Research Article Prooxidant-Antioxidant Balance in

Hindawi Publishing CorporationBiochemistry Research InternationalVolume 2013 Article ID 270545 4 pageshttpdxdoiorg1011552013270545

Research ArticleProoxidant-Antioxidant Balance in Umbilical Cord Blood ofInfants with Meconium Stained of Amniotic Fluid

Mohammad Hassan Arjmand1 Farhat Ahmad Shah2 Masoud Saleh Moghadam3

Fatemeh Tara4 Amin Jalili5 Mojtaba Mosavi Bazaz6 and Daryoush Hamidi Alamdari7

1 Department of Biology School of Sciences Payame Noor University Mashhad 91735433 Iran2Neonatal Research Center Mashhad University of Medical Sciences Mashhad 9137913316 Iran3Department of Chemistry School of Science Payame Noor University Mashhad 91735433 Iran4Department of Obstetrics and Gynecology OM-Albanin Hospital Mashhad University of Medical SciencesMashhad 9144663595 Iran

5 Department of New Science and Technologies Mashhad University of Medical Sciences Mashhad 9177948564 Iran6Department of Social Medicine Faculty of Medicine Mashhad University of Medical Sciences Mashhad 9138913131 Iran7 Stem Cell and Regenerative Medicine Research Group Department of Biochemistry Faculty of MedicineMashhad University of Medical Sciences Mashhad 9177948564 Iran

Correspondence should be addressed to Daryoush Hamidi Alamdari hamidiadmumsacir

Received 15 July 2013 Accepted 2 October 2013

Academic Editor Vladimir Uversky

Copyright copy 2013 Mohammad Hassan Arjmand et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Objective Using a novel assay termed prooxidant-antioxidant balance (PAB) assay to determine prooxidant-antioxidant balancein umbilical cord blood of infants with meconium stained of amniotic fluid (MSAF) Passage of meconium in amniotic fluid isassociated with increase of neonatal mortality and morbidity This complication occurs in about 15 of infants and is more wide-spread in postterm neonates About 15 percent of neonates with MSAF develop meconium aspiration syndromeMethod Sera of29 umbilical cord blood of infants with MSAF and 32 healthy infants (HI) were collected Both groups had nonsmoker and non-alcoholic mothers with no diseases The PAB was measured Result There was a significant increase of PAB value (328 plusmn 159HK)in umbilical cord blood of infants withMSAF in comparison to HI (245plusmn126HK) (119875 lt 005)There was no significant correlationbetween PAB value and age of mothers Conclusion The increased PAB value in infants with MSAF showed that these infants areexposed to oxidative stress Further research with larger population is needed to demonstrate the oxidative stress in infants withMSAF

1 Introduction

Meconium is composed of desquamated cells from the intes-tine and skin gastrointestinal mucin lanugo hair fatty mate-rial from the vernix caseosa amniotic fluid and intestinalsecretions It also contains blood group-specific glycopro-teins biliary acids (cholic chenodeoxycholic deoxycholicand lithocholic) copper zinc magnesium calcium ironphosphorus and plasma proteins such as alpha1-antitrypsinand phospholipase A2 Black-green color ofmeconium is dueto the presence of bile pigments [1 2] Expulsion ofmeconiumfrom the intestinal lumen into the amniotic cavity is a

consequence of increased intestinal peristalsis and of analsphincter relaxation resulting from vagal stimulation [3]

Meconium-stained amniotic fluid (MSAF) can causemechanical obstruction of airways and pulmonary air leakpneumonitis vasoconstriction of pulmonary vessels andinactivation of surfactant effect which could result in pul-monary inflammation and apoptosis 7 to 20 of deliveriesat term have meconium in the amniotic fluid which wouldreach to 40 in postterm deliveries [4] 5 of infants bornthrough MSAF develop meconium aspiration syndrome(MAS) which is a real threat to many newborns worldwidewith a case fatality rate of 5 (as much as 40) in addition

2 Biochemistry Research International

to MAS short- and long-term pulmonary and neurodevelop-mental sequelae which could occur [5 6]

In human there are the numerous prooxidants (POX)and antioxidants (AO) and a delicate balance between theproduction and the elimination of POX ismaintainedOxida-tive stress (OS) is defined as an imbalance between POX andAO in favor of POX POX (O

