Oxidant-antioxidant status in pair-matched maternal and cord blood of normo & preeclamptic patients

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ARTICLES

Oxidant唱 antioxidant status in pair唱 matched maternal and cord blood of normotensive and preeclamptic patients

Mohd Suhail ,Mohd Faizul唱 Suhail 1,2

[ Abstract] Objective Preeclampsia is associated with oxidative stress in maternal circulation.An increase in lipid peroxidation products and a decrease in antioxidant activity in preeclamptic women have been reported by us and other workers.The aim of the present study was to evaluate oxidative stress in infants born to preeclamptic mothers.Methods We investigated the concentrations of nitric oxide( NO) ,malondialdehyde( MDA) ,reduced glutathione( GSH) ,and uric acid in the cord blood of the newborns from pregnancies complicated by preeclamp唱 sia,comparing with that of normal pregnancies.Their levels were also estimated in pair唱 matched maternal blood of both normotensive and preeclamptics.Results MDA concentration was 9畅58 ±1畅89 nmol / gHb in preeclamptic maternal blood,significantly higher compared to 6畅86 ±0畅88 nmol / gHb in normotensive maternal blood,whereas its concentration was 7畅52 ±1畅25 nmol / gHb in cord,significantly low compared to pair唱 matched preeclamptic maternal blood.NO concentration, in terms of stable metabolites was 26畅46 ± 4畅65 μmol / L and 28畅15 ± 4畅20 μmol / L in preeclamptic maternal and cord plasma respectively,both significantly higher compared to pair唱 matched control plasma( maternal =21畅13 ±4畅06 μmol / L and cord =19畅22 ±3畅79 μmol / L) .GSH level was found to be 8. 74 ±1. 57 μmol / gHb in control maternal blood and 6畅96 ±2畅08 μmol / gHb in preeclamptic mater唱 nal blood.However,there was no significant difference between preeclamptic maternal and cord blood( 5畅97 ± 1畅54 μmol / gHb) .Uric acid content was 497畅67 ±44畅92 μmol / L in preeclamptic maternal plasma and 526畅3 ± 75畅3 μmol / L in cord plasma, significantly higher in comparison to their respective normal maternal (278畅5 ± 57畅0 μmol / L) and cord(301畅9 ±69畅9 μmol / L) levels.Conclusions The higher levels of NO and uric acid in cord blood and insignificant change in GSH levels in paired唱 matched preeclamptic maternal and cord blood indi唱 cate a compensatory role in neonates born to preeclamptic group.Thus,we conclude that the oxidative stress status is low in the blood of neonates compared to its level in the pair唱 matched preeclamptic mothers.Further studies are needed to explore strategies so that the normal levels of antioxidants are maintained to combat preeclampsia in women at high risk,including increased risk of fetal growth restriction and induced premature delivery due to dis唱 ease progression on the maternal or fetal side. [Key words] malondialdehyde;nitric oxide; glutathione;uric acid;maternal and cord blood;normotensive and preeclamptic women INTRODUCTION Preeclampsia( PE ) is a complex multisystem disorder that is associated with hypertension,edema and protein唱 uria during pregnancy.For the fetus,the PE syndrome could have serious consequences, including increased

risk of fetal growth restriction and induced premature delivery due to disease progression on the maternal or [1,2] fetal side .Maternal endothelial dysfunction is believed to be the common pathway leading to the [3] syndrome of PE .Nitric oxide ( NO ) mediates many

1 Department of Biochemistry,University of Allahabad,Allahabad唱 211002,India

2 City Nursing & Maternity Home Research Center,21,Minhajpur,Allahabad唱 211003,India

Correspondence to Prof.Mohd Suhail,Honorary Director,City Nursing & Maternity Home Research Center,21,Minhajpur Allahabad唱 211003,India E唱 mail:profmsuhail@gmail.com


