Science Journal of Medicine & Clinical Trials
ISSN:2276-7487
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© Author(s) 2013. CC Attribution 3.0 License.
Research Article
International Open Access Publisher
Volume 2013, Article ID sjmct-262, 6 Pages, 2013. doi: 10.7237/sjmct/262
Nitric Oxide and Antioxidant Enzyme Levels in Blood of Respiratory Distress Syndrome
Egyptian Preterms and Their Mothers
Hosny M.A. El‑Masry¹, Ahmed A. M. Nasr², Ashraf M. Al Kabeer³, H.h.amin⁴ and Hussein M.h. Eldeeb⁵
Department of Pediatrics ¹, Obstetrics & Gynecology², Internal Medicine³, Clinical Pathology⁴ and Medical Biochemistry⁵, Faculty of Medicine- Al-azhar
University-egypt - Assiut.
Accepted 22�� May, 2013
Abstract
Background: The mechanisms of tissue injury in respiratory distress
syndrome (RDS) have not been completely elucidated but the involvement
of oxidative damage due to reactive oxygen species (ROS) is important in
the pathogenesis of preterm labor and RDS.
Objectives: to assess the oxidative status in preterms with and without
RDS and in their mothers.
Patients and methods: Measuring and comparing concentrations of serum
nitric oxide (NO), and erythrocytic activities of some antioxidant enzymes
in blood of 20 preterms with RDS and 20 preterms without and their
mothers respectively.
Results: Our results confirm significant elevation of mean levels of serum
NO and reduced mean levels of erythrocytic activities of antioxidant
enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx) and
catalase(CAT), in RDS preterms and their mothers compared with controls
and their mothers. A significant positive correlations were found between
maternal and preterm serum NO (r = 0.3 & 0.6) and erythrocytic activity
of SOD (r =0.5 & 0.4) for RDS and control groups respectively. In addition,
a significant negative correlations were found between NO and SOD in all
groups
Conclusions: The existence of oxygen metabolites and lipid peroxidation
was significantly more obvious in RDS preterms and in their mothers than
those without.
Keywords:RDS,
preterm labor, oxidative stress, nitric oxide, and
antioxidant enzymes.
Introduction
Free radicals are known to be produced during normal
metabolic processes, however production is tightly
controlled by antioxidant enzymes (valco et al; 2007).
Oxidative stress during pregnancy yields free radicals and
other oxidative molecules exceeding the available
antioxidant buffering capacity in the mother and growing
fetus (Burton and Jauniaux et al; 2010) . Under diseased
conditions the regulation in the activity of any of the
enzymes may lead to accumulation of high concentration of
free radicals resulting in free radical injury. These free
radicals may damage DNA, proteins, lipids and other
biological macromolecules. Polyunsaturated fatty acids are
highly susceptible to free oxygen radicals which lead to lipid
peroxidation resulting in membrane dysfunction and
eventually cell death, which is associated not only with
preterm labor (PTL) and delivery, but also preeclampsia
(PE), preterm premature rupture of membranes (PPROM)
and intra uterine growth restriction (IUGR) as well as
several serious post-delivery complications of prematurity
such as respiratory distress syndrome (RDS). (Joshi et al.,
2008) Various actions of nitric oxide (NO) are known to
contribute both to the maintenance of pregnancy and to the
onset of labor. All three NO synthase (NOS) isoforms so far
identified (n, neuronal; i, inducible; and e, endothelial) have
been identified in human myometrium, iNOS production of
NO in the myometrium and placenta increases throughout
pregnancy before dropping sharply at term (von Mandach
et al 2003).
Inducible NOS expressed in monocytes and macrophages in
response to pro-inflammatory cytokines catalyses the
synthesis of a large amount of NO in pathological conditions
(Liu et al; 2002). NO itself comprises a nitrogen atom bound
to an oxygen atom, forming a reactive nitrogen species
depicted as "NO, a short-lived, readily diffusible molecule
that reacts with superoxide (O2.-) to form the highly reactive
and cytotoxic pro-oxidant, peroxynitrite anion (ONOO-)
(Dweik; 2005). Superoxide dismutase (SOD), glutathione
peroxidase (GPx) and catalase (CAT) function as enzymatic
antioxidants by neutralizing pro-oxidants and thus prevent
damage to the cellular structure. Information about
oxidative stress in preterms with RDS and their mothers is
lacking.
The objective of this study was to assess the oxidative
status in preterms with and without RDS and in their
mothers by measuring and comparing concentrations of
serum NO, and erythrocytic activities of SOD, GPx and CAT
in blood of preterms and their mothers.
Patients and Methods
The study was performed on forty preterm neonates (<
37weeks) admitted to neonatal unit and their mothers
admitted to obstetrics department in Al Azhar Assiut
university hospital - Egypt. They were classified according
to the presence or absence of neonatal RDS into four groups.
