatory bowel disease and found depreciated vitamin
E levels.

Kuroki Fl6 et al. (1997) studied whether vitamin E depleted in

crohns dieses and initial diagnoses and found out that the levels of
vitamin E were low in patients of crohns disease.

Plit17 et al, (1998) illustrated that concentrations of vitamin E

reduce in patients with pulmonary tuberculosis.

Geerling B J18 et al. (1999) studied on altered ascorbic acid status

in the mucosa from IBD patients and concluded that the antioxidant
enzymes and alteration in fatty acid profile in patient with prone disease.
There were decreased blood level in vitamin C and vitamin E and
decreased intestinal mucosal levels of CUZN super oxide dismutase
glutathione vitamin A,C,E and  carotene were reported for crohns
disease patients.

Lettow M19 . et al (2003) worked upon micronutrient, malnutrition

and wasting in adults with pulmonary tuberculosis in Malami and
concluded that lower dietary carotenoids, vitamin E, Vitamin A and
selenium are associated with wasting that data support the hypothesis that
wasting in tuberculosis is associated with lower plasma micronutrient
concentrations.

80
 Reddy, Y.N.20 et al (2004) studied the Severe oxidative stress
has been reported in TB patients because of malnutrition and poor
immunity. The purpose of this study was to investigate the serum lipid
peroxidation products and important free radical scavenging enzymes
i.e. superoxide dismutase (SOD), catalase and antioxidant glutathione
levels and total antioxidant status in TB patients. The subjects for this
study comprised of normal human volunteers (NHV,n=25), TB patients
(n=l00) – including, untreated (TB 1, n=55), under treatment (TB2,
n=30) and after treatment (TB3, n = 15) with anti-tuberculosis therapy
(ATT). The levels of lipid peroxidation products malondialdehyde
(MDA) were increased significantly in TB 1 & TB2 (P<0.001) and also
in TB3 (P<0.01); these levels gradually decreased with clinical
improvement with ATT. SOD, catalase, glutathione levels and total
antioxidant status were decreased significantly in TB 1 & TB2
(P<0.00l), TB3 (P<0.0l) patients in comparison with NHV, these levels
gradually increased with clinical improvement with ATT.
Oxidative stress was observed in all the TB patients (TB 1, TB2, TB3),
irrespective of treatment status. The study showed that in TB patients
free radical activity is quite high and antioxidant levels are low. A
suitable antioxidant therapy may prove beneficial and nutritional
antioxidant supplementation may represent a novel approach to fast
recovery.

Panjamurthy, K., Manoharan, S. and Ramachandran, C.R.21

(2005) investigated to assess the degree of oxidative stress in patients
with periodontitis by measuring their levels of thiobarbituric acid
reactive substances (TBARS), enzymatic antioxidants (superoxide
dismutase (SOD), catalase (CAT), glutathione peroxide (GSHPx)), and
non-enzymatic antioxidants (vitamins –E and C, reduced glutathione

81
(GSH)). This study was conducted on 25 adult chronic periodontitis
sufferers who were patients in Rajah Muthiah Dental College and
Hospital, Annamalai University. The levels of TBARS and non-
enzymatic antioxidants, and the activities of enzymatic antioxidants in
the patients' plasma, erythrocytes and gingival tissues were assayed
using specific colorimetric methods. The periodontitis sufferers had a
significantly higher TBARS level than the healthy subjects. In the
plasma, erythrocytes, erythrocyte membranes and gingival tissues of
the periodontitis sufferers, enzymatic antioxidant activities were found
to be significantly higher, whereas the levels of non-enzymatic
antioxidants were significantly lower (except for reduced glutathione
in the gingival tissues) relative to the parameters found for healthy
subjects. The disturbance in the endogenous antioxidant defense
system due to over-production of lipid peroxidation products at
inflammatory sites can be related to a higher level of oxidative stress in
patients with periodontitis.

Nwanjo, H. U. and Oze, G.O.22 (2007), studied the oxidative

imbalance and non-enzymic antioxidant status in plasma of pulmonary
tuberculosis patients in Nigeria were investigated. Forty HIV /AIDS
seronegative pulmonary tuberculosis patients with the active infection
(24 males, 16 females) aged 20-60 years diagnosed by Ziehl
Neelsonstaining/demonstration of mycobacterium tuberculosis in
sputum and sputum culture Lowenstein Jensen medium visiting Federal
Medical Centre, Owerri were selected for the study. Sixty normal
subjects free from pulmonary tuberculosis and HIV /AIDS (30 males
and 30 females) ages 20-60 years were also used as control. Patients
with complication such as renal diseases, viral and other bacterial
infections, etc. were excluded from the study. In the analysis of the

82
results using Duncan multiple range test, pulmonary tuberculosis
infected subjects presented significantly higher mean values of plasma
lipid peroxide (p<0.05) when compared with control. Also the levels of
non-enzymic antioxidants such as Vitamin C, vitamin E and reduced
glutathione in plasma were significantly depleted in the pulmonary
tuberculosis infected subjects (p<0.05) when compared with control.
This shows that pulmonary tuberculosis could probably be associated
with excess ROS production.

