Vitamin C as a modulator of oxidative stress in erythrocytes of stored blood

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Original research article/Praca oryginalna

Vitamin C as a modulator of oxidative stress in erythrocytes of stored blood

Ravikumar Soumya, Rajashekharaiah Vani *

Department of Biotechnology, Centre for Post Graduate Studies, Jain University, #18/3, 9th Main, 3rd Block, Jayanagar,Bangalore560011,India

Introduction

Bloodtransfusionisanacuteintervention,implementedto solve life- and health-threatening conditions on a short- termbasis,andingeneralitslongtermeffectstendtobeof secondaryimportance[1]. Redbloodcell(RBC)transfusion

is a key element of modern medical care. The ability to store RBCs for reasonable times clearly improves their availability and lowers their cost [2]. Wholeblood can be stored up toaperiod of35daysin anticoagulantsolution CPDA-1 (citrate, phosphate, dextrose and adenine) while erythrocytes(RBCs)possessashelflifeof42daysat48Cin SAGM(saline,adenine, glucoseandmannitol)solution[2].

article info

Articlehistory:

Received:10.03.2017 Accepted:30.08.2017 Availableonline:06.10.2017

Keywords:

Ascorbicacid

Erythrocyte

Bloodstorage

Lipidperoxidation

Proteinoxidation

abstract

Aim: TodeterminetheeffectsofVitaminC(VC-ascorbicacid)asanadditiveonerythro- cytesofstoredblood.Background: Oxidativestress(OS)playsamajorroleintheforma- tion ofstorage lesion oferythrocytes. Antioxidants,such as VCcould be beneﬁcial in combating oxidative damage during storage. Materials and methods: Blood obtained frommale Wistar rats was stored at 48C in anticoagulant solution citrate-phosphate- dextrose-adeninesolution. Bloodsamplesweredividedinto3 groups–(i) Controls, (ii) VC10 (VC ata concentration of10mM), (iii) VC30 (VC ata concentrationof 30mM).

MarkersofOSinerythrocytessuchas–hemoglobin,superoxides,antioxidantenzymes (superoxidedismutase;SOD,catalaseandglutathioneperoxidase),hemolysis,lipidperoxidation products (conjugate dienes and malondialdehyde),protein oxidation products andascorbicacidweredeterminedondays0,10and15ofstorage.Results: Additionof ascorbicacidtothestorage solutioncontributedtotheprotection oferythrocytesfrom oxidativedamage.Ascorbicacidataconcentrationof30mM decreasedSODlevelsand increasedproteinsulfhydryls(P-SH)levelsonday15showingthathigherconcentration supplementedtheinherentantioxidantdefensesystemoferythrocytesduringbloodsto- rage.Conclusion: VCprovedtobeeffectiveincombatingOSduringbloodstorage.Howe- ver,furtherexploration ofantioxidantsas additivesandtheerythrocyte storagelesion wouldresultinbettermanagementofbloodstorage.

*Correspondingauthor.

E-mailaddresses:tiwari.vani@gmail.com,vani.rs@jainuniversity.ac.in(R.Vani).

ContentslistsavailableatScienceDirect

Acta Haematologica Polonica

journal homepage:www.elsevier.com/locate/achaem

http://dx.doi.org/10.1016/j.achaem.2017.08.005

zo.o.Allrightsreserved.

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During storage, RBCs undergo structural and functional changes that may reduce function and viability after transfusion[3].ChangesaccompanyingthestorageofRBCs areknownas“storagelesion”.Duringstorageofbloodand its components, in various additive solutions, ithas been foundthat oxidativestress(OS), inﬂuencestheir function- ing,efﬁcacyandshelflife.OSisthefundamentalphenom- enon leading to the formation of storage lesion in erythrocytes[4–6].Proteinoxidationandlipidperoxidation (LPO) also occur, in turncausing vesiculation and loss of deformability[7].OSplays animportantroleintheforma- tion of storage lesions in blood and its components and there exists an inherent antioxidant system in blood to combatOS.However,duringstorage,thereisareductionin blood antioxidant capacity, leading to increased suscept- ibility to OS. Hence, there is a possibility of utilizing antioxidants to aid in protecting the erythrocytes against oxidativedamageduringstorage.