Group (I): 20 preterms with RDS, Group (II): 20 preterms
without RDS as controls, Group (A): 20 mothers of group
(l) and Group (B): 20 mothers of group (ll). Detailed base
line informations with regard to maternal (age, social status,
pregnancy spacing, medical disorders, PPROM, mode of
delivery….etc.), intrapartum and neonatal variables were
recorded. Gestational age was assessed by date of last
menstrual period, ultrasonographic findings corresponding
to the period of amenorrhea and by the method of scoring
of Ballard (Ballard et al; 1991). Hyaline membrane disease
was considered to be present on the basis of clinical and
radiological pictures. Blood gas analysis was done twice in
24 hours for all preterms with RDS. Sepsis was ruled out by
blood culture and sepsis screen investigations. Infants with
congenital heart disease and other lung diseases were
excluded. Blood sample was collected from preterms and
their mothers within 12-24 hours after labor in heparinized
Corresponding Author: Hosny M.A. El-masry
DEPARTMENT OF PEDIATRICS, FACULTY OF MEDICINE- AL-AZHAR UNIVERSITY-EGYPT - ASSIUT.
Email:hosny5219672000@yahoo.com
Science Journal of Medicine & Clinical Trials (ISSN:2276-7487)
glass tubes for the estimation of serum NO and erythrocytic
activities of antioxidant enzymes (SOD, GPX, and Catalase).
). Serum nitric oxide was determined by incubating 50-100
ul with an equal volume of griess reagent at room
temperature for 10 min. The absorbance at 550 nm was
determined. NO was determined by using sodium nitrite as
a standerd (Ding etal., 1988). SOD activity was determined
by the method of Misra and Fridovich 1972 one enzyme
unit is defined as a quantity of protein which inhibits
auto-oxidation of epinephrine by 50% under specified
conditions. GPx activity was monitored at 340 nm by
following the method of Leopold and Wolfgang 1984.
Catalase was assayed by determining the decomposition of
hydrogen peroxide (H2O2) at 230 nm by method of Aeby
1984. The biochemist conducting the analysis was not aware
of the test or control group status. Data was collected,
revised, coded, tabulated and introduced to a PC using
page 2
Statistical Package for Social Science (SPSS 15.0.1 for
windows; SPSS Inc, Chicago, IL, 2001). Descriptive analysis
was performed for demographic and clinical characteristics
of the patients. Data was represented by Mean ± SD.
Correlation coefficient (r) was used to study the correlation
between two quantitative variables. Chi- square (x²) was
used to compare between two groups in a qualitative
parameter.
Results
,
During the study period forty five preterm neonates were
delivered to forty mothers as a result of five twin
pregnancies. Out of them, five preterms not fulfilling the
inclusion criteria due to the presence of congenital heart or
lung diseases, were excluded from our results (3 from group
I and 2 from group II).
Table (1): Demographic, clinical characteristics and maternal risk factors of Controls preterm (group l) and
preterm with respiratory distress syndrome (RDS preterm group II).
Group I
Control Preterm
(n=20)
Demographic and clinical characteristics
*Gestational Age (Wks.)
34.5±1.58
Male
9 (45%)
Sex
Female
11 (55%)
*Birth Weight (Kg)
2.19±1.02
*Length (cm)
42.4±3.84
*HC in cm
31.7±1.89
Cephali
11(55%)
Fetal presentation
Breach
8(40%)
Transverse
1(5%)
12 (60%)
CS
Mode of delivery
NVD
8 (40%)
1 Min
5.6±0.50
*APGAR score
5 Min
6.8±1
Maternal risk factors
< 18
9 (45%)
Maternal age (years)
>35
11 (55%)
Pregnancy spacing < 6 months
3 (15%)
Poor socioeconomic status
12 (60%)
Multiple pregnancy
3 (15%)
Previous preterm labor
1 (5%)
Diabetes mellitus
3 (15%)
Preeclampsia
1 (5%)
Rheumatic heart disease
0 (0%)
Genito-urinary infections
1 (5%)
Antepartum Hge
5 (1%)
Incompetent cervix
0 (0%)
Polyhydramnios
2 (10%)
PROM >18 hrs.
1 (5%)
Variables
Perinatal asphyxia
2 (10%)
Group II
(RDS preterm)
(n=20)
33.9±1.64
8 (40%)
12 (60%)
2.21±0.13
41.8±2.17
30.9±1.51
12(60%)
7 (35%)
1 (5%)
14(70%)
6(30%)
4.1±0.55
5.50±1.3
10 (50%)
10 (50%)
4 (20%)
13 (65%)
2 (10%)
3 (15%)
5 (25%)
3 (15%)
1 (5%)
3 (15%)
0 (0%)
1 (5%)
2 (10%)
2 (10%)
4 (20%)
Values are given as no. (%)
* mean ± SD
NV: Normal vaginal delivery (NVD)
PROM: premature rupture of membrane
N.B: no significant differences were found between both groups.