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functions of the endothelium,including vasodilation and inhibition of platelet aggregation.Significantly, higher [4,5] levels of NO in cord blood have been reported in [6,7] PE whereas others showed no significant change [ 8] and decreased levels in their contents. Further, that the oxidative stress has been pro唱 posed as a key factor involved in the development of PE.An increased risk of hypertension in the adolescent offspring was reported as the consequence of long唱 term [9,10] effects of intrauterine exposure to PE .Oxidative stress marker, malondialdehyde ( MDA) levels change during the development of PE.Significant elevation of MDA levels in cord blood of pair唱 matched preeclamptic [11,12] mother have been reported whereas others have [ 13] concluded decline in its level or no significant [14] change .Nevertheless, elevated MDA levels both in cord and mother during preeclamptic development than [15] in normal pregnancy have also been reported .

Premature infants, with their decreased antioxidant defense,are susceptible to oxidant injury and diseases as唱 [16,17] sociated with oxidative stress . However,higher level of antioxidant,reduced glutathione(GSH)has been repor唱 [18] ted in cord blood of hypertensive mother as compared [19,20] to normotensive one.Others have reported signifi唱 cantly lower contents of GSH in the pair唱 matched cord blood of preeclamptic patients as compared to normoten唱 [14] sive women but same workers , later on,reported no significant change in cord GSH levels of the preeclamptic patients. Inconsistency in these reports has led us to take up the present study to understand and elaborate the oxi唱 dative as well as antioxidative status in the cord and ma唱 ternal blood of preeclamptic patients and compare them with those of normotensive women. Thus,it is of interest to explore whether pregnancy complications such as PE add to the imbalance between pro唱 and antioxidants in the in唱 fant. We have reported an increase in lipid peroxidation products and decrease in antioxidant activities in pre唱 [21 ~23] eclamptic women . The aim of the present study was to evaluate oxidative stress in infants born to preeclamptic mothers. For this purpose we investigated the concentra唱 tions of NO,MDA,GSH and uric acid in the cord blood of the newborns from pregnancies complicated by PE,com唱 paring with that of normal pregnancies.Present study helped us to hypothesize that antioxidant capacity of cord

blood is sufficient and placental barrier is adequate,to shield the fetus from the oxidative injury caused by higher oxidative stress of preeclamptic mother. MATERIALS AND METHODS Chemicals NADPH, FAD, 5摧 5唱 dithiobis ( 2唱 nitrobenzoic acid )

[ DTNB ], aspergillus nitrate reductase, EDTA, TBA and butylated hydroxytoluene ( BHT) were from Sigma Chemical Company(St. Louis,MO,USA).Other chem唱 icals of analytical grade were obtained from either E. Merck ( Mumbai, India ), BDH or SISCO Chemicals (Mumbai,India). Subjects The patients in our study included pregnant women with normal blood pressure ( n =24 ), severely pre唱 eclamptic women admitted to our hospital who had

been or not under regular care,and also those who were referred from private sectors or primary health centers. All the participants were within the age range of 18 ~ 36 years.The preeclamptic women were having the blood pressure ≥165 / 105 mm Hg,with urinary protein excretion over 0畅 85 g / 24 h and edema.The present study was carried out with the prior approval of the lo唱 cal ethic committee.All the patients mentioned above gave their consent in writing,and the objectives of the study were fully explained to them in detail prior to taking consent.Clinical examination and history taking excluded women addicted to tobacco,patients with dia唱 betes,ischemic heart disease,a history of stroke,kidney disorders or other conditions of known free radical eti唱 ology.The criteria for dividing women into normoten唱 sive and preeclamptic groups have been set at a blood pressure of 140 / 90 mm Hg or higher,proteinuria and edema.Body height and weight of the subjects were measured to calculate their body mass index(BMI). Sample Collection Blood samples were collected from the mothers at deliv唱 ery and cord blood was obtained immediately post par唱 tum from the umbilical vein after clamping of the cord by labor ward staff.In each case 10 ml blood were drawn into a sodium heparin vacutainer tube for separa唱


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ting plasma and stored at 4 ℃ until processed.Mater唱 nal and umbilical cord blood samples were handled identically;all samples were processed within 20 h of sampling.

ing assay, which could result in falsely elevated TBA reactivity.The addition of BHT to standard MDA did not affect the color development with TBA.MDA con唱 tents were expressed as nmol / gHb.