Seyedrezazadeh, E.23 et al. (2008) studied the increased

production of reactive oxygen species secondary to phagocyte respiratory
burst occurs in pulmonary tuberculosis (TB). The present study evaluated
the efficacy of vitamin E-selenium supplementation on oxidative stress in
newly diagnosed patients treated for pulmonary TB. A double-blind,
placebo-controlled trial including patients with newly diagnosed TB was
conducted. The intervention group (n = 17) received vitamin E and
selenium (vitamin E: 140 mg -tocopherol and selenium: 200 g) and the
control group (n = 18) received placebo. Both groups received standard
anti-TB treatment. Assessment of micronutrient levels, oxidative markers
and total antioxidant capacity were carried out at baseline and 2 months
after the intervention. Malondialdehyde levels were significantly reduced
in the intervention group (P = 0.01), while there was minimal reduction in
the control group. The mean plasma level of total antioxidants was
increased significantly (P = 0.001) in both the intervention and the
control groups. A 2-month intervention with vitamin E and selenium
supplementation reduces oxidative stress and enhances total antioxidant
status in patients with pulmonary TB treated with standard chemotherapy.

83
 Reddy, Y. Narsimha24 et al (2009) studied the the pleural fluid
was aspirated from the tuberculous patients both untreatedand under
treatment with three months anti-tuberculosis therapy. The amountof
nalondialdehyde, lactate dehydrogenase, and total protein content in
pleuralfluid of untreated tuberculous patients were found to be
significantly higher when compared with under treatment group. The
pleural fluid total antioxidant levels were significantly lower in
untreated cases in comparison to under treatment. Decrease in the total
antioxidant status was more pronounced in untreated cases, established
that antioxidants were nearly completely utilized to scavenge the free
radicals. Our findings further support the importance of antioxidants in
the treatment of tuberculous patient. This work is licensed under a
Creative Commons Attribution 3.0 License. You are free to copy,
distribute and perform the work. You must attribute the work in the
manner specified by the author or licensor phase of methanol: water
(70: 100) containing 550 mL of H3P04 with 80 nM of NaOH and 20
mL sample was injected. Catalase measurement was done based on the
ability of catalase to oxidize hydrogen peroxide (Beers and Sizer,
1952). The change in absorbance was measured for was measured at
240 nm for 3 min. The results were expressed in terms of IU/mL of
pleural fluid. For the estimation of total antioxidant status, we used a
stable free radical ,-diphenyl--picryl hydrazyl (DPPH), at the
concentration of 0.2 mM in methanol (Blios, 1958; Kalpana et al.,
2001), ascorbic acid was used as a reference standard. The standard
graph was plotted using different concentrations of ascorbic acid and
the antioxidant status values were expressed in terms of nM of ascorbic
acid. Lactate dehydrogenase is zinc containing intracellular enzyme
concerned with reversible oxidation of pyruvate to lactate, involves in
glycolytic cycle. The reaction velocity is determined by a decrease in
84
absorbance at 340 nm resulting from oxidation of NADH (Varley et al.,
1980). Pleural fluid total protein content was estimated by the method
of Lowry et al., 1951. Proteins form chromophoric complex with
phenol reagent, which was measured at 610 nm using UV-VIS
spectrophotometer (Elico, SL- 150). The protein content was calculated
from standard curve prepared with bovine serum albumin and
expressed in terms of g%. Statistical evaluation was done using student
t-test. Lipid peroxidation product and malondialdehyde level estimated
in the pleural fluid of tuberculous patients (Table 1) was significantly
decreased (p<0.001) in under treated cases when compared with
untreated cases. The levels were decreased with clinical improvement
with anti-tuberculosis therapy. The pleural fluid lipid peroxide levels
were found to be significantly high (p<0.001) in untreated cases in
comparison with under treated cases of all three categories used
different treatment regimens, the lipid peroxidation levels were more in
category 2. In the present study catalase level in pleural fluid of the
both untreated and under treated cases it was found that the difference
was not statistically significant and the catalase level was low even
after completion of antituberculosis therapy. The antioxidant levels
were increased significantly (p<0.01) in under treated cases in
comparison with untreated cases. The pleural fluid antioxidant status in
under treatment patients with different treatment regimen, in category
1 and 3 was significantly increased (p<0.01) in comparison with
untreated patients, there was no significant variation in category 2
(Table II). Lactate dehydrogenase level was decreased significantly
(p<0.01) in under treated cases in comparison with untreated cases. In
tuberculous patients based on treatment regimen, the pleural