How to Cite this Article: Hosny M.A. El-Masry, Ahmed A. M. Nasr, Ashraf M. Al Kabeer, H.h.amin, and Hussein M.h. Eldeeb “Nitric Oxide and Antioxidant Enzyme Levels in Blood
of Respiratory Distress Syndrome Egyptian Preterms and Their Mothers, Science Journal of Medicine & Clinical Trials, Volume 2013, Article ID sjmct-262, 6 Pages, 2013. doi:
10.7237/sjmct/262
Page 3
Science Journal of Medicine & Clinical Trials (ISSN:2276-7487)
Table (2): Mean ± SD levels of nitric oxide and antioxidant enzymes in the studied preterms and their mothers
Variables
Mothers of preterms (n=40)
Group A
Group B
Mothers of Mothers of
control
RDS
P
preterms preterms
(n=20)
(n=20)
11±5.6
17.2±3.3
***
Group I
Control
Preterms
(n=20)
Group II
(RDS
preterms)
(n=20)
p
5.33 ±2.96
9.97±3.6
**
SOD (units/ml)
15.4±9.8
7.8±3.5
**
20.9±5.2
11.6±4.3
**
GPx (U/g Hb)
78.7±27.1
49.9±23.8
**
111.5±13.4
93.2±11.8
***
CAT
(nmol/min/ml)
92.4 ±46.9
76.5±47.4
*
98.5±35.2
80.6±43.2
*
NO (umol/L)
Antioxidant
enzymes
Preterms (n=40)
*: significant (p<0.05)
**: highly significant (p<0.01)
RDS: Respiratory distress syndrome
SOD: Superoxide dismutase
CAT: Catalase
***: very highly significant( p<0.001)
NO: nitric oxide
GPx: Glutathione peroxidase
How to Cite this Article: Hosny M.A. El-Masry, Ahmed A. M. Nasr, Ashraf M. Al Kabeer, H.h.amin, and Hussein M.h. Eldeeb “Nitric Oxide and Antioxidant Enzyme Levels in Blood
of Respiratory Distress Syndrome Egyptian Preterms and Their Mothers, Science Journal of Medicine & Clinical Trials, Volume 2013, Article ID sjmct-262, 6 Pages, 2013. doi:
10.7237/sjmct/262
Science Journal of Medicine & Clinical Trials (ISSN:2276-7487)
Page 4
How to Cite this Article: Hosny M.A. El-Masry, Ahmed A. M. Nasr, Ashraf M. Al Kabeer, H.h.amin, and Hussein M.h. Eldeeb “Nitric Oxide and Antioxidant Enzyme Levels in Blood
of Respiratory Distress Syndrome Egyptian Preterms and Their Mothers, Science Journal of Medicine & Clinical Trials, Volume 2013, Article ID sjmct-262, 6 Pages, 2013. doi:
10.7237/sjmct/262
Page 5
Discussion
Our results confirm significant elevation of mean levels of
serum NO and reduced mean levels of erythrocytic activities
of antioxidant enzymes (SOD, GPx and catalase) in both RDS
preterms and their mothers compared with control
preterms and their mothers (table 2). Our results were found
to be in consistence with Farkas et al; (2008) who have
shown in their study increased oxidant stress markers and
reduced antioxidant defense in preterm infants with RDS.
Also in a study done by Russell; (1994) activities of
antioxidant enzymes (SOD, catalase and GPx) were found to
be depressed whereas peroxidation was significantly higher
in the newborns dying due to RDS indicating overproduction
of oxygen free radicals in these neonates. In addition Dizdar
et al; (2011) found lower levels of total antioxidant capacity
and higher levels of total oxidant species in preterms prior
to surfactant therapy. Clifford and Lovina; (2012) also
have shown in their study on preterm and term neonates
and their mothers a distinctly higher values of NO and lower
GPx activity in mothers of preterm relative to mothers of
term neonates in addition to a lower GPx activity in preterm
relative to term neonates. However in contrary to our study
conflicting findings exist that showed a similar oxidant and
antioxidant status in preterms with and without RDS
(Frosali et al; 2004 and Hamon et al;2005).
The results of our study may suggest an existence of more
oxidative stress in preterms with RDS than those without.