Estimation of Plasma Levels of NO as Stable Me唱 tabolites(Nitrite +Nitrate)

Determination of Reduced Glutathione Reduced glutathione was estimated following the meth唱

The assay was performed following the procedure of [ 24] Smarason,et al. in which NO was estimation as sta唱 ble end metabolites ( nitrite +nitrate).To 300 μl of plasma was added 300 μl of KH2 PO4 / K2 HPO4 buffer (pH 7畅 5),50 μl of 2mM NADPH,50 μl of 50 μM FAD and 50 μl of 1 unit / ml aspergillus nitrate reduc唱 tase.This was incubated at room temperature for 1 h followed by the addition of 500 μl of 0畅 2 M ZnSO4 and 70 μl of 2M NaOH to deproteinize the sample.After centrifugation,0畅 75 ml of the supernatant was added to 1 ml of 1% sulphanilic acid ( in 4 M HCl ).After

Uric Acid Estimation The estimation was made following the method of [27] Buchanan,et al. .To 4 ml N / 23 sulphuric acid, 0畅 5 ml plasma was added and mixed.0畅 5 ml 5畅 6 %

Estimation of Lipid Peroxidation Lipid peroxidation was quantified following the method [ 25] of Jain ,et al. . Packed red cells(0畅 2 ml)were used for the quantification of malondialdehyde ( MDA ) as thiobarbituric acid reactive substances ( TBARS).Ali唱

Hemoglobin Estimation [ 28] The method of Tentori and Salvati was employed for hemoglobin estimation. Hemoglobin content of the sam唱

10 min at room temperature,0畅 75 ml of freshly pre唱 pared 1% N唱 naphthylethylenediamine was also added. The resultant color developed was measured at 548 nm using spectrophotometer.Nitrite concentration was cal唱 culated using nitrite and nitrate standard solutions.

quots of 0畅 2 ml were mixed thoroughly with 0畅 8 ml of phosphate唱 buffered saline ( 8畅 1 g NaCl, 2畅 302 g Na2 HPO4 , and 0畅 194 g NaH2 PO4 / L, pH 7畅 4 ) and 25 μl of butylatedhydroxytoulene ( BHT,88 mg / 10 ml absolute alcohol)solution. After adding 0畅 5 ml of 30% trichloroacetic acid,the samples were vortexed and al唱 lowed to stand in ice for at least 2 h,and then centri唱 fuged at 2,000 g at 25 ℃ for 15 min. 1 ml of superna唱 tant was mixed with 75 μl of 0畅 1M EDTA and 250 μl of 1% thiobarbituric acid in 0畅 05 M NaOH and placed on boiling water for 15 min. After cooling to room tem唱 perature, absorbance was measured at 532 nm and 600 nm.Absorbance at 600 nm was subtracted from absorbance at 532 nm for evaluation of MDA.BHT,an antioxidant,was added to prevent MDA formation dur唱

od of Beutler . Packed red cells(0畅 2 ml) were used in the assay.The GSH was made to react with 5摧 5唱 di唱 thiobis ( 2唱 nitrobenzoic acid ) [ DTNB ], which reacts with sulfhydryl groups, to develop a stable color.The absorbance was measured at 412 nm and GSH content was expressed as μmol / gHb. [26]

sodium tungstate was then added, mixed, and centri唱 fuged. 3 ml of the decanted supernatant was placed in a test tube,to which was added 0畅 2 ml PTR( Phospho唱 tungstic acid reaction reagent),mixed well,followed by the addition of 1畅 0 ml 0畅 6 N sodium hydroxide.Read唱 ings were taken after 15 min,at 720 nm on spectropho唱 tometer.