85
 tluidlactate dehydrogenase levels were decreased
Samudram, P.25 et al (2009), investigated the case-control study
followed by a longitudinal cohort study was undertaken to
evaluate the level of lipid peroxidation product malondialdehyde
(MDA) and nitrite as an indirect measurement of nitric oxide vis-à-vis
the levels of antioxidants vitamin C and vitamin E in pulmonary
tuberculosis. Fifty-six sputum smear-positive cases or pulmonary
tuberculosis based on Ziehl-Neelsen (ZN) staining and 50
healthy controls without any systemic disease were included in this study.
Thirty-five cases were longitudinally followed up with standard
antituberculosis chemotherapy (ATT) for two months. Serum levels of
malondiadehyde (MDA), nitrite. and plasma levels of vitamins C and E
were measured. The mean serum MDA level was significantly higher
(8.1 ± 1.61 nmoles/ml) in PTB patients before commencement of ATT
as compared to healthy controls (3.45 ± 1.7 nmoles/ml) (p=0.0001) and
decreased significantly after 2 months of ATT (3.84 ± 1.28 nmoles/rnl)
(p=0.0001). The mean serum nitrite level (47.19 ± 18.44 mol/l) was
significantly elevated before A TT compared to healthy controls (32.89
±11.94 moles/l) and decreased significantly after 2 months of ATT
(27.71 ± 11.97 moles/l) (p=0.0001). The mean plasma levels of
vitamins C (0.88 ± 0.33 mg/dl) and E (0.79 ± 0.24 mg/dl) in PTB
patients before commencement of A TT were lower than healthy
controls (1.42 ± 0.38 mg/dl) and (1.35 ± 0.35 mg/dl), respectively
(p=0.001). There was a significant increase in vitamin C levels after 2
months of ATT (1.19 ± 0.40 mg/dl) compared to before A TT (0.83 ±
0.31 mg/dl) (p=0.0001), but no significant change in mean plasma
vitamin E level before and after 2 months on A TT was found. Elevated
malondialdehyde and nitrite levels with concomitant depressed vitamin
86
C and E levels are indicative of lipid peroxidation and oxidative stress.
The decrease in levels of malondialdehyde and nitrite with subsequent
increase in vitamin C levels after two months of follow-up indicate a
good response to treatment with standard ATT. Hence, the extent of
oxidative stress in PTB can be evaluated by analyzing lipid peroxidation
product, antioxidant and nitric oxide levels.

Antioxidant enzymes and Pulmonary Tuberculosis

Safarian and karapetian26 (1990) studied the serum of pul. T.B.

patients and observed an increase in SOD as compared to normal
controls.

Thomas27 et al (1992) performed a study on oxidative stress and

antioxidants in intestinal disease and found that increased oxidative stress
levels and decreased antioxidant levels.

Jacson p28 et al (1995) worked on finding the effect of

hemodialysis on total antioxidant capacity and serum antioxidants in
patients with chronic renal failure and found that there levels showed
variation.

Lih-brody L29 . et al (1996) performed a study on increased

oxidative stress and decreased antioxidant defenses in mucosa of
inflammatory bowel disease and found that and imbalance between the
increased ROS and the decreased antioxidant defenses occurs in IBD
patients.

Durak30 et al (1996) studied total cytoplasmic Cu, Zn-SoD and

mitochondrial MnSoD activities in serum and pleural fluids from patients
with lung tuberculosis, SoD activities were found to be higher
in all patients compared to control. They concluded that enzymatic

87
activity might be used as a non specific prognostic marker is assessing
cellular and mitochondrial tissue destruction.

Rock31 et al (1996) investigated that low food intake ,nutrient

malabsorption ,insufficient nutrient release from the liver ,acute phase
response and an inadequate availability of carrier molecules may
influence circulating antioxidant concentrations.

Geerling BJ32 et at. (1999) studied the relation between

antioxidant status and alteration in fatty acid profile in patient with
crohnsdisease and control.

Chan and goldkorn23 (2000) undertook a study to investigate the

role of SOD as one of the ROS that mediate lung injury.

Valerie Gouaze34 et al (2001) selectively inriestigated the

interaction of seleno-gluthathione peroxidase1(GPxno.l) with tge
eytotonic response of T470 human breast cancer cells to denorubicin
an anticancer drug known to promote production of ROs and apoptatic
mediater ceramide. Reduced glutathione and N-acetylapteine can inhit
both aorotsis abd necrosis of several cell types, suggesting a critical
role for reactive oxygen species (ROS) in cell death and results
indicated that GPx 1 can regulate deonorbicin induced cell death
signaling at least I part by interfering with the activation of the
aphingomyelin ceromide pathway.

Jingxiang Bai35 et al (2001) conducted a study on mitochondria

dysfunction induced by ROS and found that mitochondrial catalase
protects cell from oxidative injury induced by hydrogen peroxide and
antimycin A. However, it increased the sensitivity of cells tumor
necrosis factor-induced apoptosis by changing the redox-oxidatave
status in the mitochondria. They could conclude that the antioxidative

88
effectiveness of catalase when expressed In the mitochondrial
compartment is dependent upon the oxidant and the locus of ROS
production.

Jose M. Mates, Cristina Perez-Comez36 (2002) studied the

importance of antioxidant enzymes., superoxide dismutaze, glutathione
peroxidase and catalase working together in human cells against toxic
reactive oxygen species and their relationship with several
pathophysiologic process and their therapeutic implications and found
that low concentrations of ROS may be beneficial or even dispensible
in process such as intracellular signaling and defence against
microorganisms. Nevertheless, higher amounts of ROS play a role in
the ageing process as well in a number of human disease states,
including cancer, ischemia and failures in immunity and endocrine
functions. As a safeguard against the accumulation of ROS, several
non enzymatic and enzymatic antioxidant activities exist. Therefore,
when oxidative stress arises as consequence of a pathologic event, a
defense system promotes the regulation and expression of the antioxidant
enzymes.
Nobuya Ishibashi37 (2002) studied on inglammatory spouse and
glutathiones peroxidare in a model of stroke and found out that
gluthione peroxidase sensitive oxygen species play an important role in
regulation of cell death during cerebral I1R by modeling intrinsic
neuronal sensitivity as well as brain inflammatory reactions.

