Research Article Prooxidant-Antioxidant Balance in ...downloads.hindawi.com/journals/ad/2016/4343514.pdfLupus Erythematosus and Its Relationship with Clinical and Laboratory Findings

Research ArticleProoxidant-Antioxidant Balance in Patients with SystemicLupus Erythematosus and Its Relationship with Clinical andLaboratory Findings

Seyyed Mehdi Jafari12 Saeedeh Salimi12 Alireza Nakhaee12 Hamed Kalani3

Shima Tavallaie4 Farzaneh Farajian-Mashhadi25 Zahra Zakeri6 and Mahnaz Sandoughi6

1Department of Clinical Biochemistry School of Medicine Zahedan University of Medical Sciences Zahedan 9816743175 Iran2Cellular and Molecular Research Center Zahedan University of Medical Sciences Zahedan 9816743175 Iran3Department of Parasitology and Mycology School of Medicine Isfahan University of Medical Sciences Isfahan Iran4Department of Nutrition and Biochemistry Faculty of Medicine Mashhad University of Medical Sciences Mashhad Iran5Department of Pharmacology School of Medicine Zahedan University of Medical Sciences Zahedan 9816743175 Iran6Department of Internal Medicine School of Medicine Zahedan University of Medical Sciences Zahedan 9816743175 Iran

Correspondence should be addressed to Saeedeh Salimi sasalimiyahoocom

Received 19 December 2015 Accepted 11 January 2016

Academic Editor Ricard Cervera

Copyright copy 2016 Seyyed Mehdi Jafari et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Aim This study was aimed at evaluating prooxidant-antioxidant balance (PAB) in patients with systemic lupus erythematosus(SLE) and its relationship with laboratory findings and clinical manifestations Methods In this case-control study 60 patientswith SLE and 60 healthy individuals were enrolled The blood samples were collected and their sera were separated Subsequentlythe prooxidant-antioxidant balance value was evaluated using PAB assay for each sample Results The mean of PAB values in SLEpatients was significantly higher than healthy controls (1473plusmn42 versus 848plusmn322HK 119875 lt 00001) Furthermore in SLE patientsthere was a positive significant correlation between the PAB and erythrocyte sedimentation rate (ESR) (119903 = 0492 119875 lt 0001) Inaddition the PAB values in patients with alopecia discoid rash oral ulcers arthritis and nephritis were significantly higher thanthose without these manifestationsConclusionThe findings of current study showed that themean of PABwas significantly higherin SLE patients and PAB was correlated with ESR Moreover increased PAB was found in SLE patients with alopecia discoid rashoral ulcers arthritis and nephritis These findings suggest that the measurement of PAB may be useful to show oxidative stresscondition in SLE patients

1 Introduction

Systemic lupus erythematosus (SLE) is an autoimmuneinflammatory disease which can involve nearly all the bodyrsquostissues and organs such as kidneys skin cardiovascularsystem lungs joints muscles and nervous system [1] SLEis characterized by a broad spectrum of clinical presentationand the existence of multiple autoantibodies mostly involv-ing women in reproductive age [2 3] The course of thedisease is variable with periods of remission and relapse [4]Despite the fact that important advances in understanding

its etiopathogenesis have been made over the last severalyears the exact cause of lupus is unknown [4] The envi-ronmental factors and genetic and hormonal disorders arecommon factors that contribute to development of thisdisease [5] In recent years it has been demonstrated thatfree radical damage and oxidative stress play a major rolein pathogenesis and clinical symptoms in patients with SLE[6] Free radicals and reactive oxygen species (ROS) aregenerated constantly as part of cellular metabolic processesThese free radicals are deactivated by a chain reaction-breaking antioxidant defense system consisting of enzymes

Hindawi Publishing CorporationAutoimmune DiseasesVolume 2016 Article ID 4343514 5 pageshttpdxdoiorg10115520164343514

2 Autoimmune Diseases

and numerous nonenzymatic antioxidants [7] Oxidativestress is essentially an imbalance between productions ofreactive oxygen species (ROS) and antioxidant defensemech-anism [8] The increased production of free radicals disruptsredox status and can increase the expression of a wide varietyof inflammatory molecules that may be important causesof the inflammation and tissue damage [9] Several studiessuggested that oxidative stress could be a risk factor forautoimmune diseases such as SLE [8ndash10] Impaired antioxi-dant status has been reported in the saliva of SLE patients too[11]

The oxidative stress is evaluated by various methods inwhich oxidants and antioxidants aremeasured separately [12ndash14] Accordingly the PAB (prooxidant-antioxidant balance)method was developed to provide a rapid and reliabledetection of oxidant and antioxidant balance simultaneously[15] To the best of our knowledge there is no published reportabout the PAB assay in SLE patients therefore current studywas aimed at evaluating prooxidant-antioxidant balance inpatientswith SLE and its relationshipwith laboratory findingsand clinical manifestations

