International Journal of Mycobacteriology x x x ( 2 0 1 4 ) x x x –x x x

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Changes in nitric oxide synthase and nitrite and

nitrate serum levels in patients with or without
MDR-TB undergoing the intensive phase
of anti-tuberculosis therapy

Dmytro O. Butov a,*, Mikhail M. Kuzhko b, Irina M. Kalmykova c, Irina M. Kuznetsova d,

Tatyana S. Butova a, Olena O. Grinishina e, Olga A. Maksimenko f
a
Kharkiv National Medical University, Kharkiv, Ukraine
b
F.G. Yanovsky National Institute of Phtysiatry & Pulmonology, National Academy of Medical Sciences, Kiev, Ukraine
c
Regional TB Dispensary No. 1, Kharkiv, Ukraine
d
Regional TB Hospital No. 1, Kharkiv, Ukraine
e
Regional TB Dispensary No. 3, Zmeyev, Ukraine
f
Regional TB Dispensary No. 4, Izyum, Ukraine

A R T I C L E I N F O A B S T R A C T

Article history: Background: There is a paucity of published data on the effect of TB chemotherapy on nitric
Received 6 February 2014 oxide (NO) synthesis and metabolism in newly diagnosed and relapsed patients with or
Accepted 20 February 2014 without multi-drug resistant tuberculosis (MDRTB).
Available online xxxx Methods: The pattern of NO response in 140 patients with pulmonary TB, including 74 with
MDR-TB and 66 without MDR-TB has been studied and compared to the NO status of 30
Keywords: healthy donors. Patients comprised those with newly diagnosed TB and recurrent or
Pulmonary tuberculosis relapsed TB. The NO status was assessed by measuring inducible NO synthase (iNOS)
MDR-TB and nitrites and nitrates levels. This was measured prior to treatment initiation and two
MTB months after the prescribed chemotherapy.
Nitric oxide Results: Increased levels of NO indices were found in patients with tuberculosis when com-
NO pared to healthy controls. After two months of chemotherapy a significant decrease in NO
Inducible NO synthase indicators was observed in the patients with TB, particularly in those without MDR-TB and
newly diagnosed TB. The decline in NO activity was less prominent in patients with recur-
rent TB and MDR-TB, which suggests lower level of immunologic and reparative processes
in such patients.
Conclusion: Changes in serum levels of nitrites and nitrates as well as iNOS activity in neu-
trophils may serve as diagnostic criteria to differentiate various clinical forms of TB and
help as prognostic tool to predict treatment outcome.
Ó 2014 Asian-African Society for Mycobacteriology. Published by Elsevier Ltd. All rights
reserved.

