Nitric oxide and antioxidant enzymes in venous and cord blood of late preterm and term omani mothers.
ABSTRACT This study assessed the role of oxidative stress in parturition in Omani mothers following growing reports that late preterm neonates were at greater risk than term neonates of perinatal death.
Venous blood samples were collected during labour, and cord (neonatal) blood samples were taken after childbirth in late preterm and term from women at Sultan Qaboos University Hospital, Oman. Plasma nitric oxide (NO) concentrations, erythrocyte catalase (CAT). Erythrocyte glutathione peroxidase (GPx) activities were measured using spectrophotometric methods.
When compared with term mothers, late preterm mothers had markedly higher NO concentrations (μmol/L) 17.1 ± 3.3 versus 11.0 ± 5.5 (P <0.0001), and lower GPx values (U/g Hb) 94.1 ± 12.9 versus 110.4 ± 12.3 (P <0.0001). Late preterm mothers were significantly younger (P = 0.027) than term mothers and had neonates that weighed significantly less (P <0.0001) than term neonates. GPx activity was significantly reduced (P = 0.001) in late preterm neonates as compared to term neonates. CAT showed no change in activity in any comparison.
Distinctly higher values of NO and lower GPx activity were found in late preterm mothers relative to term mothers; also, lower GPx in late preterm neonates relative to term neonates suggested a pro-oxidant-antioxidant imbalance due to the greater oxidative burden in late preterm parturition.
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ABSTRACT: 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.The journal of maternal-fetal & neonatal medicine: the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstetricians 03/2014; · 1.36 Impact Factor
Sultan Qaboos University Med J, August 2012, Vol. 12, Iss. 3, pp. 300-305, Epub. 15th Jul 12
Submitted 11th Nov 11
Revision Req. 10th Mar 12, Revision recd. 22nd Mar & 21st Apr 12
Accepted 25th Apr 12
1Department of Biochemistry, College of Medicine & Health Sciences, Sultan Qaboos University, Muscat, Oman; 2Department of
Obstetrics & Gynaecology, Sultan Qaboos University Hospital, Muscat, Oman.
*Corresponding Author e-mail: firstname.lastname@example.org
مدلا في ةدسكلأل ةداضلما تايمزنلإاو كيترينلا ديسكأ
نادلولل تاينامُعلا تاهملأل يرسلا لبلحا مدو يديرولا
ينجضانلا نادلولاو نيرخأتلما نيرَسَتْبُلما
وداس�ام انيفول ،اكايبأا دروفيلك
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عمج تم :ةقيرطلا .ةَدلِولاب ِةَطيحُلما ِةَ ْ ترَفلا ُتاَيَفَو ثيح نم ينجس�انلا ةدلولا يثيدح نم بركأا رطخ في رخأاتم تقو في ناوألا لبق
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نيَسر�َتْبُلما نادلولا تاهمأا دنع كيترينلا ديس�كأا زيكرت ناك :جئاتنلاءارملحا مدلا تايركل نويثاتوللجا زيديس�كويرب طاس�ن ض�ايق تم امك
،)P <0.0001( 5.5 ± 11.0 ينجس�انلا نادلولا تاهمأا عم ةنراقلماب )ترل /لوموركيام( 3.3 ± 17.1 ظوحلم لكس�ب ىلعأا نيرخأاتلما
110.4 لباقم )ينبولغوميهلا نم مارج لكل ةدحو( 12.9 ± 94.1 ءارملحا مدلا تايركل نويثاتوللجا زيديس�كويرب طاس�نل ىندأا ميقو
)P = 0.027( ينجس�انلا نادلولا تاهمأا نم يرثكب انس� رغس�أا نيرخأاتلما نيَسر�َتْبُلما نادلولا تاهمأا تناكو .)P <0.0001( 12.3 ±
نويثاتوللجا زيديس�كويرب طاس�ن ناكو ،)P <0.0001( ينجس�انلا نادلولا نم يرثكب انزو لقأا نيرخأاتلما نيَسر�َتْبُلما نادلولا ناكو .
