Research Article The Relationship of Oxidation Sensitivity...

Research ArticleThe Relationship of Oxidation Sensitivity of Red BloodCells and Carbonic Anhydrase Activity in Stored Human BloodEffect of Certain Phenolic Compounds

Zuumlbeyir Huyut Mehmet Ramazan FekeroLlu RagJp BalahoroLluTahsin Karakoyun and Erdem Ccedilokluk

Department of Biochemistry Medical Faculty Yuzuncu Yıl University 65080 Van Turkey

Correspondence should be addressed to Zubeyir Huyut zubeyirhuyutgmailcom

Received 23 March 2016 Revised 20 May 2016 Accepted 25 May 2016

Academic Editor SivagnanamThamilselvan

Copyright copy 2016 Zubeyir Huyut et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

It has been reported that many modifications occur with the increase of oxidative stress during storage in erythrocytes In orderto delay these negative changes we evaluated whether the addition of substances likely to protect antioxidant capacity in storedblood would be useful Therefore we investigated the effects of resveratrol tannic acid and caffeic acid in lipid peroxidationand antioxidant capacity of erythrocytes in stored blood Donated blood was taken into four CPD containing blood bags Onebag was used as the control and the others were supplemented with caffeic acid (30 120583gmL) resveratrol (30120583gmL) and tannicacid (15120583gmL) respectively Erythrocyte lipid peroxidation sensitivity to oxidation glutathione levels and carbonic anhydraseglutathione peroxidase and catalase activities were measured on days 0 7 14 21 and 28 In the control group erythrocytemalondialdehyde levels and sensitivity to oxidation were increased whereas glutathione glutathione peroxidase and catalase levelswere decreased (119901 lt 005) Resveratrol and caffeic acid prevented malondialdehyde accumulation and preserved glutathioneglutathione peroxidase and catalase activities in erythrocytes We demonstrated that resveratrol caffeic acid and tannic acid instored blood could decrease the sensitivity to oxidation of erythrocytes in vitro but did not exhibit such effects on CA activity

1 Introduction

The treatment of many diseases (eg acute blood loss injuryand anemia) by blood transfusion can be successful [1]The packed red blood cells (RBCs) for transfusion can bestored for approximately up to 42 days at 2ndash6∘C [2 3] It hasbeen reported that some changes occur during the course ofstorage [4] During storage progressive morphological andbiochemical changes occur which are often related to thereduction of ATP 23-biphosphoglycerate and NADH in redblood cells (RBCs) [5 6] These changes are referred to asthe ldquostorage lesionsrdquo [5] There is also strong evidence thatoxidative stress plays a major role in the storage lesions [7]In a previous study we showed that storage time can increasesensitivity to oxidation and oxidative stress in RBCs [7] Inaddition we also revealed a decrease in levels of reducedglutathione (GSH) and activity of antioxidant protectiveenzymes such as glutathione peroxidase (GSH-Px) catalase

and superoxide dismutase (SOD) [7]These negative changesshorten the shelf lives of stored blood [3] When stored bloodcannot be used in time it is discarded therefore the loss ofbudget labour and time came into question

The carbonic anhydrase enzyme (CA EC4211) includ-ing the Zn(II) ion catalyses the reversible hydration of carbondioxide to bicarbonate and protons This enzyme has 16different isoenzymes that are currently known in human[8ndash10] CA I and CA II are the major isozymes that arepresent at high concentrations in the cytosol in RBC [11 12]CA participates in the maintenance of pH homeostasis orerythrocytes by catalysing the reversible hydration of carbondioxide

Several preservatives are added to stored blood such asantioxidants to retard these negative changes [7] The opti-mum protective solution used to protect erythrocytes pro-vides optimal oxygen release and maximum viability of RBC[1] Citrate-phosphate-dextrose (CPD) acid-citrate-dextrose

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 3057384 8 pageshttpdxdoiorg10115520163057384

2 BioMed Research International

(ACD) citrate-phosphate-dextrose-adenine (CPDA-1) andsorbitol-adenine-glucose-mannitol (SAGM) are the mostcommon combination that is added to the stored blood [313] However irrespective of the additive used in preparingthe packed cell units studies also indicate that blood keptunder proper circumstances is subject to increased lipidperoxidation and decreased antioxidant capacity due to thestorage time [14]

Antioxidants are molecules which are exogenous orendogenous These molecules neutralize the oxidative dam-age caused by oxidants by their own intra- and extracellulardefence mechanisms The extracellular defence mechanismsinclude various molecules such as albumin bilirubin trans-ferrin ceruloplasmin uric acid ascorbate and 120572-tocopherolIntracellular free radical-scavenging enzymes provide theprimary antioxidant defence mechanism These enzymesare superoxide dismutase (SOD) glutathione-S-transferaseand glutathione peroxidase (GSH-Px) glutathione reductasecatalase and cytochrome oxidase [14 15]

The antioxidant properties of phenolic compounds aremainly due to their strong reduction potential and abilityto give electrons and protons remove singlet oxygen andchelate the metal ions [16] In our study resveratrol tannicacid and caffeic acid are antioxidants from natural origins[16ndash18] Resveratrol is a natural compound acting on aseries of intracellular mediators Due to its beneficial effectson the human body it has become a focus of interest allover the world [16] Tannic acid is a naturally occurringpolyphenol content of phenolic compounds found in someplants and fruits [18] Some nutrients include tannic acidsuch as wine beer coffee black tea pears bananas black-eyed peas lentils and chocolate [19 20] It is reported thatcaffeic acid (34-dihydroxycinnamic acid) another substanceof our study acts in a protective role in the low densitylipoproteins to 120572-tocopherol [21] In previous studies it wasshown that resveratrol caffeic acid and tannic acid had betterantioxidant capacity at 30 30 and 15120583gmL concentrations invitro respectively [16ndash18]

In our study we investigated the effects of resveratrolcaffeic acid and tannic acid on lipid peroxidation sensitivityto oxidation and activity of carbonic anhydrase (CA) thatprotect pH balance in stored blood in the red blood cellsof the stored blood In addition we determined the effectsof these molecules on catalase GSH-Px and reduced glu-tathione (GSH) levels which provide the main intracellularantioxidant defence system in RBC