2

minus H2O2 OHminus etc) derive

either frommetabolic processes or from external sources andcan potentially react with the bodyrsquos ownmolecules AOmopup the excess amount of the POX before they damage theessential molecules AO consists of the soluble antioxidants(vitaminC urate etc) the lipid soluble antioxidants (vitaminE A etc) and the enzymatic antioxidants (catalase peroxi-dase dismutase etc) [7]

The concentration of either POX or AO can be measuredone by one in separate individual assays For example Ochiand Cutler invented a diagnostic plot derived from the mea-surement of 82 assays which characterize both the oxidativestress and the antioxidant profile [8] However the effect ofthe prooxidant or the antioxidant molecules in serum is anadditive effect which could lead to incorrect measurementsConsequently various methods have been developed inorder to measure the total oxidants (such as TOC and TOSassays) or antioxidants (such as FRAP and ORAC assays)separately In this context PAB assay is developed to measurethe balance of oxidants and antioxidants simultaneously inone experiment and give a redox index [7 9 10]

Growing evidence indicates that chronic and acute over-production of POX under pathophysiologic conditions isunifying mechanism for tissue damage and cell apoptosisA few studies showed that there is an oxidative stress inMSAF but more studies using different methods are neededto substantiate the PAB in MSAF

To the best of our knowledge this is the first time thatthe prooxidant-antioxidant balance is measured in umbilicalcord blood of infants with MSAF

2 Methods

21 Chemicals TMB powder (331015840551015840-Tetramethylbenzi-dine Fluka) peroxidase enzyme (Applichem 230UmgA3791 0005 Darmstadt Germany) chloramine T trihydrate(Applichem A4331 Darmstadt Germany) hydrogen perox-ide (30) (Merck) Molecular biology grade reagents wereused and preparations were done in double distilled water

22 Subjects Sera of 29 umbilical cord blood of infants withMSAF and 32 healthy infants were collected Collection ofsamples was done from July to December in 2013 Bothgroups had nonsmoker and nonalcoholic mothers withoutany special diseases The PAB was measured The study pro-tocol was approved by the Ethics Committee for Clini-cal Research of the Mashhad University of Medical Sciences(MUMS)

23 Collection of Serum Blood samples from each subjectwere collected from infantrsquos umbilical cord and then sentto the laboratory for serum separation Samples were cen-trifuged at 1500 g for 15min at room temperature to obtain

serum Hemolytic samples were excluded from analysis Serawere stored at minus80∘C

24 Method The PAB assay is the only available test thatcan measure the balance of oxidants and antioxidants simul-taneously in one experiment It uses two different kinds ofreactions one is an enzymatic reaction where the chromogenTMB is oxidized to a color cation by peroxides and the secondis a chemical reaction where the TMB cation is reduced to acolorless compound by antioxidants [7 8 11] The photomet-ric absorbance is then compared with the absorbances givenby a series of standard solutions that aremade bymixing vary-ing proportions (0ndash100) of hydrogen peroxide with uricacid [7] A low PAB valuemeans that antioxidants are presentat greater concentration than oxidants while a high PABvalue means more oxidants are present than antioxidantsThe standard solutions were prepared by mixing varyingproportions (0ndash100) of 250mM hydrogen peroxide with3mMuric acid (in 10mMNaOH)The TMB powder (60mg)was dissolved in 10mL DMSO For preparation of the TMBcation solution 400mL of the TMBDMSO solution wasadded to 20mL of acetate buffer (005M buffer pH 45) andthen 70mL of fresh chloramine T (100mM) solution wasadded The solution was mixed well and incubated for 2hours at room temperature in a dark place and then 25Uof peroxidase enzyme solution was added This mixturewas dispensed into 1mL aliquots and stored at minus20∘C TheTMB solution was prepared by adding 200 120583L TMBDMSOto 10mL of acetate buffer (005M buffer pH 58) and theworking solution was prepared by mixing 1mL TMB cationsolutionwith 10mLTMB solutionThis working solutionwasincubated for 2min at room temperature in a dark place andimmediately used Ten microliters of each sample standardor blank (distilled water) were mixed with 200120583L of workingsolution in each well of a 96-well plate which was thenincubated in a dark place at 37∘C or 12min At the end ofincubation 100120583L of 2N HCl was added to each well andthe optical density (OD) was measured with an ELISA readerat 450 nm with a reference wavelength of 620 or 570 nmA standard curve was generated from the values of thestandard samples The values of the PAB assay are expressedin arbitrary units based on the percentage of hydrogenperoxide in the standard solutionThe values of the unknownsamples were then calculated based on the values obtainedfrom the generated standard curve