* Corresponding author. Address: Department of Phtysiatry and Pulmonology, Kharkiv National Medical University, 4 Lenin Avenue,
Kharkiv 61022, Ukraine. Tel.: +380 977723777.
E-mail address: dddimad@yandex.ua (D.O. Butov).
Abbreviations: MDR-TB, multi-drug resistant TB; MTB, Mycobacterium tuberculosis; NO, nitric oxide; iNOS, inducible NO synthase; NDTB,
newly diagnosed TB; RTB, recurrent TB
http://dx.doi.org/10.1016/j.ijmyco.2014.02.003
2212-5531/Ó 2014 Asian-African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: DO Butov et al. Changes in nitric oxide synthase and nitrite and nitrate serum levels in patients with or without
MDR-TB undergoing the intensive phase of anti-tuberculosis therapy. Int. J. Mycobacteriol. (2014), http://dx.doi.org/10.1016/j.ijmyco.2014.02.003
2 International Journal of Mycobacteriology
Introduction
About one third of the world’s population is infected with
Mycobacterium tuberculosis (MTB) [1]. MTB is the leading cause
of death, which kills about 2 million people every year. In 1993
the World Health Organization declared tuberculosis (TB) as a
global epidemic [2]. Resistance of MTB to anti-mycobacterial
drugs remains one of the urgent problems all over the world
[3]. The spread of multi-drug resistant (MDR-TB) and recur-
rent TB sustains the TB epidemics; therefore, this disease re-
mains one of the main threats to global health [1]. In some
countries it is even considered to be a national security
threat. The issuance of numerous guidelines and recommen-
dations to control MDR-TB is indicative of this [4–9].
According to consensus opinion, unfavorable socio-eco-
nomic conditions are considered to be one of main causes
for TB recurrence [10–12]. However, it is also clear that
changes in the immune system and metabolite controllers
of the human body play an important role. One of these
metabolite controllers is nitric oxide (NO). NO, the product
of the arginine dependent pathway of human mononuclear
phagocytes, regulates a host of physiological functions and
pathologic processes. The discovery of the importance of
NO by Furchgott, Murad and Ignarro brought them the Nobel
Prize award. There is an evidence that NO and its metabolites,
e.g., nitrates and nitrites, can mobilize the anti-mycobacterial
defense in the host [13]. NO is synthesized in immune cells
under the influence of NO inducible synthase (iNOS), the
activity of which is influenced by various cell damage factors
[14,15]. The NO-producing phagocytic cells appear to act as
one of the critical effectors of cellular immunity to produce
the direct anti-mycobactericidal effect [16,17]. Nevertheless,
the excessive production of NO can also enhance the damag-
ing effect, negatively affecting effector cells. This results in
apoptosis and necrosis. Thus, different concentrations of
NO can either stimulate or slow down apoptosis [17].
Very few studies exist that are devoted to the evaluation of
the levels and role of NO in patients with pulmonary TB. A
consistent decrease in the level of NO in TB was noted in
some publications [13]. But in other studies, the rise of NO lev-
els in patients with pulmonary TB was noted [18].
The significance of the problem and contradictory obser-
vations led to this study. The aim of this study was to evaluate
the effect of NO synthesis and respective metabolites in pa-
tients with various forms of TB during the intensive phase
of anti-tuberculosis therapy.
Materials and methods
Patients
This study involved 140 patients who were divided as follows:
Group 1 comprised of 74 patients with MDR-TB; Group 2 com-
prised of 66 patients with TB without MDR; and Group 3 com-
prised of 30 healthy individuals as controls; all subjects were
in the 20–70 years age range. Group 1 was further divided into
two subgroups; Group 1A with 41 patients who had recurrent
TB with MDR-TB (rMDR-TB) and Group 1B with 33 patients
with newly diagnosed MDR-TB (ndMDR-TB). Group 2 was also
Please cite this article in press as: DO Butov et al. Changes in nitric oxide synthase and nitrite and nitrate serum levels in patients with or without
MDR-TB undergoing the intensive phase of anti-tuberculosis therapy. Int. J. Mycobacteriol. (2014), http://dx.doi.org/10.1016/j.ijmyco.2014.02.003
x x x ( 2 0 1 4 ) x x x –x x x
subdivided into Group 2A consisting of 15 patients with rTB
without MDR-TB and Group 2B which had 51 patients with
newly diagnosed TB without MDR (Fig. 1). All patients had
an infiltrating form of pulmonary TB. The first-line drugs were
used as standard chemotherapy: isoniazid (0.3 g), rifampicin
(0.6 g), pyrazinamide (2 g), ethambutol (1.2 g) and/or strepto-
mycin (1 g) with dose reduction after the intensive phase of
the therapy. The patients were observed and treated at the Re-
gional TB Hospital No. 1 and Regional TB Dispensary
No. 1 – both in Kharkiv city; and Regional TB Dispensaries
Nos. 3 and 4 in Zmeyev and Izyum cities of Kharkiv region,
respectively. This study has received the ethics approval of
the committee at Kharkiv TB Hospital, and all patients have
given informed consent prior to study initiation.
Nitrites, nitrates and iNOS measurement
Taking into consideration the fact that NO is an unstable mol-
ecule with the half-life of less than 5 s, the study was aimed
to measure the content of stable serum metabolites of the
NO (nitrites, nitrates) and iNOS activity in neutrophils accord-
ing to previously published spectrophotometric methods
[19,20]. Measurements of serum samples of patients were
conducted during the first days after the admission to the
hospital and 2 months after the standard anti-mycobacterial
therapy. As NO metabolites can be influenced by exogenous
factors like diet, blood samples were collected prior to the
morning meal.
Statistical evaluation
The obtained data were evaluated by the standard Student
t-test [21]. The difference was considered to be significant at
p < 0.05.
Results and discussion
Table 1 shows that the initial activity of iNOS in the neutro-
phils and the levels of NO metabolites (nitrates and nitrites)
in the venous blood of all patients with pulmonary TB was
significantly greater – almost twice that as in the healthy do-
nors (p < 0.001). This suggests that prior to treatment there is
an intensive formation of NO in the patients with TB. Simi-
larly, there is a significant difference in these indices
(p < 0.001) in patients with all forms of TB before treatment
initiation as compared with the status after two months of
anti-tuberculosis therapy. The indicators of NO synthesis
and metabolism appeared to be higher in patients without
MDR than in patients with MDR-TB. After two months of
treatment the patients with MDR had indicators higher than
those without MDR-TB, which suggests a more intensive
reparative process correlating with a significant reduction of
MTB in patients without MDR as compared with those with
MDR. The NO indices were higher in patients with newly diag-
nosed pulmonary tuberculosis (NDPTB) than in the patients
with recurrent pulmonary tuberculosis (RPTB). However, after
two months the same indices were higher in patients with
RPTB than in those with NDPTB. Therefore, the changes in
NO indices are more profound in patients with RPTB than in
 International Journal of Mycobacteriology x x x ( 2 0 1 4 ) x x x –x x x 3