لمو .ينجس�انلا نادلولا عم ةنراقلماب نيرخأاتلما نيَسر�َتْبُلما نادلولا في )P = 0.001( ظوحلم لكس�ب اس�فخنم ءارملحا مدلا تايركل
تاهمأا دنع ىلعأا امزلابلا في كيترينلا ديس�كأا ميق تناك :ةص�لالخا .ينتعومجلما ينب ءارملحا مدلا تايرك زلاتاك طاس�ن في قرف يأا رهظي
دنع ءارملحا مدلا تايركل نويثاتوللجا زيديس�كويرب طاس�ن ناك امك ،ينجس�انلا نادلولا تاهمأل اهتيرظن نم نيرخأاتلما نيَسر�َتْبُلما نادلولا
ءارملحا مدلا تايركل نويثاتوللجا زيديس�كويرب طاس�ن في ض�افخنا كلذك ظحولو .ةيناثلا ةعومجلماب ةنراقم اس�فخنم لىوألا ةعومجلما
ةدايزل ارظن اهل ةداس�لماو ةدس�كألال ةيلاولما ةنزاوملل لخ ىلع لدي يذلاو ينجس�انلا نادلولل ةبس�نلاب نيرخأاتلما نيَسر�َتْبُلما نادلولا دنع
.ناوألا لبق ةدلولا في ةدس�كألا ءبع
داهجإلا ؛ةدلولا يثيدح ،ينجس�انلا لافطألا تاهمأا ،رخأاتم َسر�َتْبُم دلو ،نويثاتوللجا زيديكويرب ،زلاتك ،كيترنلا ديس�كأا :تاملكلا حاتفم
abstract: Objectives: This study assessed the role of oxidative stress in parturition in Omani mothers following
growing reports that late preterm neonates were at greater risk than term neonates of perinatal death. Methods:
Venous blood samples were collected during labour, and cord (neonatal) blood samples were taken after childbirth
in late preterm and term from women at Sultan Qaboos University Hospital, Oman. Plasma nitric oxide (NO)
concentrations, erythrocyte catalase (CAT). Erythrocyte glutathione peroxidase (GPx) activities were measured
using spectrophotometric methods. Results: When compared with term mothers, late preterm mothers had
markedly higher NO concentrations (µmol/L) 17.1 ± 3.3 versus 11.0 ± 5.5 (P <0.0001), and lower GPx values (U/g
Hb) 94.1 ± 12.9 versus 110.4 ± 12.3 (P <0.0001). Late preterm mothers were significantly younger (P = 0.027) than
term mothers and had neonates that weighed significantly less (P <0.0001) than term neonates. GPx activity was
significantly reduced (P = 0.001) in late preterm neonates as compared to term neonates. CAT showed no change
in activity in any comparison. Conclusion: Distinctly higher values of NO and lower GPx activity were found in
late preterm mothers relative to term mothers; also, lower GPx in late preterm neonates relative to term neonates
suggested a pro-oxidant-antioxidant imbalance due to the greater oxidative burden in late preterm parturition.
Keywords: Nitric oxide; Catalase; Glutathione peroxidase; Late preterm birth; Term birth; Neonates; Oxidative
Nitric Oxide and Antioxidant Enzymes in
Venous and Cord Blood of Late Preterm and
Term Omani Mothers
*Clifford Abiaka1 and Lovina Machado2
CLINICAL & BASIC RESEARCH
Clifford Abiaka and Lovina Machado
Clinical and Basic Research | 301
Advances in Knowledge
- Oxidative stress is more a feature of parturition at late preterm than at term.
- Late preterm mothers are at greater risk of oxidative stress than term mothers.
- Late preterm neonates are more susceptible to oxidative stress than term neonates.
Application to Patient Care
- Identifying modifiable factors in the obstetric setting may be an inexpensive and noninvasive therapy for preventing preterm parturition.
- Antioxidants (including the carotenoids, and vitamins C and E) and antioxidant cofactors (such as copper, selenium, and zinc) are
capable of disposing, scavenging, or suppressing the generation of reactive oxygen species implicated in oxidative stress.
- Physicians should identify antioxidant-laden diets and educate pregnant Omani women about their benefits, and also advise them to
take daily multivitamins and microminerals throughout pregnancy and beyond.
- Regular assays of plasma nitric oxide (NO) and erythrocyte glutathione peroxidase (GPx) should be considered in suspected late
greatest number of obstetric interventions and
adverse neonatal outcomes occur at late preterm
births.4 Compared with term neonates, late
preterm neonates are at higher risk for hospital
readmissions, post-neonatal mortality, sudden
infant death syndrome, white matter injury, and
neuro-developmental problems well into the school
Nitric oxide (NO) is the product of a five-
electron oxidation of L-arginine mediated by one
of three NO synthases: endothelial, neuronal, and
inducible NO synthases (eNOS, nNOS and iNOS).9
Neuronal NOS and eNOS are responsible for the
continuous basal release of NO. Endothelial NOS
is expressed in theca cells, granulosa cells, and the
surface of oocyte during follicular development.10,11
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.12,13 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
reactive and cytotoxic pro-oxidant, peroxinitrite
anion (ONOO-).13–15 Both glutathione peroxidase
(GPx) and catalase (CAT) function as enzymatic
antioxidants by neutralising pro-oxidants and
thus prevent damage to the cellular structure. The
selenoprotein, GPx, is cytosolically located in most
tissues. It catalyses the breakdown of hydrogen
peroxide (H2O2) and organic peroxide (ROO)
supported by reduced glutathione (GSH) to form
glutathione disulphide (GSSG), water, and organic
he gestational age window termed
“late preterm birth” occurs between 34
weeks 1 day and 36 weeks 6 days.1–3 The
.-) to form the highly
alcohol (ROH). In erythrocytes from adult humans,
all GPx activity appears to be selenium-dependent.16
Catalase, a haemoprotein, specifically converts
H2O2 to yield O2, and H2O, and does not decompose
In this context, information about oxidative
stress in parturition at late preterm and term is
lacking. The objective of this study was to assess
the strength of oxidative stress at parturition
by measuring and comparing concentrations of
plasma NO, and activities of erythrocyte GPx and
CAT in venous and cord blood of Omani mothers
who delivered at late preterm and at term.