3 Statistics

The Statistical Package for the Social Sciences (SPSS) version160 was used for statistical analysis All parameters weregiven as mean plusmn SD The group comparisons were assessedby the independent t-test and also Pearson correlation wasused for correlation of age and PAB value The significancelevel was considered less than 005 with a confidence intervalof 95

4 Results

ThePAB values ofHI group andMSAF groupwere 245plusmn126(HK unit) and 328 plusmn 159 (HK unit) respectively (Figure 1)

Biochemistry Research International 3

45

0

20

40

60

80

100

120

PAB

N= 32 29

Column A Column BAmnion

48∘

1∘

lowast

Figure 1 PAB value in HI group (column A) and MSAF group(column B) (Mean and the standard errorstandard deviation ofPAB value)

HK is an arbitrary unit used by inventors of PAB method(Hamidi and Koliakos) [7] There was a significant difference(119875 value = 0027) between the PAB value of the HI groupand the MSAF group There was no significant correlationbetween PAB value and the age of mothers (Table 1)

5 Discussion

In this study we showed that there is an increased OS inumbilical cord blood of infants with MSAF in comparison tohealthy group using a novel method called PAB assay

There are two prevailing theories about mechanism ofmeconium passage in amniotic fluid at term and postterminfants one is that normal maturation of the gastrointestinaltract results inmeconiumpassage the other is that pathologicprocesses such as stress via hypoxia or some infection infetus can trigger meconium passage Previous studies haveshowed an increased OS in hypoxic fetuses and neonateswith elevated products of lipid peroxidation in expired airserum malondialdehyde serum isoprostanes serum totalhydroperoxides advanced oxidative protein products andincreased nonprotein bound iron in serum Low levels ofantioxidants have also been observed in red blood cells [1213]

Just one study showed that infants with MSAF have highconcentration of 8-iso-prostaglandin F2alpha in neonatalcord blood as a marker of lipid oxidation and suggests thatthese infants were exposed to OS [14]

It is demonstrated that there is an increased level ofproinflammatory cytokine such as IL-1120573 IL-6 tumor necro-sis factor (TNF)-120572 and IL-8 in newborns with MASF [15] Inthe other hand there is a strong correlation between proin-flammatory cytokine and OS in various diseases such as car-diovascular disease [16] diabetes mellitus [17] inflammatorybowel disease [18] obesity [19] Alzheimer [20] and alcoholicliver disease [21] OS can cause increasing cytokine pro-duction by many different mechanisms The increased POX

Table 1 Age of mothers and PAB value inMSAF and control group

Group Ages of mothers(years)lowast

Number ofmothers

PAB value(HK unit)lowastlowast

MSAF 26 plusmn 56 29 328 plusmn 159Control 258 plusmn 44 32 245 plusmn 126lowastNo significant difference between ages of mothers lowastlowastsignificant differencebetween PAB values (119875 lt 005)

levels acting similar second messengers are well known tomediate inflammatory signaling by activating various proteinkinases such as JNK PI3 K PKC and PLC These kinasescould stimulate redox sensitive transcription factors such asSTAT CREB NF-120581B AP-1 NFAT and ATF2 via a seriesof signaling events transduced by other kinases like MAPKERK and JAK The activation of transcription factors leadsto the transcriptional activation of inflammatory cytokines(TNF120572 IL-1 IL-6 IL-8 IL-18 etc) chemokines (chemoat-tractant protein-1 etc) and growth factors (transforminggrowth factor-120573 monocyte connective tissue growth factoretc) which could amplify inflammatory complications viaautocrine and paracrine pathways [22] In addition it isdemonstrated that antioxidants can downregulate the proin-flammatory cytokines through two possible mechanismsfirstly through their effect on transcription factors thatare regulated by redox status and secondly by influencingPGE2 synthesis which plays a key role in Th1 response andregulation of proinflammatory cytokines [23] Therefore itis reasonable to conclude that proinflammatory cytokine andOS could interact and provoke each other and contribute tothe development and progression of some disease