Kolanjiappan, K., Manoharan,S. and Kayalvizhi, M38. (2002),

examined the structural integrity of red blood cells in cervical cancer
patients by measuring the concentrations of thiobarbituric acid reactive
substances (TBARS), antioxidant status, cholesterol/phospholipid (C/P)
molar ratio, enzyme activity and osmotic fragility of

89
erythrocytes. Methods: This study has been conducted on 32 adult
female cervical cancer patients and an equal number of age- and sex-
matched normal subjects. Erythrocyte concentrations of lipids,
TBARS, vitamin E, reduced glutathione and enzymic activities of
catalase and Na+K+-ATPase were measured as well as plasma
concentrations of sodium and potassium. The present study also
examined the changes in erythrocyte osmotic fragility in cervical
cancer patients and normal subjects. The red cell fluidity and
permeability were determined by estimating the C/P ratio and Na+K +-
ATPase activity, respectively. Results: The release of thiobarbituric
acid reactive substances was significantly higher in cervical cancer
patients as compared to normal subjects. The increased lipid
peroxidation with concomitant decrease in antioxidants was notable in
cervical cancer patients. Red blood cells of cervical cancer patients
were more fragile than those from normal subjects. Increase in red cell
membrane C/P ratio and Na +K+-ATPase activity was noticed in
cervical cancer patients as compared to normal subjects. Conclusions:
Increased lipid peroxidation, insufficient antioxidant potential and
changes in C/P molar ratio as well as activity of Na +K+-ATPase cause
structural and functional abnormalities in the erythrocytes of cervical
cancer patients.

Reddy Y, N39. (2003) conducted a study on the effect of

antioxidant on patients suffering from leprosy and reported that
antioxidant therapy is suitable to reduce the oxidative stress.

Blankenberg40 (2003) worked on patients with suspected

coronary artery disease to asses the sist of cationascular events
associated with bare lie erythcyte glutathione peroxidase 1 ad
superoxide dismtancare activity and found that was among thestogest

90
univariate predictors of therisl of catiorasalar events whereas SOD
hasdno association with rick based on the background that cellular
antioxidants enzymes such as GPx 1 and superoxide dismetase have
acentral role in the control of reactive oxygen species and data and
studies in animals models suggest that there enzymes may protect
against arherosclerons but little is known about their releave to
hinadiseas.

James D. Crapo41 et at (2003) investigated that neutrophils are

second line of defence when alveolar macrophages fail and the
accumulationof eosinophills in the lung is mediated by chemokines , he
performed a study in which he deduced that presence of inflammatory
cells such as eosinophills or neutrophills in the lungs is associatedwith
increased oxidant injury ,lung is uniquely designed to control oxidative
stress and lung linning fluids are enriched in the antioxidantglutathione
which reaches a higher level.

Joppa, P. Petrasova,D. Stancak, B42-43. et at (2006-07)

investigated that xidative stress plays an important role in the
pathogenesis of chronic obstructive pulmonary disease (COPD).
Oxidant/antioxidant imbalance has also been reported in various forms of
pulmonary hypertension. The present study aimed to assess systemic
oxidative stress, as reflected by serum malondialdehyde (MDA)
concentrations and activities of antioxidant enzymes in erythrocytes
[glutathione peroxidase (GPX), superoxide dismutase (SOD) and catalase
(CAT)) in patients with and without pulmonary hypertension secondary
to COPD. Seventy-five patients (58 male) with COPD (mean age 65.l ±
1.2 years; mean smoking history 35.6 ± 3.8 pack-years) were studied.
Twenty-one healthy non-smokers served as a control group. Pulmonary
function was evaluated with body plethysmography; mean and systolic

91
pulmonary artery pressures (Ppa) were assessed with Doppler
echocardiography.Serum concentrations of MDA and activities of GPX,
SOD and CAT in washed red blood cells were measured using
spectrophotometry. Pulmonary hypertension was present in 28 patients
with COPD (systolic Ppa: 46.4 ± 2.3 mmHg; mean Ppa: 26.0 ± 1.9
mmHg) and absent in 47 (systolic Ppa: 22.9 ± 0.8 mmHg; mean Ppa: 13.4
± 0.6mmHg). Compared with the healthy control group, all the patients
(with or without pulmonary hypertension) had higher serum MDA
concentrations(1.5 ± 0.1 versus 2.3 ± 0.1 versus 2.3 ± 0.1 nmol/mL,
ANOYA, P < 0.001) and lower erythrocyte GPX activity (51.3 ± 3.2
versus 42.2 ± 2.0 versus 41.3 ± 2.5 U/g Hb, P = 0.029), whereas SOD
(1121.1± 29.0 versus 1032.6 ± 21.8 versus 1032.7 ± 36.2 U/g Hb, P =
0.063)and CAT activities (4.9 ± 0.2 versus 4.6 ± 0.1 versuS 4.7 ± 0.2 U/g
Hb; p= 0.454) were similar. No differences were observed in serum
MDA concentrations or activities of GPX, SOD and CAT in erythrocytes
between COPD patients with and without pulmonary hypertension.