VitaminC (VC)or ascorbicacidisfoundinerythrocytes andcanscavenge awidevariety offreeradicalsdirectlyin theaqueousphase.VCreducessuperoxideandlipidperoxyl radicals, a synergistic agent for Vitamin E [8–10] and protects membrane [11]. Ascorbic acid serves as both an antioxidantandaprooxidant.Asaprooxidant,it generates cofactorsofactivatedoxygenradicalsduringthepromotion ofLPO,inpresenceofFe³⁺andCu²⁺ions[12].Studiesonthe effectsof ascorbicacidonerythrocytesduring storagehave focusedonbiochemicalparameters,hemolysisandfragility [13–19].

Ratmodelsprovideaplatformforrapidexperimentation, in turn forming a basis for further studies on human models. A comparative study between rat and human erythrocytesrevealedthatrat erythrocyteswhenstoredfor 1weekinCPDA-1solution,developastoragelesionequiva- lenttothatofalesionformedinhumanerythrocytesstored for 4 weeks [20]. Our previous work has elucidated that reactiveoxygenspeciesaremaximumondays10and15of storage [21] and hence, our current study focuses on the storagelesionondays0,10and15ofstorage.Thereforethis studyaimstoevaluatetheeffectsof VConerythrocytesof storedratbloodthroughthefollowing3aspects–(i)oxidant levels of erythrocytes in terms of hemolysis, LPO and proteinoxidationproducts,(ii)antioxidantenzymesand(iii) VCasanadditiveinstoragesolution.

Materials and methods

Animal care and maintenance was in accordance with ethicalcommitteeregulations.

Chemicals

Hemoglobin (Hb) reagent was obtained from Coral Clinical Systems,Goa,India.Thiobarbituric acid(TBA),Epinephrine, Glutathione reductase (GR), Glutathione (GSH) and Bovine Serum Albumin (BSA) werepurchased from Sigma–Aldrich Chemicals [St. Louis, MO, USA]. All other chemicals used wereofreagentgradeandorganicsolventswereofspectral grade.

Bloodsampling

Animals were lightly anaesthetized with ether and restrained indorsalrecumbancy asdescribedearlier [6]. In brief,asyringeneedlewasinsertedjust belowthe xyphoid cartilage and slightly to the left of midline. Four to ﬁve milliliter of blood was carefully aspirated from the heart into 5mLplastic collecting tubes with anticoagulant solution,citrate-phosphate-dextrose-adenine-1(CPDA-1).

Experimentaldesign

BloodwasdrawnfromWistarrats(4monthsold)andstored at 48C in anticoagulant solution, CPDA-1. Blood samples weredividedinto3groups–(i)Controls,(ii)VC10(samples withVCasadditiveataconcentrationof10mM),(iii)VC30 (samples withVCasadditiveat aconcentrationof 30mM).

Erythrocytes were isolated from stored blood and the biomarkersofOSwerestudied.

Erythrocyteseparation

Erythrocytes were isolated by centrifugation for 20min at 1000gat48C,plasmaandbuffycoatwereremovedusing a micropipette. Cell pellet was washed 3 times and sus- pended in an equal volume of isotonic phosphate buffer [22].Thisconstitutedtheerythrocytesuspension.

Hbestimation

Hb was measured using Hemocor-D Kit [Coral Clinical Systems, Goa, India], which utilizes Cyanomethemoglobin method [23]. Whole blood was incubated with Hb reagent for 3min at room temperature and absorbance was measured colorimetrically at 540nm. Hb concentration was representedintermsofg/dL.

Superoxide

Superoxide generated was determined by the method of OlasandWachowisz[24].Cytochromec(160mMconcentra- tion) was addedto equal volumeof sampleand incubated at 378C for 5–15min. Samples were then centrifuged at 3500rpm for 5min. Absorbance of the supernatant was measuredat550nm.