Our early results showed that infants with MSAF areexposed with OS However larger population is neededto confirm these early results In addition to that furtherresearch will be necessary to precisely determine the corre-lation between proinflammatory cytokines and OS in infantswith MSAF

Abbreviations

AP1 Activator protein 1JNK c-Jun N-terminal kinasePI3K Phosphatidylinositol 3-kinaseNF-120581B Nuclear factor-120581-binding proteinMAPK Mitogen-activated protein kinasePKC Protein kinase CPLC Phospholipase CERK Extracellular signal-regulated kinasesJAK Janus kinaseSTAT Signal transducer and activator of

transcriptionNFAT Nuclear factor of activated T-cellsATF Activating transcription factorPGE2 Prostaglandin E2IL2 Interleukin-2CREB cAMP response element-binding proteinTNF120572 Tumor necrosis factor alphaPGE2 Prostaglandin E2


TOC Total oxidant capacityTOS Total oxidant statusFRAP The ferric reducing ability of plasmaORAC Oxygen radical absorbance capacity

Acknowledgments

The authors thank the Vice Chancellor for Research ofMash-had University of Medical Sciences for funding and supportsThis paper is the outcome of the MS thesis of MohammadHassan Arjmand (Master of biochemistry Department ofBiology School of Sciences Payam Noor University Mash-had Iran) The authors thank Mrs Afsharpour and MrsNasoti for their collaboration in this study

References

[1] S Rapoport and D J Buchanan ldquoThe composition of meconi-um isolation of blood-group-specific polysaccharides Abnor-mal composition ofmeconium inmeconium ileusrdquo Science vol112 no 2901 pp 150ndash153 1950

[2] R H Cote and J P Valet ldquoIsolation composition and reac-tivity of the neutral glycoproteins from human meconiumswith specificities of the ABO and Lewis systemsrdquo BiochemicalJournal vol 153 no 1 pp 63ndash73 1976

[3] E H Hon Modern Trends in Human Reproductive PhysiologyButterworth amp Co London UK 1963

[4] F C Miller and J A Read ldquoIntrapartum assessment of thepostdate fetusrdquo American Journal of Obstetrics and Gynecologyvol 141 no 5 pp 516ndash520 1981

[5] T EWiswell ldquoAdvances in the treatment of themeconium aspi-ration syndromerdquo Acta Paediatrica vol 90 no 436 pp 28ndash302001

[6] G M Cleary and T E Wiswell ldquoMeconium-stained amnioticfluid and the meconium aspiration syndrome an updaterdquo Pedi-atric Clinics of North America vol 45 no 3 pp 511ndash529 1998

[7] D H Alamdari K Paletas T Pegiou M Sarigianni C Befaniand G Koliakos ldquoA novel assay for the evaluation of theprooxidant-antioxidant balance before and after antioxidantvitamin administration in type II diabetes patientsrdquo ClinicalBiochemistry vol 40 no 3-4 pp 248ndash254 2007

[8] H Ochi and R G Cutler ldquoUse of oxidative stress diagnosticplot as a health indicator for assessing oxidative stress and itscontrol in humansrdquo United States Patent 6569 683 B1 2003httppatftusptogovnetahtmlPTOsearch-boolhtml

[9] O Erel ldquoA new automated colorimetric method for measuringtotal oxidant statusrdquo Clinical Biochemistry vol 38 no 12 pp1103ndash1111 2005

[10] O Erel ldquoA novel automated method to measure total antiox-idant response against potent free radical reactionsrdquo ClinicalBiochemistry vol 37 no 2 pp 112ndash119 2004

[11] D H Alamdari M Ghayour-Mobarhan S Tavallaie et alldquoProoxidant-antioxidant balance as a new risk factor in patientswith angiographically defined coronary artery diseaserdquo ClinicalBiochemistry vol 41 no 6 pp 375ndash380 2008

[12] L Ciccoli V Rossi S Leoncini et al ldquoIron release in erythro-cytes and plasma non protein-bound iron in hypoxic and nonhypoxic newbornsrdquo Free Radical Research vol 37 no 1 pp 51ndash58 2003

[13] G Buonocore S Zani S Perrone B Caciotti and R BraccildquoIntraerythrocyte nonprotein-bound iron and plasma malon-dialdehyde in the hypoxic newbornrdquo Free Radical Biology andMedicine vol 25 no 7 pp 766ndash770 1998