Study population
N=140

Group 1 Group 2 Group 3

N=74 N=66 N=30
With MDR-TB Without MDR-TB Healthy controls

Subgroup 1B Subgroup 2A Subgroup 2B

Subgroup 1A N=33 N=15 N=51
N=41 Newly diagnosed Recurrent TB Newly diagnosed TB
Recurrent MDR-TB MDR-TB

Fig. 1 – Composition of study population according to underlying diagnosis.

Table 1 – Nitric oxide synthase (iNOS) activity and levels of nitrates and nitrites in patients with pulmonary
tuberculosis prior to and after 2-month TB chemotherapy as compared with healthy controls (Mean ± SD).
Groups and Nitrates Nitrites iNOS
subgroups (lmol/L) (lmol/L) (pmole/min/mgB)
Before After 2 months Before After 2 months Before After 2 months
treatment of treatment treatment of treatment treatment of treatment

1 MDR-TB (N = 74) 62.89 ± 1.42a,b 46.65 ± 1.04a,b,d 5.626 ± 0.15a,b 4.21 ± 0.13a,b,d 231.6 ± 6.65a,b 114.9 ± 3.2a,b,d
1A recurrent MDR-TB 59.29 ± 1.79b,e,h 49.38 ± 1.3b,d,e,h 5.027 ± 0.17b,e,h 4.42 ± 0.14b,d,f,h 208.4 ± 8.26b,e,h 125.3 ± 4.5b,d,e,h
(N = 41)
1B new Dx MDR-TB 67.36 ± 2.03b,g,k 43.26 ± 1.5b,d,g,k 6.371 ± 0.19b,g,l 3.93 ± 0.13c,d,g,k 260.4 ± 8.56b,g,k 102 ± 3.53b,d,g,k
(N = 33)
2 TB (N = 66) 72.02 ± 1.43b 35.65 ± 1.06c,d 6.747 ± 0.17b 3.67 ± 0.09c,d 286.3 ± 5.92b 91.4 ± 2.53b,d
b,i
2A recurrent TB (N = 15) 66.26 ± 1.89 40.53 ± 1.83c,d,i 5.686 ± 0.2b,i
4.04 ± 0.19c,d,i 260.9 ± 14.12 b,i
106.7 ± 5.2b,d,i
2B new Dx TB (N = 51) 73.72 ± 1.71b,j 34.22 ± 1.19b,d,j 7.059 ± 0.19b,j 3.56 ± 0.1c,d,j 293.7 ± 6.13b,j 86.7 ± 2.59c,d,j
3 Controls (N = 30) 37.98 ± 1.3 3.83 ± 0.093 81.03 ± 2.36
a Discrepancy is significant (p < 0.001) when Groups 1 and 2 are compared.
b Discrepancy is significant (p < 0.05) when compared with Group 3.
c Discrepancy is not significant (p > 0.05) when compared with Group 3.
d Discrepancy is significant (p < 0.001) when compared before treatment and after two months levels among Subgroups.
e Discrepancy is significant (p < 0.05) when Subgroup 1A and 2A are compared.
f Discrepancy is not significant (p > 0.05) between Subgroups 1A and 2A.
g Discrepancy is significant (p < 0.01) between Subgroups 1B and 2B.
h Discrepancy is significant (p < 0.05) between Subgroups 1A and 1B.
i Discrepancy is significant (p < 0.05) between Subgroups 2A and 2B.
j Discrepancy is significant (p < 0.001) between Subgroups 1A and 2B.
k Discrepancy is not significant (p > 0.05) between Subgroups 2A and 1B.
l Discrepancy is significant (p < 0.05) between Subgroups 2A and 1B.