2 Material and Methods

The study protocol was performed in accordance with theHelsinki Declaration as revised in 2000 The study wasalso approved by the Ethics Committee of the Yuzuncu YılUniversity and all patients provided their consent Bloodsamples were collected into quaternary paediatric pouchsystems (Kansuk Turkey) containing citrate-phosphate-dextrose (CPD) from 10 healthy male volunteers between 20and 30 years of age Blood pouches contained 500mL bloodand 70mL CPD solution Each 500mL blood sample wasseparated into 4 paediatric pouches connected to the main

pouch The first group was used as the control group withno substance added to the blood A total of 30 120583gmL caffeicacid and 30 120583gmL resveratrol and 15 120583gmL tannic acid wereadded to the second third and fourth group respectivelyNext 10mL blood samples were taken in each group on day0 and on the 7th 14th 21st and 28th days respectively andcentrifuged at 2500 g and the plasma was separated Equalvolumes of isotonic solution andRBCwere added to the tubesand centrifuged at 2500 gThe supernatant was aspirated andthis washing procedure was repeated three timesMDA levelsand the susceptibility to oxidation were quantified from RBCsuspension on the same days The RBC packages were storedat minus20∘C until catalase and GSH-Px and GSH levels weremeasured

21 Haemoglobin Measurement of Erythrocyte PackagesHaemoglobin was measured by the method of Fairbanksand Klee which utilizes cyanomethemoglobin [22] For thispurpose 5mL Drabkin solution and 20120583L haemolysate wereadded to tubes and incubated for 10 minutes at room tem-perature The absorbance was measured colorimetrically at540 nm in spectrophotometer (ShimadzuUVmini 1240) andhaemoglobin concentrationwas represented in terms of gdL

22 Measurement of MDA MDA levels of RBC suspensionswere analyzed by high-performance liquid chromatography(HPLC) as described by Khoschsorur et al [23]Themixture(50 120583L erythrocytes suspension 044M and 750 120583L H

3PO4

and 42mMand 250120583LTBA)was incubated for the formationof the MDA-thiobarbituric acid (TBA) coloured complexMDA-TBA complex was detected fluorometrically at an exci-tation wavelength of 527 nm and at an emission wavelengthof 551 nm We used Agilent 1200 series HPLC systems withan autosampler a gradient pump a column frame a fluo-rescence detector (FLD) (Agilent Technologies WaldbronnGermany) and RP-C18 analytical column (150 times 46mm5 120583m particle size ACE Aberdeen Scotland) Phosphatebuffer (pH 68 50mM) andmethanol (vv 46) were used asmobile phase by adding them to each other The MDA-TBAadduct peakwas calibrated using a 1133-tetraethoxypropane(Sigma-Aldrich USA) standard solution and theMDA levelswere expressed in nmolgHb

23 Susceptibility to Oxidation of Erythrocyte The methodof Stocks et al was used to measure the sensitivity of RBCto in vitro oxidation induced by H

2O2(in concentration

003 in buffer saline solution) in RBC suspension [24]In this method the RBC suspension (5mL) was preparedusing azide buffer and RBC Next 55mL of H

2O2solution

was added to the same cell suspension and it was incubatedat 37∘C for 2 h The MDA level was measured again withTBA as mentioned above (Section 22) (2 h) Haemoglobinconcentration of the erythrocyte suspension was also mea-sured asmentioned above (Section 21) andMDA levels wereexpressed as nmolgHb

24 Measurement of GSH-Px Activity Erythrocyte GSH-Px activity was determined using the method described byPaglia and Valentin [25] In the presence of glutathione

BioMed Research International 3

reductase and NADPH the oxidized glutathione is imme-diately converted to the reduced form with concomitantoxidation of NADPH to NADP+ The decrease in absorbanceat 340 nm in spectrophotometer (Shimadzu UV mini 1240)was measured every 30 seconds six times for 3 minutesThe (Δ119860) absorbance change was calculated and the obtainedresults were expressed as IUgHb

25 Measurement of Catalase Activity Erythrocyte catalaseactivity was measured according to the colorimetric methodof Goth [26] The 02mL sample was incubated with 1mLH2O2(65 120583molL in phosphate buffer pH 74) for oneminute

and the reaction was stopped by adding 1mL ammoniummolybdate solution (324mmolL) The absorbance of theyellow complex formed by molybdate and H

2O2was mea-

sured at 405 nm spectrophotometrically in spectrophotome-ter (Shimadzu UV mini 1240) The obtained results areexpressed as kIUgHb

26 Measurement of Reduced GSH RBC glutathione levelwas measured according to the spectrophotometric methodof Fairbanks and Klee [27] Virtually all of the nonproteinsulfhydryl groups of erythrocytes are in the form of reducedGSH 551015840-Dithiobis(2-nitrobenzoic acid) (DTNB) is a disul-phide chromogen that is readily reduced by sulfhydryl com-pounds to an intensely yellow compound The absorbanceof the reduced chromogen is measured at 412 nm in spec-trophotometer (Shimadzu UV mini 1240) and is directlyproportional to the GSH concentration Although the DTNBassay measures all free sulfhydryl groups in protein andnonprotein it is considered that the level of free sulfhydrylgroups reflects the level of GSH in RBC because the GSHlevel in erythrocytes is notably high The obtained results areexpressed as mggHb

27 Measurement of Carbonic Anhydrase Activity Total CAenzyme activity in RBCwasmeasured by using themethod ofRickly et al that ismodified byWilbur andAnderson [28 29]The enzymatic reaction in a total volume of 17mL contained1mL veronal buffer (pH 82) 01mL bromine thymol blue(004) 055mL pure water and 005mL enzyme solutionA reference measurement was obtained by preparing thesame cuvette without enzyme solution Carbonic anhydraseactivity (CO

2-hydratase activity)was assayed by following the

hydration of CO2 CO2-hydratase activity as an enzyme unit

(EU) was calculated by using the equation 1199050minus 119905119888119905119888 where 119905

0

and 119905119888are the times for pH change of the nonenzymatic and

the enzymatic reactions respectively [30]