CV Balasubrahmanya prasad44 et al (2007) conducted a study on

erythrocytic superoxide dismutase (AD) and (catalase) (CAT) activities
and hydrogen peroxide peroxide induced lipid peroxidation in leprosy
and found the induced peroxidation was significantly high and the
enzyme activities were significantly in leprosy total patients) as compared
to controls. A progressive increase in peroxidation was detected along the
leprosy spectrum from tuberculid leprosy to lepromatous leprosy and the
increase was significant in Bordieline leprosy, Borduline lepromotous,
leprosy and lepromatous leprosy as compared to controls.
GA Jacobseon45 et al (2007) investigated the plasma glutathione
peroxidase concentration GPX activity plasma selenium and oxidative
stress in acute severe asthma and that the levels of MDA increased but

92
no differences in plasma selenium levels or GPx activity. GPx levels
measured by Elisa were elevated in severe asthma. These results are
consistent with an adaptive up regulation of GPx to protect against
oxidative stress.
Halil Ciftci, Ayhan verit, Ercan yeni, Murat Saves46 (2008)
found that in the urine of patients with UTI have higher total
antioxidant spacity (TAC) and lower total peroxide and oxidative
stress, index levels, However TAC and plasma Vitamin C
concentration and higher total peroxide and as 1 levels wer obser ed in
UTI. This condition may be a factor which facilitates the development
of the infection on is secondary to UTI.
Naguva Abdallah ismail47 et al (2009) studied the oxidative stress
status in children with glycogen storage disease by determining
activities of GPx SOD and CAT in liver tissue. They concluded that
oxidative stress could play an important role in the pathogenesis of
glycogen torage disease and increased levels of SOD and GPx were
observed in the study.

ADA and Pulmonary Tuberculosis

Giblet48 et al (1972) observed the relationship between

immunological dys functions and ADA deficiency by pointing out the
ADA deficiency concepts for the first time.
Stranfcinga49 et al (1987) found that serum ADA level in pleural
fluid were significantly higher than serum ADA levels in both
tuberculosis and non tuberculosis pleural effusion.Adenosine deaminase
in the diagnosis of pulmonary tuberculosis and monitoring the efficienty
of therapy. The ADA activity was (mean ± SD) 21.77 ± 8.51 u/h in
pulmonary tuberculosis, 6.24 ±3.25 u/Lin old tuberculosis patients, 8.58 ±

93
4.38 u/L in healthy control subjects, whereas the mean for the patients
with bronchial cancer was 18.51 ± 7.85. In pulmonary tuberculosis
patients ADA activities were determined both before and after treatment
and significant decrease was observed in ADA activation after treatment
and serum ADA activity is increased in pulmonary tuberculosis patients,
it may be helpful parameter for monitoring therapy.

Segura50 et al (1989) investigated ADA activity in body fluid and

found out that it is a useful diagnostic tool in tuberculosis.

Gupta D.K51. (1990) revealed that 53 cases of pleural effusion out

of which 36 were of tuberculosis etiology. The mean ADA level in
tuberculous was 50.57 U/L while in malignant and parapneumonic
effusion it was 14.47 U/L and 28.65 U/L respectively. The sensitivity and
specificity for diagnosing tuberculosis were 100% and 94.1%
respectively.

Jeronimo Jaqueti52 et al (1990) reported that ADA reported that

ADA enzyme, is essential for the differentiation of lymphoid cells and
has been used for monitoring several diseases in which immunity
is altered. Excess of intracellular reactive oxygen species result in an
environment that may modulate gene expression or damage cellular
molecules.

Ida53 et al (1990) observed that abnormally high levels of serum

ADA activity were seen in patients with pulmonary tuberculosis
,indicating that serum ADA is a good diagnostic tool for tuberculosis.

Bhargava et al And Al-shamrnary54 et al (1990) concluded from

their study that the ADA level in the serum of children with T.B
was significantly higher than that of healthy children and in the TB
diagnosis the cut off value of serum ADA LEVEL WAS DECLARED

94
AS 78, 12, 32, 8 respectively in comparison to the cut off value of 53, 76
U/L as found by other studies.

Mukesh Kumar Agarwal55 et al (1991) found that the mean

serum ADA levels in patients of pulmonary tuberculosis was 38.48 +/-
1.5634 U/Lt. The value was significantly higher than the mean value
for healthy controls.
Meftun Unsal56 et al (1992) undertook a study to assess the levels
of ADA in patients suffering with active and inactive pulmonary
tuberculosis and derived the result that average serum ADA levels were
found to be higher in active disease patient group than in the inactive
patients group there was no stastical difference between serum ADA
levels of two groups .
Lakshmi57 et al (1992) found that the average serum ADA values
in 61 patients with active pulmonary tuberculosis patients in
their study as follows: sputum –ve PPD patient group 13.13+1/-5.97l .
Sputum +ve (PPD)+/- patient group 33.52+/-15.22 and they stated that
serum ADA values in patients that may have active pulmonary
tuberculosis can be helpful for diagnosis.
Burgess L. J58. (1995) showed ADA activity in tuberculous
effusion was higher than in any other diagnostic group. At a level of 50
U/L the sensitivity and specificity for the identification of tuberculosis
was 90% and 89% respectively. Voight studied 41 cases which were
bacteriologically confirmed tuberculosis cases with other causes the
mean ADA levels according to tubercular etiology was 99.8 U/L with
sensitivity and specificity for diagnosis tubercular ascities was 95 %
and 98% respectively. DwivediM studied on 49 patients with ascities
of which 19 were of tubercular etiology with mean ADA level of 98.8

95
U/L. At an ADA level >33 U/L. The sensitivity, specificity, positive
negative predictive values were 100%, 96, 95% and 100 %
respectively.

Kelbel59 et al (1995) deduced from their study that serum ADA

was a selective marker of immune stimulation in tuberculosis but not in
cancer when compared the serum ADA activity of pulmonary
tuberculosis patients with the patients of lung cancer pre- treatment and
healthy individuals .