Ethical approval was granted for the study by the
research review board at the authors’ institution.
Enrollment was voluntary, and all participants
signed a consent form previously approved by the
institution’s ethics committee for the protection of
human subjects in research. A total of 37 mothers
who delivered at late preterm were registered, as
well as 37 mothers who delivered at term. They were
non-smokers, normotensive, and had singleton
gestations ranging from 34 to 42 weeks. Gestational
age at entry was determined by an obstetric estimate
of the last menstrual period, uterine size, and
ultrasound examination.17 Late preterm mothers
delivered between 34 weeks and <37 weeks and
their ages ranged from 14–44 years (mean 28 years).
Term mothers delivered between 37 and 42 weeks
and were aged 22–45 years (mean 33 years). No
medical complications were encountered during
pregnancy and all deliveries occurred vaginally
and were free of artificial support. The cord blood
was deemed neonatal. Late preterm neonates (20
Nitric Oxide and Antioxidant Enzymes in Venous and Cord Blood of Late Preterm and Term Omani Mothers
302 | SQU Medical Journal, August 2012, Volume 12, Issue 3
males, 17 females) born to late preterm mothers,
had a mean ± SD birth weight of 2,463 ± 415 g.
Term neonates (17 males, 20 females) born to term
mothers, had a mean ± SD birth weight of 3,292 ±
Maternal venous blood samples were taken
during labour from the median cubital vein into
evacuated lithium heparinised tubes. Samples of
umbilical cord blood were collected immediately
after delivery by aseptic puncture of the umbilical
veins connected to the maternal placenta into
evacuated lithium heparinised tubes. The specimens
were centrifuged at 4o C; cord and venous plasma
were separated for assay of NO. Haemolysates were
prepared from the packed cells for the assays of
GPx and CAT. Measurement of NO concentration
was performed in a 96-well microtitre plate
using reagents from Cayman (Cayman Chemical
Company, Michigan, USA) and was a two-step
process based on the method of Green et al.18
The first step is the conversion of nitrate to nitrite
utilising nitrate reductase; the second is the addition
of the Greiss reagents which, on reaction with
nitrite, develop a deep purple azo chromophore.
Absorbance is read after 10 minutes at 540 nm using
a plate reader, the Thermo Labsystem Multiskan
Spectrum (Thermo Electron Corporation, Zantaa,
Finland). Standards, controls and samples were
measured in triplicate and expressed in µM
Haemolysates were prepared from the remaining
packed cells as previously described for the GPx
assay, using Ransel kits.19 Ransel controls (Randox
Laboratories, Crumlin, UK) were used to monitor
the GPx activity.
Catalase activity in the haemolysates and
controls was assayed in a 96-well microtitre
plate, using catalase assay kits (Cayman Chemical
Company, Michigan, USA), which utilise the
peroxidative function of CAT for determination
of enzyme activity. The method is based on the
reaction of CAT with methanol in the presence
of an optimal concentration of H2O2 to form
formaldehyde, measured with purpald (4-amino-
3-hadrazino-5- mercapto-1,2,4-triazole) as the
chromogen.20,21 Purpald specifically forms a bicyclic
heterocycle with aldehydes, which upon oxidation
changes from colourless to a purple colour.19,20
The absorbance was read at 540 nm using a plate
reader, the Thermo Labsystem Multiskan Spectrum
(Thermo Fisher Scientific, Massachusetts, USA).
The imprecision study was done using the
internal quality control sera supplied in the
commercial kits. Analyses of twelve aliquots of
the controls revealed an intra-assay coefficient of
variations (%) of 2.9, 3.6 and 4.1 for NO, GPx and
CAT, respectively; the inter-assay CV values (%)
performed over six days were 3.6, 5.0 and 5.4 for
NO, GPx and CAT, respectively.
Each data set was confirmed to have Gaussian
distribution by the one-sample Kolmogorov-
Smirnoff test and, additionally, by histogram plots.
Then all data were presented as mean ± SD. The
mean differences between more than two groups
were determined by one-way analysis of variance
(ANOVA) using Scheffe’s post-hoc test for a multiple
comparison. Probability (P) values were two-tailed
and P <0.05 was considered to be significant.