[14] B Y Liu C CWang T K Lau et al ldquoMeconium-stained liquorduring labor is associated with raised neonatal cord blood8-iso-prostaglandin F2120572 concentrationrdquo American Journal ofObstetrics and Gynecology vol 192 no 1 pp 289ndash294 2005

[15] A J De Beaufort A C Bakker M J D Van Tol B J PoorthuisA J Schrama and H M Berger ldquoMeconium is a source of pro-inflammatory substances and can induce cytokine productionin cultured A549 epithelial cellsrdquo Pediatric Research vol 54 no4 pp 491ndash495 2003

[16] Y Mizuno R F Jacob and R P Mason ldquoInflammation andthe development of atherosclerosismdasheffects of lipid-loweringtherapyrdquo Journal of Atherosclerosis and Thrombosis vol 18 no5 pp 351ndash358 2011

[17] E T De Lemos J Oliveira J P Pinheiro and F Reis ldquoRegularphysical exercise as a strategy to improve antioxidant andanti-inflammatory status benefits in type 2 diabetes mellitusrdquoOxidative Medicine and Cellular Longevity vol 2012 Article ID741545 15 pages 2012

[18] H Zhu andY R Li ldquoOxidative stress and redox signalingmech-anisms of inflammatory bowel disease updated experimentaland clinical evidencerdquo Experimental Biology and Medicine vol237 no 5 pp 474ndash480 2012

[19] B Tran S Oliver J Rosa and P Galassetti ldquoAspects of inflam-mation and oxidative stress in pediatric obesity and type 1diabetes an overview of ten years of studiesrdquo ExperimentalDiabetes Research vol 2012 Article ID 683680 7 pages 2012

[20] F De Oliveira C A Veloso J A Nogueira-Machado et alldquoAscorbic acid alpha-tocopherol and beta-carotene reduceoxidative stress and proinflammatory cytokines in 7 mononu-clear cells of Alzheimerrsquos disease patientsrdquo Nutritional Neuro-science vol 5 no 2 pp 148ndash156 2012

[21] A Ambade and P Mandrekar ldquoOxidative stress and inflamma-tion essential partners in alcoholic liver diseaserdquo InternationalJournal of Hepatology vol 2012 Article ID 853175 9 pages 2012

[22] K V Ramana ldquoAldose reductase new Insights for an oldenzymerdquo Biomolecular Concepts vol 2 no 1-2 pp 103ndash114 2011

[23] S N Han and S N Meydani ldquoAntioxidants cytokines andinfluenza infection in aged mice and elderly humansrdquo Journalof Infectious Diseases vol 182 no 3 supplement 1 pp S74ndashS802000

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Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


to MAS short- and long-term pulmonary and neurodevelop-mental sequelae which could occur [5 6]

In human there are the numerous prooxidants (POX)and antioxidants (AO) and a delicate balance between theproduction and the elimination of POX ismaintainedOxida-tive stress (OS) is defined as an imbalance between POX andAO in favor of POX POX (O

2

minus H2O2 OHminus etc) derive

either frommetabolic processes or from external sources andcan potentially react with the bodyrsquos ownmolecules AOmopup the excess amount of the POX before they damage theessential molecules AO consists of the soluble antioxidants(vitaminC urate etc) the lipid soluble antioxidants (vitaminE A etc) and the enzymatic antioxidants (catalase peroxi-dase dismutase etc) [7]

The concentration of either POX or AO can be measuredone by one in separate individual assays For example Ochiand Cutler invented a diagnostic plot derived from the mea-surement of 82 assays which characterize both the oxidativestress and the antioxidant profile [8] However the effect ofthe prooxidant or the antioxidant molecules in serum is anadditive effect which could lead to incorrect measurementsConsequently various methods have been developed inorder to measure the total oxidants (such as TOC and TOSassays) or antioxidants (such as FRAP and ORAC assays)separately In this context PAB assay is developed to measurethe balance of oxidants and antioxidants simultaneously inone experiment and give a redox index [7 9 10]

Growing evidence indicates that chronic and acute over-production of POX under pathophysiologic conditions isunifying mechanism for tissue damage and cell apoptosisA few studies showed that there is an oxidative stress inMSAF but more studies using different methods are neededto substantiate the PAB in MSAF