Antioxidantenzymes

Superoxidedismutase[SOD,EC1.15.1.1]

SOD was measured by the method of Misra and Fridovich [25]. Hemolysate was added to carbonate buffer [0.05M].

Epinephrine was added to the mixture and measured [ELICO, Model SL 159, India] at 480nm. SOD activity is expressedastheamountof enzymethatinhibitsoxidation ofepinephrineby50%.

Catalase[CAT,EC1.11.1.6]

CAT was determined by the method of Aebi [26]. Brieﬂy, hemolysatewithabsolutealcoholwasincubatedat08C.An aliquotwastakenupwith6.6mMhydrogenperoxide(H2O2)

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and decrease in absorbance was measured at 240nm. An extinctioncoefﬁcient of43.6Mcm¹wasusedtodetermine enzymeactivity.

Glutathioneperoxidase[GSH-Px,EC.1.11.1.9]

GSH-Px wasanalyzedby themethod ofFlohe and Gunzler [27]. Fifty microliters of 0.1M phosphate buffer (pH 7.0), 100mLenzymesample,100mLGR(0.24units),and100mLof 10mM GSH were mixed and pre-incubated for 10min at 378C followed by addition of 100mLof 1.5mM b-nicotina- mideadeninedinucleotidephosphatereducedtetrasodium salt (NADPH) in 0.1% sodium bicarbonate (NaHCO3). The overallreactionwasstartedbyadding100mLofpre-warmed tert-butyl hydroperoxide and the decreasein absorptionat 340nmwasmonitoredfor3min.

Hemolysis

A5%suspensionofpackederythrocytesinbufferwasmixed withequal volumeof 8mMH2O2. Mixtureswereincubated at 378C in an incubator. Hemolysis was determined by measuring released Hb into the supernatant of induced samplesat540nmandexpressedonthebasisofmaximum absorbance [100%] in aliquots of erythrocytes completely hemolysedindistilledwater[28].

LPO

Conjugatedienes

Primary marker of LPO,conjugate dienes, was assessed by the method of Olas and Wachowisz [24]. Brieﬂy, samples werediluted5timesinether:ethanol(1:3,v/v),vortexedfor 1min, centrifuged at 8000rpm for 20minand supernatant wasanalyzedinaspectrophotometerat234nm.Conjugate dienes produced was calculated using molar extinction coefﬁcientof29500M¹cm¹.

Malondialdehyde(MDA)

MDA, a product of LPO was determined according to the method of Ohkawa et al. [29]. In brief, hemolysate was added to 8.1% sodium dodecyl sulfate (SDS), vortexed and incubated at room temperature. This was followed by the addition of 20% acetic acid and 0.6% TBA, and placed in boiling water bath. Samples were allowed to cool and butanol-pyridine was added and centrifuged. Absorbance was measured at 532nm with 1, 1, 3, 3-tetramethoxy propane as a standard. MDA concentration was expressed asnmol/mgprotein.

Proteinoxidation(proteinsulfhydryls(P-SH))

P-SH was measured as described by Habeeb [30]. In brief, 0.08mol/L sodium phosphate buffercontaining 0.5mg/mL of ethylene diamine tetra acetic acid disodium salt (Na2- EDTA),and2%SDSwereadded toeachassaytube.0.1mL of 5,5⁰-dithiobis-(2-nitrobenzoic acid) (DTNB) was also added. Absorbance was measured at 412nm. P-SH was calculated from net absorbance and molar absorptivity, 13600mol/Lcm¹.

Ascorbicacid

Ascorbic acid levels were estimated by the method of Omaye et al. [31]. Brieﬂy, Samples weremixed with equal volumesof10%trichloroaceticacid(TCA)andcentrifugedat 3500g for 20min. Supernatant was mixed with DTC reagent (2,4-dinitrophenylhydrazine/thiourea/copper sulfate solution) and incubated for 3h at 378C. Sixty ﬁve percent ice coldsulfuric acidwasaddedandsampleswereallowed to stand at room temperaturefor 30min. Absorbance was measuredat520nm.

Proteindetermination

Protein was determined in lysate and membrane by the methodofLowryetal.,usingBSAasstandard[32].