ple was measured using cyanmethemoglobin method by mixing 20 μl of blood and 5 ml of(1∶ 251 diluted)ferri唱 cyanide reagent [ K3 Fe( CN) 6 ,200 mg;KCN 50 mg; K2 HPO4 , 140 mg; appropriate amount of detergent Triton X唱 100 dissolved and raised to one liter,pH 7畅 4] and allowing to stand for at least 3 min. Afterwards,ab唱 sorbance was read at 540 nm using water as blank. Statistical Analysis SPSS version 15畅 0 for Windows( SPSS Inc. ,Chicago, IL,USA)software package was used to analyze the data and to obtain the box唱 plots for various parameters.The results were statistically analyzed using paired唱 samples t唱 test to compare both maternal / cord blood of normo唱 tensive pregnant and preeclamptic patients groups.The


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t唱 test statistical significance was set at P≤ 0畅 05.Val唱 ues were expressed as mean ±standard deviation. RESULTS We compared MDA,NO,GSH and uric acid contents in maternal and cord blood of two groups of pair matched maternal and neonates. Group A consisted of 24 uncom唱 plicated, normotensive唱 pregnancies ( called controls ) and group B comprised of 24 severely preeclamptic pa唱

tients,all singleton pregnancies.Group A had three, whereas group B had six caesarian sections,because of prolonged labor.The clinical characteristics of these groups are shown in Table 1.The values of mean ± standard deviation of all the variables in pair唱 matched normotensive & preeclamptic maternal and cord plasma are presented in Table 2. Newborns from these mothers weighed significantly ( P <0.001 ) less than others at birth as evident from Table 1.

Table 1 Clinical characteristics for the patient groups Parameters

Number of maternal / neonatal pairs

Normotensive control group

Severely preeclamptic group

24

24

Maternal age( years)

P倡

28畅4 ±5畅8

27畅6 ±6畅6

0畅658

21畅8 ±2畅8

20畅8 ±3畅6

0畅288

Gestational age( weeks)

34畅6 ±4畅2

32畅8 ±5畅4

0畅204

115畅2 ±14畅6

168畅6 ±15畅8

<0畅001

68畅8 ±10畅2

110畅8 ±12畅6

<0畅001

72畅6 ±2畅8

74畅2 ±3畅6

0畅093

3 428 ±330

2 112 ±466

<0畅001

Nil

1畅18 ±0畅98

++in all cases

BMI at delivery( kg / m ) BP at delivery,systolic( mm Hg)

BP at delivery,diastolic( mm Hg) Pulse rate( beats / min) Birth weight( g)

Proteinuria( g / d) Edema

Nil

Note:Values are expressed as mean ±SD;BMI唱 body mass index;BP唱 blood pressure,P

=Two唱 Samples T唱 test probability

Table 2 Oxidant and antioxidant contents in pair唱 matched normotensive & preeclamptic maternal and cord blood Parameters MDA( nmol / gHb) GSH( μmol / gHb) NO as stable

metabolites( μmol / L) Uric acid( μmol / L)

Normotensive maternal ( N =24)

Normotensive cord

Preeclamptic maternal

( N =24)

( N =24)

Preeclamptic cord ( N =24)

6畅86 ±0畅88

6畅91 ±1畅05

9畅58 ±1畅89

7畅52 ±1畅25

8畅74 ±1畅57

7畅97 ±1畅62

6畅96 ±2畅08

5畅97 ±1畅54

21畅13 ±4畅06

19畅22 ±3畅79

26畅46 ±4畅65

28畅15 ±4畅20

278畅5 ±57畅0

301畅9 ±69畅9

497畅67 ±44畅92

526畅3 ±75畅3

Note:Values are expressed as mean ±SD;MDA唱 malondialdehyde,GSH唱 reduced glutathione,NO唱 nitric oxide as stable metabolites ( NOx,nitrite +ni唱 trate) ,N唱 respective numbers

These data suggest a need for interest in monito唱

were significantly lower ( P <0.001) in cord as com唱

period,especially taking into account that the oxidative

ma. The variations in its levels in pair唱 matched normo唱

ring the oxidative state of mothers during the pregnancy

level could be involved in later risks of metabolic dis唱 eases for both mother and newborn.