28 Statistical Analysis Descriptive statistics for continuousvariables according to the obtained result were expressedas the mean plusmn standard deviation Variance analysis (two-way ANOVA) was used to compare the groups and Duncanrsquosmultiple comparison was used to compare intergroups 119901values less than 005 were considered to be significant SPSSstatistics package program (SPSS Inc IL USA version 15)was used to perform statistical analysis

3 Results and Discussion

31 Lipid Peroxidation and Susceptibility to Oxidation inErythrocytes Packages A major contributing factor to thedecreasing lifespan of the stored erythrocytes could be adecrease in the antioxidant defence system or an increasein oxidative stress Numerous studies have shown that aftertransfusion RBCs substantially lose their viability (approxi-mately 30) due to storage time [3 7] Erythrocytes are richin nonconjugated polyunsaturated fatty acids with reactivemethylene groups that are susceptible to hydrogen atomabstractionThe oxidation of these fatty acids by free radicalsincreases MDA levels which are the end product of lipidperoxidation [31 32] MDA is the end product of lipidperoxidation and formed due to the effect of free radicalsthat modify lipid and lipid derivative molecules Oxidantmolecules such as superoxide anion (O

2

∙minus) H2O2 and the

hydroxyl radical (HO∙minus)may occur during the storage periodby the influence of some factors They may cause damage tothe protein and lipid structures of erythrocytes [32]

It has also been reported that the functions of membranephospholipids and proteins are negatively affected by MDA[7 32] A decrease in antioxidant capacity and an increasein lipid peroxidation of the erythrocytes in the stored bloodduring the storage period have been reported in variousstudies [32 33] Sekeroglu et al [7] reported that MDA andGSH levels and GSH-Px SOD and catalase activities weredecreased by lipid peroxidation time-dependently in the RBCof the stored blood Additionally Aslan et al [14] showed thatMDA levels in stored blood decreasedThe additive solutionsused in preparing packed cell units were not initially designedto reduce oxidative stress per se most were designed to tryto maintain 23-DPG levels pH and so forth Some of thecomponents do however have antioxidant properties Therehave been numerous studies to increase the storage timeof the blood by adding preservative substances to reduceoxidative stress or increase the antioxidant capacity of theRBC [3 7] In the literature there has been no other studythat has examined the protective effect of resveratrol caffeicacid and tannic acid on oxidative stress and the antioxidantcapacity of RBC in stored blood

In our study comparisons in all groups compared tobaseline MDA levels of RBC were higher from the 7th day(119901 lt 005) However MDA levels of caffeic acid group onthe 14th 21st and 28th days were lower than MDA levels ofother groups on the same days (Table 1119901 lt 005) In additionwe also found that erythrocyte MDA levels increased withstorage time in the stored blood This finding is consistentwith previous studies and supports the view that oxidativedamage of the erythrocytesmembrane during storage rendersthem more susceptible to further oxidative stress

We also tested the sensitivity to in vitro oxidation ofstored RBC induced by hydrogen peroxide The effects ofcaffeic acid tannic acid and resveratrol on the sensitivity tooxidation of RBC 2 hours after adding the solution of H

2O2

are shown in Table 2 Intragroup comparisons showed thatthe sensitivity to oxidation of RBC increased from the 7th daycompared to the baseline (119901 lt 005) However the sensitivityto oxidation of RBC in the caffeic acid and resveratrol groups


Table 1The inhibition effects of caffeic acid resveratrol and tannic acid on erythrocyteMDA levels in stored whole blood time-dependently

MDA (nmolgHb)Control Caffeic acid Resveratrol Tannic acid

Baseline (time 0) 2233 plusmn 637 2128 plusmn 513 2197 plusmn 808 2129 plusmn 6497th day 3570 plusmn 289Ł 2964 plusmn 167Łlowast 3109 plusmn 479Łlowast 3143 plusmn 301Łlowast

14th day 3736 plusmn 528Ł 3220 plusmn 276Łlowastlowast 3691 plusmn 198Ł 3768 plusmn 520Ł

21st day 3994 plusmn 695Ł 3386 plusmn 291Łlowastlowast 4026 plusmn 492Ł 4441 plusmn 602Łlowast


Significant differences (119901 lt 005) between the control and treated groups are indicated by lowastSignificant differences (119901 lt 005) between the control and resveratrol and tannic acid are indicated by lowastlowastSignificant differences (119901 lt 005) in each group from time 0 (baseline) are given by Ł

Table 2 The inhibition effects of caffeic acid resveratrol and tannic acid on erythrocyte MDA levels after the two-hour incubation with thesolution of H

2

O2

(sensitivity to oxidation of RBC)

2-hour MDA (nmolgHb)Control Caffeic acid Resveratrol Tannic acid

Baseline 10196 plusmn 914 10356 plusmn 1440 10796 plusmn 914 10881 plusmn 11387th day 27475 plusmn 2193Ł 30919 plusmn 2820Łlowast 22128 plusmn 1569Ł 26793 plusmn 2471Łlowast

14th day 39754 plusmn 2338Ł 39010 plusmn 3271Ł 29809 plusmn 2989Ł 42170 plusmn 2715Łlowastlowastlowast

21st day 43784 plusmn 1996Ł 40785 plusmn 3835Łlowast 31402 plusmn 1841Łlowastlowastlowast 43326 plusmn 1882Ł

28th day 49969 plusmn 1260Ł 41151 plusmn 2015Łlowast 33885 plusmn 1583Łlowastlowastlowast 44173 plusmn 2017Ł

Significant differences (119901 lt 005) between the control and treated groups are indicated by lowastSignificant differences (119901 lt 005) between the control and caffeic acid and tannic acid are indicated by lowastlowastSignificant differences (119901 lt 005) in each group from time 0 (baseline) are given by Ł

on the 21st and 28th days was lower than in the control group(119901 lt 005) In addition MDA levels of resveratrol groupon the 21st and 28th days were lower than MDA levels ofthe control tannic acid and caffeic acid groups (Table 2119901 lt 005) These results showed that the susceptibility tooxidation of RBC is protected with the addition of caffeic acidand resveratrol This property of resveratrol caffeic acid andtannic acid is consistent with the findings of previous studiesin vitro [16ndash18]