To the best of our knowledge this is the first time thatthe prooxidant-antioxidant balance is measured in umbilicalcord blood of infants with MSAF

2 Methods

21 Chemicals TMB powder (331015840551015840-Tetramethylbenzi-dine Fluka) peroxidase enzyme (Applichem 230UmgA3791 0005 Darmstadt Germany) chloramine T trihydrate(Applichem A4331 Darmstadt Germany) hydrogen perox-ide (30) (Merck) Molecular biology grade reagents wereused and preparations were done in double distilled water

22 Subjects Sera of 29 umbilical cord blood of infants withMSAF and 32 healthy infants were collected Collection ofsamples was done from July to December in 2013 Bothgroups had nonsmoker and nonalcoholic mothers withoutany special diseases The PAB was measured The study pro-tocol was approved by the Ethics Committee for Clini-cal Research of the Mashhad University of Medical Sciences(MUMS)

23 Collection of Serum Blood samples from each subjectwere collected from infantrsquos umbilical cord and then sentto the laboratory for serum separation Samples were cen-trifuged at 1500 g for 15min at room temperature to obtain

serum Hemolytic samples were excluded from analysis Serawere stored at minus80∘C

24 Method The PAB assay is the only available test thatcan measure the balance of oxidants and antioxidants simul-taneously in one experiment It uses two different kinds ofreactions one is an enzymatic reaction where the chromogenTMB is oxidized to a color cation by peroxides and the secondis a chemical reaction where the TMB cation is reduced to acolorless compound by antioxidants [7 8 11] The photomet-ric absorbance is then compared with the absorbances givenby a series of standard solutions that aremade bymixing vary-ing proportions (0ndash100) of hydrogen peroxide with uricacid [7] A low PAB valuemeans that antioxidants are presentat greater concentration than oxidants while a high PABvalue means more oxidants are present than antioxidantsThe standard solutions were prepared by mixing varyingproportions (0ndash100) of 250mM hydrogen peroxide with3mMuric acid (in 10mMNaOH)The TMB powder (60mg)was dissolved in 10mL DMSO For preparation of the TMBcation solution 400mL of the TMBDMSO solution wasadded to 20mL of acetate buffer (005M buffer pH 45) andthen 70mL of fresh chloramine T (100mM) solution wasadded The solution was mixed well and incubated for 2hours at room temperature in a dark place and then 25Uof peroxidase enzyme solution was added This mixturewas dispensed into 1mL aliquots and stored at minus20∘C TheTMB solution was prepared by adding 200 120583L TMBDMSOto 10mL of acetate buffer (005M buffer pH 58) and theworking solution was prepared by mixing 1mL TMB cationsolutionwith 10mLTMB solutionThis working solutionwasincubated for 2min at room temperature in a dark place andimmediately used Ten microliters of each sample standardor blank (distilled water) were mixed with 200120583L of workingsolution in each well of a 96-well plate which was thenincubated in a dark place at 37∘C or 12min At the end ofincubation 100120583L of 2N HCl was added to each well andthe optical density (OD) was measured with an ELISA readerat 450 nm with a reference wavelength of 620 or 570 nmA standard curve was generated from the values of thestandard samples The values of the PAB assay are expressedin arbitrary units based on the percentage of hydrogenperoxide in the standard solutionThe values of the unknownsamples were then calculated based on the values obtainedfrom the generated standard curve

3 Statistics

The Statistical Package for the Social Sciences (SPSS) version160 was used for statistical analysis All parameters weregiven as mean plusmn SD The group comparisons were assessedby the independent t-test and also Pearson correlation wasused for correlation of age and PAB value The significancelevel was considered less than 005 with a confidence intervalof 95

4 Results

ThePAB values ofHI group andMSAF groupwere 245plusmn126(HK unit) and 328 plusmn 159 (HK unit) respectively (Figure 1)


45

0

20

40

60

80

100

120

PAB

N= 32 29


48∘

1∘

lowast



5 Discussion







Number ofmothers





Abbreviations





Acknowledgments


References































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


45

0

20

40

60

80

100

120

PAB

N= 32 29


48∘

1∘

lowast



5 Discussion







Number ofmothers





Abbreviations





Acknowledgments


References































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Acknowledgments


References































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Prooxidant-Antioxidant Balance in