Statisticalanalyses

Results are represented as meanstandard error (SE).

Values between the groups were analyzed by two-way analysisofvariance(ANOVA)andwasconsideredsigniﬁcant at P<0.05. Bonferroni’s Post Test was performed for all assaysusingGraphPadPrism5software.

Results

Hb

Changes in Hb were insigniﬁcant during storage in all sampleswithrespecttoday0.

Superoxide

Superoxide levels changed signiﬁcantly as storage progressed.Superoxidelevelsreducedwithstorageby70%(day 10) and80%(day15)againstday0incontrols.Decrements of 75% (day 10) and 90% (day 15) were observed in VC 10 withday0.VC30 alsoshoweddecreasesinsuperoxidesby 85%(days10and15)againstday0.

Changesbetweenantioxidantswereinsigniﬁcant(Fig.1).

Antioxidantenzymes

SOD

Signiﬁcant changes in SOD activity were observed in all groups with storage. Increments of 150% and 40% were observedincontrolsondays10and15(day0).SODactivity in VC 10 increased by 83% and 110% on days 10 and 15 againstday0.VC30showedanincreaseintheSODactivity onday10by63%withrespecttoday0.

SOD varied signiﬁcantly between antioxidants. SOD increasedinVC10by65%thancontrolwhileVC30showed lowerlevelsofSOD(40%)thanVC10onday15(Fig.2).

CAT

OurresultsshowedsigniﬁcantchangesinCATactivitywith storage.Anincreaseof190%(day10)and43%(day15)was observedincontrolsagainstday 0.CAT increasedby 100%

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and32%inVC10whileitincremented by55%inVC30on days10and15.

Changes in CAT between antioxidants weresigniﬁcant.

Reductionsof60%(days10 and15)wereobservedinVC10 and 70% (day 10) wereobservedin VC 30 againstcontrols (Fig.3).

GSH-Px

GSH-Pxactivity was signiﬁcant withstorage period. There was an elevation of 220% and 110% on days 10 and

15 respectively in controls (day 0). GSH-Px increased by 52%(day10)and280%(day15)withrespecttoday0inVC 10.GSH-Pxactivitywashigher inVC30by30%ondays10 and15.

Changes between antioxidants were signiﬁcant. VC 10 showed a reduction in GSH-Px by 65%, 85% and 40% in comparison to respective controls on days 0, 10 and 15.

LevelsofGSH-Pxreducedby73%and57%inVC30ondays 10and15,againstcontrols(Fig.4).

Fig.1–Superoxideinerythrocytesofstoredblood.Values areexpressedasmeanWSEfrom5animals.VC10– VitaminC(10mM);VC30–VitaminC(30mM).Changes wereanalyzedbytwo-wayANOVAfollowedby

Bonferroni’spost-testusingGraphPadPrismSoftware.

P<0.05wasconsideredsignificant.Changesbetweenthe groupsarerepresentedinuppercase,whilechanges withinagrouparerepresentedinlowercase.Thosenot sharingthesamelettersaresignificant

Fig.2–Superoxidedismutaseinerythrocytesofstored blood.ValuesareexpressedasmeanWSEfrom5animals.

VC10–VitaminC(10mM);VC30–VitaminC(30mM).

Changeswereanalyzedbytwo-wayANOVAfollowedby Bonferroni’spost-testusingGraphPadPrismSoftware.

Fig.3–Catalaseinerythrocytesofstoredblood.Valuesare expressedasmeanWSEfrom5animals.VC10–VitaminC (10mM);VC30–VitaminC(30mM).Changeswere analyzedbytwo-wayANOVAfollowedbyBonferroni’s post-testusingGraphPadPrismSoftware.P<0.05was consideredsignificant.Changesbetweenthegroupsare representedinuppercase,whilechangeswithinagroup arerepresentedinlowercase.Thosenotsharingthesame lettersaresignificant

Fig.4–Glutathioneperoxidaseinerythrocytesofstored blood.ValuesareexpressedasmeanWSEfrom5animals.

VC10–VitaminC(10mM);VC30–VitaminC(30mM).