32 Changes of Some Antioxidant Enzyme and Protein Valuesin Erythrocyte Packages Dumaswala et al [33] showed thatmembrane lipids and proteins were damaged due to thereduction of RBC antioxidants such as GSH and GSH-Px instored blood These researchers also showed accumulationof MDA band 3 protein aggregation and carboxylation ofband 41 protein in stored blood which could adversely affecterythrocyte survival

One of the most important reasons for the increase inoxidative stress in stored blood is the reduction in the antiox-idant capacity The major antioxidant systems in humanerythrocytes are glutathione SOD GSH-Px and catalase [7]It has been reported that GSH GSH-Px and catalase act byremoving H

2O2in RBC [34] Gaetani et al [35] showed that

GSH-Px and catalase have equal active roles in the detoxifi-cation of H

2O2in human erythrocytes It was reported that

catalase and NADPH protect RBC against acute and highexogenous levels of H

2O2 while GSH protects against the

continuously produced low level of endogenous H2O2[36]

Dumaswala et al [37] showed that increasing the glutathioneconcentration in the stored blood protected the erythrocytes

against free radical damage Numerous studies have shownthat antioxidant enzyme activity and antioxidant capacitydecrease in stored blood in a time-dependent manner [7 14]Ziouzenkova et al [38] showed that free haemoglobin levelincreased during blood storage and caused oxidative stressSekeroglu et al [7] showed that erythrocyte GSH GSH-Pxand catalase levels were decreased during blood storage timeIn this study erythrocyte catalase activitywas increased in thecaffeic acid and resveratrol groups whereas it was preservedin the tannic acid group on the 7th 14th 21st and 28th days(119901 lt 005) RBC catalase activity was decreased in the controlgroup on the 28th day (119901 lt 005) RBC catalase activity ofthe caffeic acid and resveratrol groups was higher than of thecontrol group on the 7th 14th 21st and 28th days and of thetannic acid group on the 14th 21st and 28th days In additioncatalase activity of the caffeic acid group was higher than ofthe other groups on the 21st day (Table 3 119901 lt 005)

In intragroups comparisons GSH levels of RBCdecreased on the 21st and 28th days in the control group andwere preserved in the tannic acid group On the contrarythe levels increased in the caffeic acid group on the 21st and28th days and on the 7th 14th 21st and 28th days in theresveratrol group respectively (119901 lt 005) GSH levels werehigher in the resveratrol caffeic acid and tannic acid groupsthan in the control group on the 21st and 28th days (Table 4119901 lt 005) In addition GSH levels were higher in the caffeicacid group than in the control and tannic acid groups on the28th day (119901 lt 005)

There was a similar situation in GSH-Px activity Inintragroups comparisons erythrocyte GSH-Px activity wasincreased on the 7th 14th 21st and 28th days in control


Table 3The protective effects of caffeic acid resveratrol and tannic acid on catalase activity of RBC in stored whole blood time-dependently

Catalase (kUgHb)Control Caffeic acid Resveratrol Tannic acid

Baseline 1747 plusmn 212 1796 plusmn 449 1802 plusmn 676 1912 plusmn 1537th day 1652 plusmn 304 3012 plusmn 997Łlowast 3864 plusmn 1132Łlowast 1952 plusmn 33614th day 1445 plusmn 156 4094 plusmn 533Łlowast 4096 plusmn 597Łlowast 2026 plusmn 299lowast

21st day 1270 plusmn 151 5622 plusmn 842Łlowastlowastlowast 5015 plusmn 1357Łlowast 2089 plusmn 208lowast

28th day 916 plusmn 152Ł 4884 plusmn 1162Łlowast 4757 plusmn 732Łlowast 2047 plusmn 173lowast


Table 4The protective effects of caffeic acid resveratrol and tannic acid on the GSH levels of RBC in stored whole blood time-dependently

GSH (mggHb)Control Caffeic acid Resveratrol Tannic acid

Baseline 1072 plusmn 068 980 plusmn 244 994 plusmn 069 1034 plusmn 1597th day 1051 plusmn 115 1096 plusmn 152 1286 plusmn 120Ł 1027 plusmn 12014th day 1036 plusmn 063 1061 plusmn 072 1409 plusmn 176Łlowast 1100 plusmn 17521st day 853 plusmn 194Ł 1252 plusmn 229Łlowast 1246 plusmn 091Łlowast 1071 plusmn 144lowast

28th day 796 plusmn 059Ł 1232 plusmn 154Łlowastlowastlowast 1267 plusmn 052Łlowast 1057 plusmn 131lowast

Significant differences (119901 lt 005) between the control and treated groups are indicated by lowastSignificant differences (119901 lt 005) between the control and tannic acid are indicated by lowastlowastSignificant differences (119901 lt 005) in each group from time 0 (baseline) are given by Ł

group and on the 28th day in tannic acid group In thecaffeic acid and resveratrol groups the activity of GSH-Pxwas increased on the 7th and 14th days and then decreasedback to the baseline levels (Table 5 119901 lt 005) In addition thecomparison between groups showed that the GSH-Px levelsof resveratrol caffeic acid and tannic acid groupswere higherthan of the control group on the 7th 14th 21st and 28th days(119901 lt 005)

The levels of antioxidant enzyme and protein such asGSH GSH-Px and catalase were significantly increased atthe beginning of the storage period and throughout thestorage time which was consistent with previous studies [39]However we observed that the addition of resveratrol caffeicacid and tannic acid protects the levels of GSH GSH-Pxand catalase in stored blood and activates them during thefirst two weeks of storage time We did not find any previousstudy in the literature concerning how phenolic compoundsaffect the erythrocyte GSH GSH-Px and catalase levels inin vitro human or animal models But it was shown thattannic acid and resveratrol decreased lipid peroxidation andincreased SODGSHGSH-Px and catalase activity in nickel- lead- and alcohol-induced oxidative stress in tissue ofkidney and liver as well as in vivo [40ndash42]This situationmaybe caused by free radical-scavenging properties of phenoliccompounds