The mechanisms of tissue injury in RDS have not been
completely elucidated but the involvement of oxidative
damage due to reactive oxygen species is important in the
pathogenesis. Pulmonary surfactant normally contains
significant amounts of SOD and catalase, suggesting that
antioxidant enzymes are present to help prevent lipid
peroxidation and surfactant inactivation by ROS (Davis and
Auten; 2010). The rapid transition of the fetus from a
relatively hypoxic to a relatively hyperoxic environment at
birth gives rise to oxidative stress. However, oxidant defense
mechanisms are induced late in gestation. Antioxidant
enzyme systems are up regulated primarily during the latter
part of gestation and at the same time non-enzymatic
antioxidants cross the placenta in increasing quantities. The
exposure of the preterm lung to increased oxygen, which is
often necessary for survival, contributes to excessive
production of ROS in the respiratory system. Thus,
premature infants are more vulnerable to this oxidative
stress (Dizdar et al; 2011). The highly reactive oxidant
species led to damage to cellular macromolecules including
proteins, carbohydrates, lipids and nucleic acid as well as
surfactant. The resulting damage promotes inflammation.
ROS also have important effects on different lung cells as
regulators of signal transduction, activators of key
transcription factors and modulators of gene expression and
apoptosis, resulting in a number of inflammatory pulmonary
diseases including RDS (Gitto et al;2001).
Pregnancy is a state of oxidative stress arising from
increased placental mitochondrial activity and production
of reactive oxygen species (ROS), mainly superoxide anion.
The placenta also produces other ROS including nitric oxide,
carbon monoxide, and peroxynitrite which have pronounced
effects on placental function including trophoblast
proliferation and differentiation and vascular reactivity.
Excessive production of ROS may occur at certain windows
Science Journal of Medicine & Clinical Trials (ISSN:2276-7487)
in placental development and in pathologic pregnancies,
such as those complicated by preeclampsia and/or IUGR,
overpowering antioxidant defenses with deleterious
outcome (Myatt L and Cui X;2004).
In the current study, a significant positive correlations were
found between maternal and preterm serum NO levels (r =
0.3 & 0.6 for RDS and control groups respectively) figure (1),
also a significantly positive correlations were found between
maternal and preterm erythrocytic activity of SOD (r =0.5 &
0.4 for RDS and control groups respectively) figure (2).
These results could be explained by some earlier studies
which confirm increase nitrate plus nitrite levels in amniotic
fluid and maternal plasma between early and late normal
and abnormal pregnancy, together with a slight decline
shortly before delivery (von Mandach et al;2003). Also
other studies reported a greater expression of the isoforms
of NOS (eNOS, iNOS) in the myometrium of mothers of
preterm compared with non-pregnant women or pregnant
women at term (Bansal et al; 1997 and Clifford and
Lovina et al 2012). The placenta synthesizes, on a weightfor-weight basis, more NO than the fetal membrane or the
myometrium and iNOS promotes inflammation, and
inflammation in turn promotes pro-oxidant formation that
induce preterm labor and RDS (Angelidou et lal; 2002).
Superoxide dismutase is the vanguard cytoprotective
enzyme in that it disproportionates superoxide anion to O2
and H2O2, the latter being a substrate for GPx and CAT.
Glutathione peroxidase and CAT are both predominantly
intracellular cytoprotective enzymes, but in addition to
scavenging H2O2, GPx and not CAT, also scavenges lipid
hydroperoxides (Clifford and Lovina et al; 2012).
In this context the high prooxidant levels, coupled with
reduced antioxidant enzymes, signify a disturbance of the
pro-oxidant-antioxidant balance and this may explain the
significant negative correlations that were found between
NO and SOD in all studied groups (figure 3).
Conclusion and Recommendations
As per the above observations there is possibility that
oxidative stress in preterms and their mothers could play, a
significant role in tissue damage of RDS. Preterm newborns
are also capable of counteracting some of these free radicals
by the consumption of antioxidant enzymes. Thus, it would
be significant to use the key antioxidants as a preventive
measure to reduce the state of injury in the preterm labor
and RDS.
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How to Cite this Article: Hosny M.A. El-Masry, Ahmed A. M. Nasr, Ashraf M. Al Kabeer, H.h.amin, and Hussein M.h. Eldeeb “Nitric Oxide and Antioxidant Enzyme Levels in Blood
of Respiratory Distress Syndrome Egyptian Preterms and Their Mothers, Science Journal of Medicine & Clinical Trials, Volume 2013, Article ID sjmct-262, 6 Pages, 2013. doi:
10.7237/sjmct/262
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How to Cite this Article: Hosny M.A. El-Masry, Ahmed A. M. Nasr, Ashraf M. Al Kabeer, H.h.amin, and Hussein M.h. Eldeeb “Nitric Oxide and Antioxidant Enzyme Levels in Blood
of Respiratory Distress Syndrome Egyptian Preterms and Their Mothers, Science Journal of Medicine & Clinical Trials, Volume 2013, Article ID sjmct-262, 6 Pages, 2013. doi:
10.7237/sjmct/262