The MDA concentrations were significantly higher

pared to their pair唱 matched preeclamptic maternal plas唱 tensive and preeclamptic maternal as well as cord plas唱 ma have been shown in Figure 1.

The nitric oxide concentrations in terms of stable

( P <0畅 001 ) in preeclamptic maternal plasma com唱

metabolites( NOx, nitrite +nitrate) were significantly

non唱 significantly higher level ( P =0畅 124 ) in pre唱

cord plasma compared to pair唱 matched control.How唱

pared to normotensive maternal plasma;whereas it was eclamptic cord plasma.However, the concentrations

( P <0畅001) higher in preeclamptic both maternal and ever,there was no significant difference( P =0畅194 )


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in preeclamptic maternal and cord levels.Their com唱 parative levels have been depicted in Figure 2. The reduced glutathione level was found to be sig唱 nificantly ( P =0畅 003 ) low in preeclamptic maternal plasma as compared to control maternal plasma, but唱 there was no significant ( P =0畅 06 ) change between preeclamptic maternal and cord plasma,as evident from

Uric acid content was significantly ( P <0畅 001 ) higher in preeclamptic maternal plasma and cord plas唱 ma in comparison to their respective normal maternal and cord plasma.But,there was no significant change in between normal maternal and cord plasma and pre唱 eclamptic maternal and cord plasma,which is shown e唱 laborately in Figure 4.

Figure 1 MDA concentrations in pair唱 matched normotensive &

Figure 2 NOx concentrations in pair唱 matched normotensive &

Figure 3 Reduced glutathione concentrations in pair唱 matched

Figure 4 Uric acid concentrations in pair唱 matched normoten唱

DISCUSSION NO plays an important role in the pathogenesis of PE as the enhanced level of oxidative stress increases the con唱 centration of superoxide that reacts with NO to produce peroxynitrite( ONOO唱 ),reducing the level of available

NO. Peroxynitrite is a potentially harmful reactive oxy唱 gen species( ROS) as it causes nitrosylation of tyrosine residues, leading to changes in protein conformation and its inactivation. PE is associated with increased ni唱 trotyrosine residues in placental villous vascular endo唱

Figure 3 .

preeclamptic maternal and cord blood

normotensive & preeclamptic and cord blood.

preeclamptic maternal and cord blood

sive & preeclamptic maternal and cord blood.


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thelium . The effect of elevation in peroxynitrite and reduced bioavailability of NO,resulting from enhanced production of free radicals and the resultant placental oxidative state,may contribute to the elevation of ma唱 ternal blood pressure, proteinuria, platelet dysfunction and alteration in the level of thromboxane,prostacyclin [ 30] and endothelin characteristic of PE .Another com唱

that reduces H2 O2 to water.The role of reduced gluta唱 thione in protection of macromolecules against oxidative damage is clearly evident from our findings since the level of GSH was significantly higher in normotensive women compared with preeclamptics. Reduced glutathi唱 one provides resistance to cells against oxidative insult with sufficient intracellular concentration of GSH.Dur唱

circulation . Although,the levels of NO in both maternal and cord plasma in preeclamptics were significantly high but may be the bioavailability of NO might have been blocked,as there was an increase of 39畅 7% in MDA [21,22] contents. Our previous reports have shown signifi唱 cant elevation in the levels of MDA or TBARS during the development of PE. This may result in a greater po唱 tential for endothelial damage ultimately leading to en唱 [32] hanced diastolic pressure which further aggravates the condition of preeclamptic patients.Enhanced ROS

compared to normotensive cord plasma,which may ren唱 der higher antioxidant capacity in preeclamptic mater唱 nal and cord plasma providing increased resistance to [ 31] lipid peroxidation. Yanik,et al. have reported that in preeclamptic patients there might be a compensatory increase in nitric oxide production during labor in order to maintain the systemic circulation.Glutathione levels might also be increased to compensate for the marked oxidative stress. From our results it is evident that the levels of NO and uric acid are significantly higher in preeclamptic