Decreasing of catalase andGSH levels is caused by increas-ing formation of H

2O2or free radicals time-dependently

The addition of resveratrol caffeic acid and tannic acidmay be responsible for the increased activities of GSH-Pxand catalase in the first two weeks of this study because oftheir properties of scavenging the free radicals or activatingthe catalase Moreover the molecular properties of phenolic

compounds may lead to an increase in GSH levels Resver-atrol caffeic acid and tannic acid may scavenge the freeradicals which occur in the stored blood For this reasonresveratrol caffeic acid and tannic acid may become asso-ciated with the conversion of oxidized glutathione (GSSG) tothe reduced form

33 Changes of Total Carbonic Anhydrase Enzyme Activitiesin Erythrocytes Packages CA enzyme catalyses the hydration(H+) of carbon dioxide (CO

2) to bicarbonate (HCO

3

minus)and the corresponding dehydration of bicarbonate in acidicmedium with regeneration of CO

2 in a two-step reaction

[43] It is clearly an important role in ensuring proper pHenvironment to sustain antioxidant capacity and the life of theerythrocytes Therefore the preservation of the CA activityis very important for pH balance in the stored blood orerythrocytes There is no other study in the literature thathas determined time-dependent change in CA activity ofthe RBC in stored blood In our study erythrocyte total CAactivity in the control group decreased time-dependentlybut it is of no statistical significance (Table 6) ErythrocyteCA activities of the resveratrol tannic acid and caffeic acidgroups were significantly higher on the 14th day compared tothe respective baseline levels (119901 lt 005 Table 6) Howeverthe activity of CA decreased back to the baseline over timeThis increase only continued past the 21st day in the caffeicacid group (119901 lt 005)

pH value is another parameter associated with CA activ-ity If the pH changes the erythrocyte membrane integrity isimpaired and an increase in erythrocyte oxidation is seen [1]In our study baseline (time 0) pH values were similar in allgroups (in the range of 7037 plusmn 0073 and 708 plusmn 0114) and


Table 5The protective effects of caffeic acid resveratrol and tannic acid onGSH-Px activity of RBC in stored whole blood time-dependently

GSH-Px (IUgHb)Control Caffeic acid Resveratrol Tannic acid

Baseline 5035 plusmn 238 4892 plusmn 373 4914 plusmn 281 5029 plusmn 4247th day 4547 plusmn 289Ł 5093 plusmn 336lowast 5209 plusmn 225Łlowast 5082 plusmn 181lowast


21st day 3705 plusmn 204Ł 5120 plusmn 326lowast 5035 plusmn 232lowast 4976 plusmn 151lowast

28th day 2869 plusmn 391Ł 4665 plusmn 303lowast 4890 plusmn 238lowast 4530 plusmn 174Łlowast

Significant differences (119901 lt 005) between the control and treated groups are indicated by lowastSignificant differences (119901 lt 005) in each group from time 0 (baseline) are given by Ł

Table 6 The protective effects of caffeic acid resveratrol and tannic acid on carbonic anhydrase activity of RBC in stored whole bloodtime-dependently

Carbonic anhydrase activity (IUgHb)Control Caffeic acid Resveratrol Tannic acid

Baseline 3744705 plusmn 661071 3545950 plusmn 687523 3306122 plusmn 332876 3959214 plusmn 4641807th day 3575953 plusmn 584147 4048928 plusmn 585781 4021321 plusmn 755289Ł 4312478 plusmn 1107250lowast

14th day 3488180 plusmn 691069 4405582 plusmn 1054732Łlowast 4607218 plusmn 1185002Łlowast 4328998 plusmn 761990lowast

21st day 3482220 plusmn 257016 4760152 plusmn 1616080Łlowastlowastlowast 3689759 plusmn 475526 3827701 plusmn 66533028th day 3371445 plusmn 476006 4018218 plusmn 650396 3685838 plusmn 573433 3731928 plusmn 435700Significant differences (119901 lt 005) between the control and treated groups are indicated by lowastSignificant differences (119901 lt 005) between the control and resveratrol and tannic acid are indicated by lowastlowastSignificant differences (119901 lt 005) in each group from time 0 (baseline) are given by Ł

there was no statistical difference between the groupsThe pHlevels gradually dropped as time progresses (119901 lt 005 controlgroup 6583plusmn0055) But pH values of the 28th day in groupsof resveratrol and caffeic acid were higher compared to thecontrol group (119901 lt 005 6590 plusmn 0051 and 6627 plusmn 0073resp)

These results demonstrated that pH levels decreasedtime-dependently but the resveratrol and caffeic acid par-tially protected pH changes This situation may be causedfrom partially protective effect of resveratrol and caffeic acidon the CA activity

4 Conclusions

This study demonstrated that lipid peroxidation and sen-sitivity to oxidation increased and the antioxidant capacitydecreased in RBC of the stored blood time-dependentlyHowever the caffeic acid resveratrol and tannic acid par-tially prevented the lipid peroxidation in RBC Also inRBC they decreased the sensitivity to oxidation of RBC byprotecting antioxidant enzyme activities such as GSH-Pxcatalase and GSH In addition caffeic acid and resveratrolpartially prevented the CA activity and pH change time-dependently This situation explains why resveratrol andcaffeic acid can better prevent lipid peroxidation in RBCFurthermore because the phenolic compounds are able toremove the free radicals forming in the stored blood time-dependently resveratrol tannic acid and caffeic acid mayhave increased the activity of GSH GSH-Px and catalaseBut nevertheless in vitro kinetic studies are needed toexplain the molecular effects of resveratrol caffeic acid and

tannic acid on the increase of erythrocyte GSH-Px GSH andcatalase activities

Based on these results resveratrol caffeic acid and tannicacid may provide a positive contribution to the extensionof the shelf life of stored blood and the conservation ofthe antioxidant capacity in RBC But their influence onfactors such as osmotic fragility total antioxidant capacitytotal oxidative stress and protein carbonyl was investigatedAdditionally to minimise their possible side effects such asacidosis they must be used carefully and their dosages mustbe adjusted correctly