Rohani M. Y 60 et al (1995) performed a study on the CSF from

patients with meningitis and other conditions with CN system
symptoms and ADA activity was deermined .T.B meningitis patients
had ADA activity greater than the cut off value of 9.0I/L .ADA is an
adjunctive rapid marker for tuberculosis.

Ferrer61 et al (1997) found that ADA measurement is commonly

used in European and asian countries where there is a high incidence of
tuberculosis.

Palival R. Shahikr62 (1998) conducted a study to evaluate the

efficiency and usefulness of serum ADA activity for diagnosis of
pulmonary tuberculosis and other common non tuberculosis
respectively conditions. Serum ADA levels were determined in 10
healthy individuals and 90 patients which included 65 patients with
pulmonary tuberculosis, 15 patients with supportive lung disease
and 10 patients with lung carcinoma. It was fond that serum ADA levels
were significantly higher in patients with pulmonary tuberculosis.

Collazos63 et al (1998) found in his study that the serum ADA

level of 52% of active pulmonary tuberculosis patients was high.

96
 Kuyucu64 et al (1999) conducted a study on serum ADA activity
in childhood pulmonary tuberculosis serum ADA levels were measured
in 20 children diagnosed with pulmonary tuberculosis (group 1) and 150
children (group 2) including 128 with tuberculosis infection and 22
healthy children . In group 1 the mean serum ADA activity was
74.06±18.5 which was significantly higher than that of group 2 (40.36±
12.00). So it was found serum ADA activity was a useful diagnostic tool
in childhood pulmonary tuberculosis.

Raintawan65 et al ,1999 ,Valdes66 at al 1996, Burgess67 et al

1996, Aggarwal68 et al 1999 & Roth69 suggested that ADA diagnostic
test has 90 to 100% specificity and isinexpensive. Collazazos70 et al,
1998; Bansal71 et al ,1991; Segura72 et al,1989;Conde73 et al, 2002 in
their studies have suggested the use of serum adenosine deaminase
levels for the diagnosis of pulmonary tuberculosis.

Mishra74 et al (2000) noticed raised serum adenosine deaminase

activity in a group of tuberculosis cases compared with healthy
individuals.

According to Yash P Kataria and Imtiyaz Khurshid75 (2001)

the reliability of early diagnsis of plural TB has been greatly improved
by the use of biochemical markers such as ADA interferon  and
lysozyme. The determination of ADA level in the suspected pleural
fluid appears to the most promising marker because of care, rapidly
and cost-effectiveness of the ADA assay. The determination of ADA
activity was first proposed as a serologic diagnostic matter for lung
cancer in 1970.

Dilmac A 2002, leolu K76 et al (2002) found the diagnostic value

of ADA activity in sputum in pulmonary tuberculosis.

97
 Conde M.B.77 et al. (2002) studied ADA levels in serum for the
diagnosis of tuberculosis and ADA levels have been followed during the
tuberculosis treatment 14 is accepted as cut off value for ADA. Bhargave
et al and al shamary et al accepted the cut off value of serum as a level as
78, 12 and 32, 8.
Verma78 et al (2004) studied the serum ADA activity in serum and
pleural fulid in patiens affected with pulmonary tuberculosis and other
common non tubercular chronic respiratory diseases. The study was
carried out on patients suffering from various pulmonary diseases, the
study revealed that the serum ADA activity was higher in patients of
pulmonary tuberculosis and pleural diseases and non tuberculosis
pulmonary diseases than in control subjects. The mean serum ADA
activity in the patients group was 35.5±6.93l as compared to
16.60±2.85.
K. Pratibha79 et al (2004) reported that patients with liver
disease showed significantly higher levels of serum bilirubin and
enzymes as compared with control group , a comparison between
patients and controls for ADA ,5'NT and MDA,all three parameters were
found to be significantly higher in patient group
Sharma, S.K80 . et al (2006) evaluated that diagnostic accuracy
and cost-effectiveness of ascitic fluid interferon- (IFN-) and
adenosine deaminase (ADA) assays in the diagnosis of tuberculous
ascites. Ascitic fluid from patients with proven tuberculosis (TB) (n =
31) and non-TB ascites (n = 88) was analyzed for IFN- and ADA
levels. Areas under the receiver operative characteristic (ROC) curves
(AUCs) for the two biologic markers were compared. Levels of ascitic
fluid IFN-, median (range): 560 (104-1600) pg/mL vs. 4.85 (0–320)
pg/mL (p < 0.001), and ADA, median (range): 58 (16-331) IU/L vs. 10

98
(0-59) IU/L (p = 0.001), were significantly different between TB and
non-TB groups. IFN- and ADA assays showed equal sensitivity (0.97)
and differed marginally in specificity (0.97 vs. 0.94). Difference in
AUCs was not significant (0.99 vs. 0.98, p < 0.62). For differentiating
TB from non- TB ascites, optimal cutoff points were 112 pg/mL for
IFN- and 37 IU/L for ADA. The accuracy of the ADA assay was similar
to that of the IFN- assay in differentiating of TB from non-TB ascites.
Because both material and human costs of the ADA assay are
far less than those of the IFN- assay, the former is probably the most
appropriate diagnostic test for analysis of peritoneal fluid in resource
limited settings.