The data comparisons (mean ± SD) of the
parameters of late preterm versus term neonates
are presented in Table 1. Late preterm neonates
weighed significantly (P = <0.0001) less and had
lower GPx activity (P = 0.001) as compared to
term neonates. NO concentrations and CAT
activity were similar in both groups of neonates.
Table 2 represents maternal data (mean ± SD) of
age, NO, GPx and CAT. Late preterm mothers were
significantly younger (P = 0.027), had strikingly
higher NO concentrations (P <0.0001), and a
markedly lower level of GPx activity (P = <0.0001)
relative to term mothers. Catalase activities of both
mothers were similar.
Table 1: Comparisons of parameters (mean ± SD) of
birth weights, nitric oxide, glutathione peroxidase, and
catalase of term neonates versus late preterm neonates
(n = 37)(n = 37)
Birth weight (g)
2,463 ± 4153,292 ± 491<0.0001
5.32 ± 3.675.07 ± 2.960.910
GPx (U/g Hb)
48.8 ± 22.777.6 ± 26.90.001
96.5 ± 44.391.3 ± 45.80.993
Note: Statistical significance was considered at P <0.05.
Legend: NO = nitric oxide; GPx = glutathione peroxidase; CAT=
Clifford Abiaka and Lovina Machado
Clinical and Basic Research | 303
Data on the effect of gender on neonatal
parameters are omitted because the differences did
not attain statistical significance.
Earlier studies carried out on animal and
human tissues generally reported decreasing NO
synthesis concomitant with declining NOS activity
as gestation approached term.22–28Higher NO values
found in late preterm mothers compared with term
mothers Table 2 complement those reports.22–28
Some earlier studies reported a greater expression
of the isoforms of NOS (eNOS, iNOS) in the
myometrium of the preterm ‘not in labour’ group
compared with all other groups: non-pregnant
women, pregnant women in the early third trimester,
and pregnant women at term (both before and after
labour).27,28 Since iNOS promotes inflammation,
and inflammation in turn promotes pro-oxidant
formation, this may explain why late preterm
mothers and not term mothers had more elevated
plasma NO concentrations and reduced erythrocyte
antioxidant (GPx) activity Table 2.12 High pro-
oxidant levels, coupled with reduced antioxidants,
signify a disturbance of the pro-oxidant-antioxidant
balance otherwise known as oxidative stress.29,30
The placenta synthesises, on a weight-for-weight
basis, more NO than the foetal membrane or
the myometrium.26 From early pregnancy, the
mitochondria replete placenta influences maternal
homeostasis and consumes about 1% of the basal
metabolic rate of the pregnant woman.31 The
placenta in addition, is highly vascular and, on
exposure to high maternal oxygen tension, generates
the mitochondrial respiratory chain seep out
of the mitochondria.31 Superoxide dismutase is
.-); about 5% of all electrons in
the vanguard cytoprotective enzyme in that it
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 (organic) hydroperoxides
(ROOH). Hence, it follows that GPx, because of
its greater antioxidant burden as compared to CAT
was the cytoprotective enzyme most utilised and
consistently depleted in this study [Tables 1 and
2]. According to a literature search, there are no
previous studies related to the effect of GPx and
CAT on late preterm and term labour or parturition
to which it is possible to make comparisons.
.- to O2 and H2O2 the latter
The findings of the present study suggest that
oxidative stress was more a feature of childbirth
at late preterm than at term. It is anticipated
that this information would be of use to the
medical community in general, and particularly,
to obstetricians, midwives, and neonatologists
who deal with neonatal morbidity, high neonatal
hospital readmissions, and post-neonatal mortality,
including sudden infant death syndrome among
The authors would like to thank Ms. Barbara Swales
(head midwife) and the midwives of the delivery
ward at SQUH for their cooperation and assistance
in sample collection for this study.
CONFLICT OF INTEREST
The authors declared no conflict of interest.
Table 2: Comparisons of parameters (mean ± standard deviation) of age, nitric oxide, glutathione peroxidase, and
catalase of term mothers versus late preterm mothers
(n = 37)
Late Preterm MothersTerm Mothers
(n = 37)
Age (years)27.5 ± 6.532.5 ± 6.00.027
NO (µmol/L)17.1 ± 3.311.0 ± 5.5<0.0001
GPx (U/g Hb)94.1 ± 12.9110.4 ± 12.3<0.0001
CAT (nmol/min/mL)93.9 ± 32.696.3 ± 32.30.998
Note: Statistical significance was considered at P <0.05.
Legend: NO = nitric oxide; GPx = glutathione peroxidase; CAT= catalase.
Nitric Oxide and Antioxidant Enzymes in Venous and Cord Blood of Late Preterm and Term Omani Mothers
304 | SQU Medical Journal, August 2012, Volume 12, Issue 3
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