Ethical Approval

The study protocol was carried out in accordance with theHelsinki Declaration as revised in 2000 The study protocolwas approved by the local ethics committee and informedconsent was obtained from each subjectThe Ethics Commit-tee of Yuzuncu Yıl University approved this study (REC no0525122014)

Disclosure

Zubeyir Huyut is the guarantor for this researchThe authorssolely are accountable for the content and writing of thispaper

Competing Interests

The authors declare that they have no competing interests


Authorsrsquo Contributions

Zubeyir Huyut and Mehmet Ramazan Sekeroglu researchedthe literature and conceived the study Zubeyir Huyut TahsinKarakoyun and Ragıp Balahoroglu were involved in protocoldevelopment gaining ethical approval patient recruitmentand data analysis Zubeyir Huyut and Erdem Cokluk wrotethe first draft of the paper All authors reviewed and editedthe paper and approved its final version

Acknowledgments

This study was supported by the Presidency of the IndividualResearch Projects Yuzuncu Yıl University (2013-TF-B006)

References

[1] E Beutler ldquoPreservation and clinical use of erythrocytes andwhole bloodrdquo inWilliamsHematology A LMarshall B ErnestJ K Thomas S Uri K Kenneth and T P Josef Eds pp 2159ndash2163TheMcGraw-Hill Companies New York NY USA 2006

[2] C LrsquoAcqua S Bandyopadhyay R O Francis et al ldquoRed bloodcell transfusion is associated with increased hemolysis andan acute phase response in a subset of critically ill childrenrdquoAmerican Journal of Hematology vol 90 no 10 pp 915ndash9202015

[3] I Mustafa A Al Marwani K M Nasr N A Kano andT Hadwan ldquoTime dependent assessment of morphologicalchanges leukodepleted packed red blood cells stored in SAGMrdquoBioMed Research International vol 2016 Article ID 4529434 7pages 2016

[4] B W Gabrio A R Stevens Jr and C A Finch ldquoErythrocytepreservation III The reversibility of the storage lesionrdquo TheJournal of Clinical Investigation vol 33 no 2 pp 252ndash256 1954

[5] S Proffitt SThomas I Swann et al ldquoStorage of washed or irra-diated red cells in AS-7 improves their in vitro characteristicsrdquoVox Sanguinis vol 109 no 3 pp 203ndash213 2015

[6] C Lei and L-Z Xiong ldquoPerioperative red blood cell transfu-sion what we do not knowrdquo Chinese Medical Journal vol 128no 17 pp 2383ndash2386 2015

[7] M R Sekeroglu Z Huyut and A Him ldquoThe susceptibility oferythrocytes to oxidation during storage of blood effects ofmelatonin and propofolrdquo Clinical Biochemistry vol 45 no 4-5 pp 315ndash319 2012

[8] S B Ozturk Sarikaya F Topal M Senturk I Gulcin and C TSupuran ldquoIn vitro inhibition of 120572-carbonic anhydrase isozymesby some phenolic compoundsrdquo Bioorganic amp Medicinal Chem-istry Letters vol 21 no 14 pp 4259ndash4262 2011

[9] C Yenikaya M Sari H Lkimen M Bulbul and OBuyukgungor ldquoSynthesis and characterization of a novel aminosalicylato salt and its Cu(II) complex and their inhibitionstudies on carbonic anhydrase isoenzymesrdquo Polyhedron vol30 no 3 pp 535ndash541 2011

[10] M Senturk I Gulcin A Dastan O I Kufrevioglu and C TSupuran ldquoCarbonic anhydrase inhibitors Inhibition of humanerythrocyte isozymes I and II with a series of antioxidantphenolsrdquo Bioorganic amp Medicinal Chemistry vol 17 no 8 pp3207ndash3211 2009

[11] D Ekinci H Cavdar O Talaz M Senturk and C T Supu-ran ldquoNO-releasing esters show carbonic anhydrase inhibitory

action against human isoforms I and IIrdquo Bioorganic and Medic-inal Chemistry vol 18 no 10 pp 3559ndash3563 2010

[12] S Lindskog ldquoStructure and mechanism of Carbonic Anhy-draserdquo Pharmacology and Therapeutics vol 74 no 1 pp 1ndash201997

[13] C R Valeri D A Valeri A Gray A Melaragno R C Dennisand C P Emerson ldquoViability and function of red blood cellconcentrates stored at 4∘C for 35 days in CPDA-1 CPDA-2 orCPDA-3rdquo Transfusion vol 22 no 3 pp 210ndash216 1982

[14] R Aslan M R Sekeroglu M Tarakcıoglu and H KoyluldquoInvestigation of malondialdehyde formation and antioxidantenzyme activity in stored bloodrdquo Haematologia vol 28 no 4pp 233ndash237 1997

[15] N Altan A S Dincel and C Koca ldquoDiabetes mellitus veoksidatif stresrdquo Turkish Journal of Biochemistry vol 31 no 2pp 51ndash56 2006

[16] I Gulcin ldquoAntioxidant properties of resveratrol a structure-activity insightrdquo Innovative Food Science amp Emerging Technolo-gies vol 11 no 1 pp 210ndash218 2010

[17] I Gulcin ldquoAntioxidant activity of caffeic acid (34-dihydroxycinnamic acid)rdquo Toxicology vol 217 no 2-3 pp213ndash220 2006

[18] I Gulcin Z Huyut M Elmastas and H Y Aboul-EneinldquoRadical scavenging and antioxidant activity of tannic acidrdquoArabian Journal of Chemistry vol 3 no 1 pp 43ndash53 2010

[19] K-T Chung T Y Wong C-I Wei Y-W Huang and Y LinldquoTannins and human health a reviewrdquo Critical Reviews in FoodScience and Nutrition vol 38 no 6 pp 421ndash464 1998

[20] A King and G Young ldquoCharacteristics and occurrence ofphenolic phytochemicalsrdquo Journal of the American DieteticAssociation vol 99 no 2 pp 213ndash218 1999

[21] J Laranjinha O Vieira V Madeira and L Almeida ldquoTworelated phenolic antioxidants with opposite effects on vitaminE content in low density lipoproteins oxidized by ferrylmyo-globin consumption vs regenerationrdquo Archives of Biochemistryand Biophysics vol 323 no 2 pp 373ndash381 1995