Changeswereanalyzedbytwo-wayANOVAfollowedby Bonferroni’spost-testusingGraphPadPrismSoftware.

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Hemolysis

Signiﬁcant changes in hemolysis were observed in all samples against storage. Hemolysis increased by 46% and 65%incontrols ondays10and15duringstorage.Levelsof hemolysisincreasedinVC10andVC30by39%and43%on days10and15respectively.

Changesbetweenantioxidantswereinsigniﬁcant.

LPO

Conjugatedienes

Changesbetweencontrolsandexperimentalsduringstorage wereinsigniﬁcant.

MDA

MDA was assayed as a measure of LPO and insigniﬁcant changes were observed during storage period. Changes betweengroupswereinsigniﬁcant.

Proteinoxidation(proteinoxidation(P-SH))

Lysate

P-SH was signiﬁcant with storage. Increases of 86% and 130% were observed on days 10 and 15 in controls. P-SH reducedby40%onday10whileitincreasedby35%onday 15inVC10andVC30.

VC 30 showed rises of 370% (day 0) with control. P-SH incremented inVC 30by 200%and 95% onday 15 against controlandVC10respectively(Fig.5).

Membrane

P-SHwasinsigniﬁcantwithstorageand betweengroupsin membrane.

Ascorbicacid

Ascorbic acid varied signiﬁcantly withstorageperiod.Con- trols showed increased levels of ascorbic acid on day 10 (150%) andday 15 (30%)againstday 0.Ascorbicacid levels were higher in VC 10 by 50% on day 10. VC 30 showed higher ascorbic acid on days 10 and 15 (70% and 160%

respectively)againstday0.

Ascorbicacidlevelsweresigniﬁcantamongantioxidants.

Ascorbicaciddecrementedby35%inVC30againstcontrol on day 10 while it incremented by85% on day 15 against respectivecontrolandVC10(Fig.6).

Discussion

Hb,theoxygencarryingmoleculepresentinerythrocytes,is exposedtoaconstantstateofoxygen.Iron(ferrous)present in Hb,upon exposure tooxygenundergoes autoxidationto MetHb,whichisunabletobindand carryoxygen[33]. This reaction is a reversible one and Hb can be restored by several antioxidant mechanisms [34]. Changes in Hb are indicativeofsuccessfulconversionofMetHb(formedduring storage)toHb.

Superoxides(O2)areproducedinerythrocyteseitherby auto-oxidationofHborbyexogenoussources[35,36].These radicalscandirectlyorindirectlyaffectredcellmembranes [37]. Levels of O2 decreased in all groups with storage, indicative of successfulscavenging by SODand interaction of superoxide in reactive oxygen species (ROS) cascade to formH2O2,hydroxylandperoxynitrite[11].

Various endogenous and exogenous mechanisms are present inordertocombatthedeleterious effectsof OSby ROS [38]. SOD, CAT and GSH-Px play an important role in the protection of erythrocytes againstoxygen toxicity[39].

The ﬁrst barrier of defense against superoxide radicals is SOD – an enzyme which facilitates the dismutation of

Fig.5–Proteinsulfhydryls(hemolysate)inerythrocytesof storedblood.ValuesareexpressedasmeanWSEfrom5 animals.VC10–VitaminC(10mM);VC30–VitaminC (30mM).Changeswereanalyzedbytwo-wayANOVA followedbyBonferroni’spost-testusingGraphPadPrism Software.P<0.05wasconsideredsignificant.Changes betweenthegroupsarerepresentedinuppercase,while changeswithinagrouparerepresentedinlowercase.

Thosenotsharingthesamelettersaresignificant

Fig.6–Ascorbicacidinerythrocytesofstoredblood.Values areexpressedasmeanWSEfrom5animals.VC10– VitaminC(10mM);VC30–VitaminC(30mM).Changes wereanalyzedbytwo-wayANOVAfollowedby

Bonferroni’spost-testusingGraphPadPrismSoftware.