[29]

mon source of placental oxidative damage is the peroxi唱 dation of membrane phospholipids that exerts a variety of effects within placenta.Our results on NO showed that its concentration was 25% higher in preeclamptic maternal plasma as compared to that of normotensive mothers,and its level was 46. 5% higher in preeclamp唱 tic cord plasma in comparison to normal cord plasma. Our results on NO concentrations are in harmony with [4,5] those of other reports .In preeclamptic patients, such higher levels in nitric oxide production might be a compensatory action in order to maintain the systemic [31]

in turn can oxidize many other important biomolecules including erythrocyte membrane phospholipids.The in唱 teresting finding of the present study is 21.5% lower level of MDA concentrations in the cord plasma as compared to maternal plasma in preeclamptics.Our present findings on MDA concentrations are consistent [ 13] with those of Orhan,et al. . Our results on NO / MDA show that the preeclamptic mother is under oxidative stress rather than the pair唱 matched neonate. GSH is the most abundant thiol唱 based antioxi唱 dant,and is found primarily in the reduced form,with intracellular concentration up to 11 mM and provides sulfhydryl唱 buffering capacity. It also conveys an antioxi唱 dant power through the direct inactivation of ROS or by acting as an electron donor for glutathione peroxidase

ing oxidative insult,GSH is oxidized to GSSG,as a con唱 sequence of which level of GSSG gets enhanced.Our results showing significantly lower level of GSH in pre唱 eclamptic maternal and cord plasma support the reports [19] [20] of Jean唱 Baptiste,et al. and Ta爧tekin,et al. .It showed 20% and 25% lower concentrations in mater唱 nal and cord plasma as compared to their levels in nor唱 motensive maternal and cord plasma.However, uric acid levels were 78畅 7% higher in preeclamptic mater唱 nal plasma as compared to normotensive maternal plas唱 ma and 74畅 3% higher in preeclamptic cord plasma as

cord blood,but no significant difference between pre唱 eclamptic maternal and cord blood.This may render higher antioxidant level to the preeclamptic cord blood. Further,that the elevated level of NO might be a com唱 pensatory action in order to maintain the systemic cir唱 culation in neonate.Thus,we conclude that the oxida唱 tive stress status is low in the blood of neonates com唱 pared to that in the pair唱 matched preeclamptic mothers and may be sufficient to combat the oxidative assault caused by higher oxidative stress of preeclamptic moth唱 er.Further studies are needed to explore strategies so that the normal levels of antioxidant are maintained to combat preeclampsia in women at high risk,including increased risk of fetal growth restriction and induced premature delivery due to disease progression on the


JOURNAL of CHINESE CLINICAL MEDICINE VOLUME 4 | NUMBER 5 | May 2009 maternal or fetal side. Abbreviations

NM, normotensive maternal; NC, normotensive cord; PM,preeclamptic maternal;PC,preeclamptic cord;PE,

Preeclampsia;NO, nitric oxide; ONOO唱 , peroxynitrite; MDA,malondialdehyde;ROS,reactive oxygen species; TBA, thiobarbituric acid; TBARS, thiobarbituric acid reactive substances; Hb, hemoglobin; BP, Blood pres唱 sure; SBP, Systolic Blood Pressure; DBP, Diastolic Blood Pressure;NADPH,nicotinamide adenine dinucle唱 otide reduced; FAD, flavin adenine dinucleotide; DT唱 NB,5摧 5唱 dithiobis (2唱 nitrobenzoic acid); EDTA, ethyl唱 enediaminetetraacetic acid;BHT,butylated hydroxytol唱 uene. ACKNOWLEDGMENT We are highly grateful to those patients of our hospital

who volunteered to donate their blood when needed for this project.The authors would like to thank Dr.Safia Suhail for managing and monitoring the patients groups. Our thanks are also due to the paramedical staff of this hospital for their assistance in collecting and maintaining blood samples.

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