[22] V Fairbanks and G Klee ldquoMeasurement of hemoglobin conce-tration in whole bloodrdquo in Text Book of Clinical Chemistry pp1532ndash1534 W B Saunders 1986

[23] G A Khoschsorur BMWinklhofer-Roob H Rabl T Auer ZPeng and R J Schaur ldquoEvaluation of a sensitive HPLCmethodfor the determination of malondialdehyde and application ofthe method to different biological materialsrdquo Chromatographiavol 52 no 3-4 pp 181ndash184 2000

[24] J Stocks E L Offerman C B Modell and T L DormandyldquoThe susceptibility to autoxidation of human red cell lipids inhealth and diseaserdquo British Journal of Haematology vol 23 no6 pp 713ndash724 1972

[25] D E Paglia and W N Valentine ldquoStudies on the quantitativeand qualitative characterization of erythrocyte glutathione per-oxidaserdquo The Journal of Laboratory and Clinical Medicine vol70 no 1 pp 158ndash169 1967

[26] L Goth ldquoA simple method for determination of serum catalaseactivity and revision of reference rangerdquo Clinica Chimica Actavol 196 no 2-3 pp 143ndash151 1991

[27] V F Fairbanks andM D Klee ldquoBiochemical aspect of hematal-ogyrdquo in Tietz Textbook of Clinical Chemistry C A Burtis and ER Ashwood Eds pp 1974ndash2072 WB Saunders PhiladelphiaPa USA 1994

[28] E E Rickly S A S Ghazanfar B H Gibbons and J T EdsallldquoCarbonic anhydrase from human erythrocytesrdquoThe Journal ofBiological Chemistry vol 239 pp 1065ndash1078 1964


[29] K M Wilbur and N G Anderson ldquoElectrometric and colori-metric determination of carbonic anhydraserdquo The Journal ofBiological Chemistry vol 176 pp 147ndash154 1948

[30] T H Maren ldquoCarbonic anhydrase chemistry physiology andinhibitionrdquo Physiological Reviews vol 47 no 4 pp 595ndash7811967

[31] T Sugihara W Rawicz E A Evans and R P Hebbel ldquoLipidhydroperoxides permit deformation-dependent leak of mono-valent cation from erythrocytesrdquo Blood vol 77 no 12 pp 2757ndash2763 1991

[32] G M Wagner D T-Y Chiu J-H Qju R H Heath andB H Lubin ldquoSpectrin oxidation correlates with membranevesiculation in stored RBCsrdquo Blood vol 69 no 6 pp 1777ndash17811987

[33] U J Dumaswala L Zhuo D W Jacobsen S K Jain and KA Sukalski ldquoProtein and lipid oxidation of banked humanerythrocytes role of glutathionerdquo Free Radical Biology andMedicine vol 27 no 9-10 pp 1041ndash1049 1999

[34] F Gultekin M Akdogan I Altuntas N Delikbas and MKaptanagasi ldquoChanges in erythrocyte lipid peroxidation andantioxidant potential rupings storage of blood and protectiveeffect of melatoninrdquo Turkish Journal of Biochemistry vol 25 no3 pp 83ndash91 2000

[35] G F Gaetani S Galiano L Canepa A M Ferraris and HN Kirkman ldquoCatalase and glutathione peroxidase are equallyactive in detoxification of hydrogen peroxide in human erythro-cytesrdquo Blood vol 73 no 1 pp 334ndash339 1989

[36] J W Eaton ldquoCatalases and peroxidases and glutathione andhydrogen peroxide mysteries of the bestiaryrdquo The Journal ofLaboratory and Clinical Medicine vol 118 no 1 pp 3ndash4 1991

[37] U J Dumaswala M J Wilson Y L Wu et al ldquoGlutathioneloading prevents free radical injury in red blood cells afterstoragerdquo Free Radical Research vol 33 no 5 pp 517ndash529 2000

[38] O Ziouzenkova L Asatryan and A Sevanian ldquoOxidativestress resulting from hemolysis and formation of catalyticallyactive hemoglobin protective strategiesrdquo International Journalof Clinical Pharmacology andTherapeutics vol 37 no 3 pp 125ndash132 1999

[39] H Carl A Chandni K Neha S Trishna and R VanildquoCurcumin as a modulator of oxidative stress during storageA Study on Plasmardquo Transfusion and Apheresis Science vol 50no 2 pp 288ndash293 2014

[40] L Pari and A Prasath ldquoEfficacy of caffeic acid in preventingnickel induced oxidative damage in liver of ratsrdquo Chemico-Biological Interactions vol 173 no 2 pp 77ndash83 2008

[41] L Pari and K Karthikesan ldquoBeneficial effect of caffeic Acid onalcohol-induced alterations in lipid peroxidation and antioxi-dant defense in ratsrdquo Toxicology Mechanisms and Methods vol17 no 9 pp 527ndash534 2007

[42] W M Chen L H Shaw P J Chang et al ldquoHepatoprotectiveeffect of resveratrol against ethanol-induced oxidative stressthrough induction of superoxide dismutase in vivo and in vitrordquoExperimental and Therapeutic Medicine vol 11 no 4 pp 1231ndash1238 2016

[43] S Beydemir M Ciftci O I Kufrevioglu and M EBuyukokuroglu ldquoEffects of gentamicin sulfate on enzymeactivities of carbonic anhydrase from human erythrocytes invitro and from rat erythrocytesrdquo Biological and PharmaceuticalBulletin vol 25 no 8 pp 966ndash969 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


(ACD) citrate-phosphate-dextrose-adenine (CPDA-1) andsorbitol-adenine-glucose-mannitol (SAGM) are the mostcommon combination that is added to the stored blood [313] However irrespective of the additive used in preparingthe packed cell units studies also indicate that blood keptunder proper circumstances is subject to increased lipidperoxidation and decreased antioxidant capacity due to thestorage time [14]