patients with NDPTB. When RPTB patients with MDR are com-
pared to patients with relapsed TB without MDR, the more
reliable indicators of NO were observed in TB patients without
MDR than in those with MDR before treatment (p < 0.05). After
two months of chemotherapy the indices were higher in TB
patients with MDR than in TB patients without MDR. The
same outcome was observed in patients with NDPTB with
or without MDR. The levels of nitrites after the two months
in the RPTB patients with MDR and without MDR were not
affected in a significant manner (p > 0.05). The comparison
of indices of RPTB with MDR and those of NDPTB without
MDR reveals a significant difference between these subgroups
(p < 0.05). Moreover, when RPTB without MDR is compared
with NDPTB with MDR, the differences were insignificant
(p > 0.05) both prior to treatment as well as after the two
months, which suggests that the clinical course is more se-
vere in patients with relapsed TB.
When NO indicators in healthy controls were compared
with treated TB patients, practically the same nitrite levels
were observed (p > 0.05) in the following subgroups: 1B, 2B

Please cite this article in press as: DO Butov et al. Changes in nitric oxide synthase and nitrite and nitrate serum levels in patients with or without
MDR-TB undergoing the intensive phase of anti-tuberculosis therapy. Int. J. Mycobacteriol. (2014), http://dx.doi.org/10.1016/j.ijmyco.2014.02.003
4 International Journal of Mycobacteriology
and 2A. The nitrate levels were the same in Groups 2 and 2A
and iNOS activity in Subgroup 2B. This suggests the
normalization of the NO metabolism in the above Groups
and Subgroups. In the remaining groups of patients the differ-
ences in NO indices were significant (p < 0.05) as compared
with healthy controls.
Conclusions
In patients with pulmonary TB, significantly higher levels of
NO activity were observed as compared with the levels in
healthy individuals. In patients with recurrent TB and MDR-
TB, significantly lower levels of NO indicators were observed
by comparison with patients with newly diagnosed pulmon-
ary tuberculosis.
After two months on chemotherapy, a significant decrease
in iNOS activity and NO metabolites was observed in patients
with pulmonary TB, but the decrease of NO indicators was
manifested mostly in the NDPTB patients and patients with-
out MDR as opposed to patients with recurrent TB and
MDR-TB, which suggests lower levels of immunologic and
reparative processes in such patients.
Therefore, the levels of nitrites and nitrates as well as iNOS
activity may serve as additional diagnostic criteria to differen-
tiate MDR-TB from non-resistant TB in patients with relapsed
and newly diagnosed TB. Easily assessed NO-related markers
can also serve as predictors of treatment outcome since pa-
tients with drug-susceptible strains had lower NO output
approaching levels found in controls. Further studies are war-
ranted to elucidate the relationship between NO and resis-
tance of mycobacteria to TB drugs.
Author contributions
Conceived and designed the trial: D.O. Butov, M.M.
Kuzhko. Carried out the trial: D.O. Butov, I.M. Kalmykova,
I.M. Kuznetsova, T.S. Butova, O.O. Grinishina, O.A. Mak-
simenko. Analyzed the data: D.O. Butov, M.M. Kuzhko. Wrote
the paper: D.O. Butov, M.M. Kuzhko, T.S. Butova.
Funding
The study was supported by a grant from the Ukrainian Min-
istry of Education. The funder had no role in study design,
data collection and analysis, decision to publish, or prepara-
tion of the manuscript.
Competing interests
The authors have declared that no competing interests exist.
Conflict of interest
We have no conflict of interest to declare.
Acknowledgements
We thank all volunteers who participated in this study. The
wholehearted support of clinicians, nurses and lab personnel
Please cite this article in press as: DO Butov et al. Changes in nitric oxide synthase and nitrite and nitrate serum levels in patients with or without
MDR-TB undergoing the intensive phase of anti-tuberculosis therapy. Int. J. Mycobacteriol. (2014), http://dx.doi.org/10.1016/j.ijmyco.2014.02.003
x x x ( 2 0 1 4 ) x x x –x x x
who contributed their effort made this study possible. Our
gratitude is expressed to many experts in TB and immunology
fields who kindly shared with us their opinions and critiques
prior to and after this study was completed.
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Please cite this article in press as: DO Butov et al. Changes in nitric oxide synthase and nitrite and nitrate serum levels in patients with or without
MDR-TB undergoing the intensive phase of anti-tuberculosis therapy. Int. J. Mycobacteriol. (2014), http://dx.doi.org/10.1016/j.ijmyco.2014.02.003