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superoxideradicalsusingthemetalionlocated initsactive site [40]. SOD activity was higheron day 10 in accordance with higher levels of ROS. Decrease of SOD activity upon progressionofstorageperiodcanbeattributedtoreduction in free radicals which can bedue to effective antioxidant system of erythrocytes. Ascorbic acid itself reacts with superoxide radicals[41], hence assisting SODin quenching superoxide radicals,asobservedintheresultsof VC30on days10and15.

GSH-Px efficientlyprotects thecell membranefrom LPO andcatalyzesreactionsofhydroperoxideswithGSHtoform glutathione disulfide(GSSG)[42]. GSH-Px enzymecompetes withCATfor H2O2 asasubstrate and isamajorsource of protection againstlow levels of OS[43]. CAT mediatesthe decomposition of H2O2 through catalytic or peroxidative mechanisms at high levels of OS [44]. Our study shows activation of CAT and GSH-Px inall groups in accordance withSODactivity,tocombatH2O2. High levels of CATand GSH-Px wereobserved on day10 incontrolsinaccordance withhighlevelsofROS[21].Decreaseinantioxidantenzyme activity in control on day 15, may be due to free radical scavengingactivityofantioxidantenzymesandtheirinhibi- tion by freeradicalspecies [45, 46]. GSH-Px and CAT were significantlylowerinexperimentalgroupsandthiscouldbe attributedtoradicalscavengingeffectsofVC[8–10],thereby reducingH2O2formation.

Erythrocytemembraneishighlysusceptible toperoxida- tivemodiﬁcationsduetolargeamountsofpolyunsaturated fattyacids, oxygen richenvironmentand ironrichHb [11].

Hemolysis is a reliable marker of erythrocyte membrane stabilityandincreasedinallgroupsindicatingthatVCcould notprotect the membranefrom oxidation completely. LPO is a process that is accelerated by ROS cascade [47] and causes loss of membrane deformability and integrity, in turn leading tocell death [11]. Conjugate dienes and MDA areprimaryand secondarymarkersof LPOrespectively. As storage progressed, LPO was insigniﬁcant demonstrating thatVCmaintainedconjugatedienesandMDA.

Proteinoxidation occurs due toOS and inerythrocytes, P-SH are one of the protein oxidation products of great importance. P-SHcan bereversibly orirreversibly modiﬁed based on the extent of oxidative modiﬁcations [48]. P-SH levelsincreasedonday15of storageinVC30,indicativeof the antioxidant effect of ascorbic acid. VC successfully maintainedsulfhydryllevelsduringstorage.

Ascorbicacid was analyzedto determine theutilization of the added VC during storage. Ascorbic acid levels increased on day 15 inVC 30, showingearly utilization of VCinerythrocyteantioxidantdefense.

Conclusion

VCassistederythrocytesbyscavengingfreeradicals,activat- ing antioxidant enzymes and maintaining Hb, LPO and proteinoxidationproducts.VCataconcentrationof30mM decreasedSOD levels and increased P-SH levels on day 15 showingthathigherconcentrationsupplementedtheinher- ent antioxidant defense system of erythrocytes and pro- tectedthemagainstOSduringstorage.Ourﬁndingsgivean

insightintotheeffectsofVCasanadditiveinbloodstorage solutions.Thisstudyformsabasisforfurtherinvestigations ofeffectivebloodstoragesolutions.

Authors’ contributions/Wkład autorów

Accordingtoorder.

Conﬂict of interest/Konﬂikt interesu

Nonedeclared.

Financial support/Finansowanie

Nonedeclared.

Ethics/Etyka

The work described in this articlehas been carried out in accordance with The Code of Ethics of the World Medical Association (Declarationof Helsinki)for experiments invol- ving humans; EU Directive 2010/63/EU for animal experiments;UniformRequirementsformanuscriptssubmittedto Biomedicaljournals.

Acknowledgements/Podziękowanie

Theauthors acknowledgeDr. Leela Iyengar,Ms. Manasa K, Mr. Carl Hsieh and Jain University for their support. The authors alsoacknowledge theaward of INSPIRE-Fellowship fromtheDepartmentofScienceandTechnology(DST),India toMs.Soumya.

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