Mohammed A Aezoghaibi81 et al (2007) performed the study on

lipid peroxide in patients with inflammatory bowel diseases
increases level of plasma MDA which were an important indication of
oxidative stress. Patients with crohns diseases are more susceptible to
oxidative stress than the patient with ulserative colitis.

Cimen, FiIiz. and Ulukavak Ciftci, Tansu82 . et al (2008)

aimed to investigate serum ADA levels in patients with tuberculosis and
its relation with drug resistance and tuberculosis categories. The
study involved 51 pulmonary tuberculosis patients and eleven healthy
controls. All patients classified according to the World Health
Organization (WHO) and 60.8% of the patients were category I, 21.6%
were category II and 17.6% were category IV pulmonary tuberculosis.
Serum ADA levels of the sensitive cases to isoniazid (H), rifampicin
(R), ethambutol (E) and streptomycin (S) were compared with those of
the resistant cases. Serum ADA levels were higher in Rand E sensitive
group then in the resistant group (p=0.046, p=0.045). Serum ADA

99
levels were similar in Hand S sensitive group and resistant group
P>0.05). Serum ADA levels were higher in category I group when
compared with the levels of the healthy group (p: 0.03). Comparison
between the serum ADA levels of the groups of category I, II and IV
with each other showed that the values of category I were significantly
higher than values of category II (p=0,038). Although there were no
statistically significant differences, it was shown that when the number
of resistant drugs increased, the mean serum ADA level tend to
decrease. In conclusion, serum ADA levels of category I patients were
higher than healthy group and while the number of resistant drugs
increasing, ADA levels were decreasing.

Bandyopadhyay, D. and Gupta, S83. (2008) studied 179 cases

of EPIB were analysed for acid-fast bacilli (AFB) smear, adenosine
deaminase activity (ADA) and multiplex polymerase chain reaction
(PCR). Although estimation of ADA is helpful, its sensitivity and
specificity varies widely. On the other hand, a multiplex PCR using
amplicons such as IS6110, dnaJ gene and hsp65 genes has high
sensitivity (60-88%) and specificity (81-100%). On comparing AFB
and ADA results with PCR, the PCR is clearly more effective than
AFB (P < 0.001) and ADA estimation (P < 0.02) in CSF. The same
result was observed with peritoneal fluid (P < 0.001 vs. P < 0.05) and
pleural fluid (P < 0.001 and P < 0.05). The study shows that multiplex
PCR remains the best tool and is a much better marker for diagnosing
EPTB.
Kamaldeen Baba84 et al (2008) found in their study that ADA is
sensitive marker of tunerculous pleuritis even in HIV patients with
very low CD4 counts in a high TB endemic region .ADA assay is
inexpensive ,rapid and simple to perform and is of great value for the

100
immediate diagnosis of TB pleuritis while waiting for culture result and
this has a positive impact on patients routine.

Lipid Peroxidation and Pulmonary Tuberculosis

Letbowitz85 et al (1973) found that tuberculosis pleural effusion is

thought to result from delayed sensitivity reaction that occurs in
response to the presence mycobacterial antigens in pleural space.

Mead86 (1976) one of the accepted hypo thesis to explain the

molecular mechanism of cell death is lipid peroxidation. In addition to
oxidative alternation of complex lipids free radical mediated events may
also affect other cellular components such as protein carbohydrate
compound and nucleic acid.

May and Spagnuolo 87 (1987) found in their study that

mycobacterium can induce reactive oxygen species production by
activating phagocytes.

Halliwell B.88 (1991) deduced from his study that thiobarbituric

acid reactive substances primarily reflect malondialdehyde ,a
breakdown product of lipid peroxides and are commonly used as the
measure of oxidative stress.

DJ Silver Man 89 et al (1992) in their study cells infective by

ricketia rickettsii, the causative agent of rocky mountain spotted fever,
display unusual intracellular morphological changes characterized by
dilation of the membranes of the endoplasmic reticulum and outer
nuclear envelope. This observation suggest that the increased peroxide
in infected cell may be lipid peroxide degradation products of free
radical attack on poly enoic fatty acid.

J A North90 et al (1994) found that the extent of lipid radical

101
formation in response to oxidative stress can be influence by changes
in the poly unsaturated fatty acid composition of cell lipid and suggest the
possibility that carbon centered lipid radicals may interact with
extracellular structure.

Jack91 et al (1994) and Grimble92 (1994) investigated that

although an important part of host defense against mycobacteria and
enhenced ROS phagocytes generation may promote tissue injury and
inflammation that also leads to immunosuppression.

Raviglione93 et al (1995) in his study describe T.B as a chronic

granulo matus disease caused by mycobacterium tuberculosis with
various manifestation, involving most commonly the lung but other
system as well.

Sen and packer94 (1996) found that reactive oxygen species

generated as a result of ischemic-reperfusion associated with
haemorrhage has been proposed to contribute to the progress of lung
Injury.

Christopher K. W. Lai95 et al (1997) found that the clinical

manifestations of IB depend on cellular immune responses to the
tubercle bacilli characterized by the accumulation of monocytes
,macrophages ,lymphocytes and polymorphonuclear leukocytes in
tuberculous lesions .These responses are initiated on sensitization of T
lymphocytes by the bacterial antigen with the release of cytokines that
regulate macrophage function .Activation of T lymphocytes in TB is
supported by the previous findings that the serum concentration of
soluble interlukin (II -2 )receptor in patients with active disease was
markedly raised. CD4+ Tcells are likely to be the pivot cells in
orchestrating the immunologic defence against the mycobacteria as

102
depletion of these cells is associated with increased susceptibility to TB
in both animals and humans.