Antioxidants are molecules which are exogenous orendogenous These molecules neutralize the oxidative dam-age caused by oxidants by their own intra- and extracellulardefence mechanisms The extracellular defence mechanismsinclude various molecules such as albumin bilirubin trans-ferrin ceruloplasmin uric acid ascorbate and 120572-tocopherolIntracellular free radical-scavenging enzymes provide theprimary antioxidant defence mechanism These enzymesare superoxide dismutase (SOD) glutathione-S-transferaseand glutathione peroxidase (GSH-Px) glutathione reductasecatalase and cytochrome oxidase [14 15]

The antioxidant properties of phenolic compounds aremainly due to their strong reduction potential and abilityto give electrons and protons remove singlet oxygen andchelate the metal ions [16] In our study resveratrol tannicacid and caffeic acid are antioxidants from natural origins[16ndash18] Resveratrol is a natural compound acting on aseries of intracellular mediators Due to its beneficial effectson the human body it has become a focus of interest allover the world [16] Tannic acid is a naturally occurringpolyphenol content of phenolic compounds found in someplants and fruits [18] Some nutrients include tannic acidsuch as wine beer coffee black tea pears bananas black-eyed peas lentils and chocolate [19 20] It is reported thatcaffeic acid (34-dihydroxycinnamic acid) another substanceof our study acts in a protective role in the low densitylipoproteins to 120572-tocopherol [21] In previous studies it wasshown that resveratrol caffeic acid and tannic acid had betterantioxidant capacity at 30 30 and 15120583gmL concentrations invitro respectively [16ndash18]

In our study we investigated the effects of resveratrolcaffeic acid and tannic acid on lipid peroxidation sensitivityto oxidation and activity of carbonic anhydrase (CA) thatprotect pH balance in stored blood in the red blood cellsof the stored blood In addition we determined the effectsof these molecules on catalase GSH-Px and reduced glu-tathione (GSH) levels which provide the main intracellularantioxidant defence system in RBC

2 Material and Methods

The study protocol was performed in accordance with theHelsinki Declaration as revised in 2000 The study wasalso approved by the Ethics Committee of the Yuzuncu YılUniversity and all patients provided their consent Bloodsamples were collected into quaternary paediatric pouchsystems (Kansuk Turkey) containing citrate-phosphate-dextrose (CPD) from 10 healthy male volunteers between 20and 30 years of age Blood pouches contained 500mL bloodand 70mL CPD solution Each 500mL blood sample wasseparated into 4 paediatric pouches connected to the main

pouch The first group was used as the control group withno substance added to the blood A total of 30 120583gmL caffeicacid and 30 120583gmL resveratrol and 15 120583gmL tannic acid wereadded to the second third and fourth group respectivelyNext 10mL blood samples were taken in each group on day0 and on the 7th 14th 21st and 28th days respectively andcentrifuged at 2500 g and the plasma was separated Equalvolumes of isotonic solution andRBCwere added to the tubesand centrifuged at 2500 gThe supernatant was aspirated andthis washing procedure was repeated three timesMDA levelsand the susceptibility to oxidation were quantified from RBCsuspension on the same days The RBC packages were storedat minus20∘C until catalase and GSH-Px and GSH levels weremeasured

21 Haemoglobin Measurement of Erythrocyte PackagesHaemoglobin was measured by the method of Fairbanksand Klee which utilizes cyanomethemoglobin [22] For thispurpose 5mL Drabkin solution and 20120583L haemolysate wereadded to tubes and incubated for 10 minutes at room tem-perature The absorbance was measured colorimetrically at540 nm in spectrophotometer (ShimadzuUVmini 1240) andhaemoglobin concentrationwas represented in terms of gdL

22 Measurement of MDA MDA levels of RBC suspensionswere analyzed by high-performance liquid chromatography(HPLC) as described by Khoschsorur et al [23]Themixture(50 120583L erythrocytes suspension 044M and 750 120583L H

3PO4

and 42mMand 250120583LTBA)was incubated for the formationof the MDA-thiobarbituric acid (TBA) coloured complexMDA-TBA complex was detected fluorometrically at an exci-tation wavelength of 527 nm and at an emission wavelengthof 551 nm We used Agilent 1200 series HPLC systems withan autosampler a gradient pump a column frame a fluo-rescence detector (FLD) (Agilent Technologies WaldbronnGermany) and RP-C18 analytical column (150 times 46mm5 120583m particle size ACE Aberdeen Scotland) Phosphatebuffer (pH 68 50mM) andmethanol (vv 46) were used asmobile phase by adding them to each other The MDA-TBAadduct peakwas calibrated using a 1133-tetraethoxypropane(Sigma-Aldrich USA) standard solution and theMDA levelswere expressed in nmolgHb

23 Susceptibility to Oxidation of Erythrocyte The methodof Stocks et al was used to measure the sensitivity of RBCto in vitro oxidation induced by H

2O2(in concentration

003 in buffer saline solution) in RBC suspension [24]In this method the RBC suspension (5mL) was preparedusing azide buffer and RBC Next 55mL of H

2O2solution

was added to the same cell suspension and it was incubatedat 37∘C for 2 h The MDA level was measured again withTBA as mentioned above (Section 22) (2 h) Haemoglobinconcentration of the erythrocyte suspension was also mea-sured asmentioned above (Section 21) andMDA levels wereexpressed as nmolgHb

24 Measurement of GSH-Px Activity Erythrocyte GSH-Px activity was determined using the method described byPaglia and Valentin [25] In the presence of glutathione





2O2was mea-







0






2






2O2











2

O2









































3




















4 Conclusions




Ethical Approval


Disclosure


Competing Interests





Acknowledgments


References





















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





2O2was mea-







0






2






2O2











2

O2









































3




















4 Conclusions




Ethical Approval


Disclosure


Competing Interests





Acknowledgments


References





















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










2

O2









































3




















4 Conclusions




Ethical Approval


Disclosure


Competing Interests





Acknowledgments


References





















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





















3




















4 Conclusions




Ethical Approval


Disclosure


Competing Interests





Acknowledgments


References





















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















4 Conclusions




Ethical Approval


Disclosure


Competing Interests





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

Research Article The Relationship of Oxidation Sensitivity...

Documents

Transcript of Research Article The Relationship of Oxidation Sensitivity...