Subrahmanyam M96 . et al (1998) in their study B on D effects of

topical application on honey on burn wound healing they took 100 burn
patients and were divided into two special to be treated with
honey dressing or with silver sulphadiazine gauze dressing in honey
treated group wounds healed earlier seem lipid peroxide levels were
revised in the immediate post burn period in both group. Dacleval
cultured revealed that 90% were rendered sterile in the honey treated
group, where as in the silver-sulphadiazine treated group there was
persistent infection.

Kwiatkowska 97 et al (1999) showed that malondialdehyde and

lipid peroxidation product levels in sputum smear positive patients
with advanced pul tuberculosis and sputum smear negative patients with
small radiographic changes were significantly higher than those of
healthy control group.

J Bhattacharya and AG Datta98 (2001) conducted a study on

effects of lipopoly saccharide on lipid peroxidation of erthyrocyte and
its reversal by mannital and glyceraol.

Makinskii99 et al (2002) showed that high pretreatment levels of

free radicals had decreased to normal at the end of the treatment in pul.
T.B. cases.

S Kastenbauer, U Koedal100 et al (2002) conducted study on

oxidative stress in bacterial meningitis in humans and found that
tyrosine nitration was strongly increased during meningitis. It was
most evident in inflammatory cells and blood vessels in the sub

103
arachnoid space. High CSF nitrotyrosine concentration were associated
with an unfavorable outcome according to the Glasgow outcome score
in CSF the increase in nitrotyrosine was accompanied by a depletion of
the antioxidant ascorbic acid and an increased oxidation of natural
peroxinitrite scavenger uric acid to allantoin.

Peter Barany101 et al (2003) worked on inflammation and

resistance to erythropoises. Stimulating agents - links to oxidative
stress and cardiovascular mortality and found that patients with chronic
kidney diseases are thought to have reduced capacity to one thing is there.

Figen Deveei and Nevin Ilhan102 (2003) studied on plasma

malondialdehyde and serum trace element concentrations in patients with
active pulmonary tuberculosis and found that increased amount
reactive oxygen species that a produce as a consequence of a
phagocytic respiratory burst during pulmonary inflammation are
responsible for the increase circulating levels MDA.

Yildiz Guney103 et al (2004) investigated the serum

malondialdehyde levels and superoxide dismutase activities in
pulmonary tuberculosis and lung cancers and came up with the
conclusion that serum MDA level in the whole group of patient (lung
cancer and TV were much higher than in healthy control) as well as from
species of exogenous ori...... such as cigarette.

Kiranjeet Kaur104 (2005) reported that there IS an oxidative

stress and decreased antioxidant activity in patient of pulmonary
tuberculosis which correlate with radiological extent sputum grading
and cavity status.

104
 Ceylan105 et al (2005) found that in TB patients the levels of
serum reactive oxygen metabolites were increased as compared to
normal controls ,therefore building up oxidative stress in the patients.

Walsh106 et al (2006) have primarily shown that TBARS highly

correlate with 8-isoprostane and thus it accurately reflects lipid
peroxides and that MDA can affect the membrane proteins by cross
linkage, rendering them useless as receptors or enzymes.

Silvia Carraro107 et al (2008) conducted a study of exhaled

breath condensate that is a safe and easy technique that enables several
biomarkers of lung disease. EBC has been applied in the study of
various sepiraty diseases in children (mostly asthma and cystic fibrosis,
but also other diseases such as primary ciliary dyskinesia). Several bio
markers of airway inflammation and oxidative stress have been
detected in the EBC of these patients, demostaling the role of different
inflammatory pathways in the pathophysiology of respiratory diseases.

Critical Appraisal of Studies Reviewed

On analyzing the review of studies in the area of medicinal

chemistry, the following observations may be made parameter vise that
are considered in the study.

 Most of the researches on ADA are concentrated on drug

resistance, nutritional management, anti-oxidation, infection
disease, immune stimulation, and gene expression etc. very few
researches were found in pulmonary tuberculosis and variation of
biochemical parameters in as sputum +ve and sputum –ve patients.
 Studies conducted on lipid preoxidation in the context of

105
 meningitis, honey, cardovas cular disease, poli unsaturated fat,
defence mechanism, mico bacterium, lung injury, but hardly any
study is traced as is under consideration.
 Researches in the area of antioxidants have been conducted on
leprosy, mental disorders, several falinic , hapetic apurecrosis,
immunology, asthma, but not even few researches are found
related to pulmonary tuberculosis with the combination of
parameters as considered in this study.
 Studies on glycoprotein have been reported on levels of
glycoprotein, glycoprotein pattern, immuno compromised. Very
few studies have been observed that too with the variation from
this study.
 Vitamin E has been studied in case of inflammatory disease IDB
patients micronutrients, T cells, reactive oxygen species and
vitamin E selenium etc. Hence the studies on vitamin E and
sputum +ve and sputum -ve pulmonary tuberculosis are very few to
report.

Consequently as may be derived from the review of researches in

the area of medicinal chemistry, no research with the parameters that
are taken in this study, has been reported. Therefore this research study
will make a significant contribution in the area of early diagnosis of
pulmonary tuberculosis wherein is so frame in its' consequences.

106
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