2 Materials and Methods

21 Participants This study was confirmed by the UniversityResearch Ethics Committee (UREC) of the Zahedan Univer-sity of Medical Science In this case-control study 60 patientswith SLE (54 women and 6 men) with a mean age of 3057 plusmn774 years as a test group and 60 healthy individuals (54women and 6 men) with a mean age of 3107 plusmn 75 years asa control group were enrolled All participants completed theinformed consent form Patients in this study were selectedin accordance with the American College of Rheumatology(ACR) criteria for SLE Moreover the participants in thecontrol group had no systemic diseases and their antinuclearantibody (ANA) test was negative

22 Sampling Two-milliliter fasting blood samples werecollected from all the subjects The blood samples werecentrifuged immediately and their sera were separated andthen stored at minus80∘C until use

23 Chemicals 331015840551015840-Tetramethylbenzidine (TMB) pow-der and chloramine T trihydrate were obtained from Appli-Chem (Darmstadt Germany) peroxidase enzyme and uricacid were obtained from Sigma-Aldrich (St Louis MO) andhydrogen peroxide and DMSO were obtained from MerckCompany (Darmstadt Germany)

24 Prooxidant-Antioxidant Balance (PAB) Assay The PABassay was performed in accordance with the methodexplained in detail by Alamdari et al [16] Briefly the PABmethod was used to determine the prooxidant-antioxidantbalance value

241 Preparation of Solutions 60mg TMB powder wasweighed and 10mL DMSO was added The TMB cation wasprepared as follows 20mL of acetate buffer [005M buffer

pH 45] 400 120583L of TMBDMSO and 70 120583L of fresh chlo-ramine T (100mM) solution were mixed and incubatedat room temperature for 2 hours in dark place 25U ofperoxidase enzyme solution was added into 20mL TMBcation dispensed into 1mL aliquots and frozen at minus20∘C theTMB solution was prepared with 200120583L of TMBDMSO in10mL of acetate buffer [005M buffer pH = 58] in orderto prepare working solution 1mL TMB cation was added in10mL of TMB solution incubated at room temperature for2min in a dark place and used freshly

242 Procedure 10 120583L of 200120583L of working solution in eachwell of a 96-well plate each sample standard or blank wasadded and the mixture was allowed to incubate at 37∘C for12min in a dark place at the end of the incubation time100 120583L of 2N HCl was added to each well the absorbance at450 nm was read against distilled water Hydrogen peroxidewas considered as a standard in different concentrations (0ndash100) After drawing the standard curve the PAB valuewas calculated for each unknown sample according to thestandard curve using OD obtained for each sample in the testand control groups

25 Data Analysis All the statistical analysis was carriedout by SPSS software version 16 Data have been presentedas mean plusmn SD as well as percentage Data in each groupshowed a normal distribution by Kolmogorov-Smirnov (K-S) statistical test Comparison between test and controlgroups was performed using the independent sample 119905-testIn addition Pearsonrsquos correlation coefficient test was used todetermine the correlation among different factors

3 Results

Table 1 shows demographic and laboratory data related to thepatients and control groups The data analysis showed thatthere was statistically a significant difference between meansof RBC hemoglobin WBC platelets hematocrit and ESR intwo independent groups

As shown in Figure 1 the mean of PAB values in SLEpatients (1473 plusmn 42HK) was higher than the healthy group(848 plusmn 322HK) and it was highly significant statistically(119875 lt 00001)

In addition Table 2 shows the correlation between thePAB value and laboratory findings in the patients and controlgroups In SLE patients there was a positive significant corre-lation between the PAB value and erythrocyte sedimentationrate (ESR) (119903 = 0492 119875 lt 0001)

Moreover as has been shown in Table 3 the PAB valueswere significantly different in SLE patients with and withoutsome clinicalmanifestations such as alopecia (164plusmn42 versus132 plusmn 36 119875 = 0002) discoid rash (176 plusmn 42 versus 140 plusmn39 119875 = 0005) oral ulcers (158 plusmn 42 versus 132 plusmn 38 119875 =0002) arthritis (158 plusmn 39 versus 112 plusmn 29 119875 = 00001) andnephritis (182plusmn29 versus 134plusmn40119875 = 00002)Therewere nocorrelations between PAB values and photosensitivity malarrash anemia serositis neurological involvement ANA andanti-dsDNA manifestations

Autoimmune Diseases 3

Table 1 Demographic and laboratory characteristics of SLE patientsand healthy controls

Parameters SLE patients119899 = 60

Healthycontrols119899 = 60

119875 value

Sex (MF) 546 537 1Age (years) 306 plusmn 78 311 plusmn 75 07Hemoglobin (gdL) 111 plusmn 1 127 plusmn 17 lt00001RBC (times1000000mL) 43 plusmn 06 47 plusmn 064 00002WBC (times1000mL) 71 plusmn 23 73 plusmn 18 06Platelets (times1000mL) 1996 plusmn 674 2238 plusmn 487 003Hematocrit () 341 plusmn 28 378 plusmn 47 lt00001ESR (mmh) 361 plusmn 159 186 plusmn 73 lt00001PAB (HK unit) 1473 plusmn 42 848 plusmn 322 lt0001F femaleMmale Hb hemoglobin RBC red blood cellsWBCwhite bloodcells ESR erythrocyte sedimentation rate

Table 2 Correlation of PAB values and laboratory findings inpatients with SLE and healthy controls

ParametersPAB (HK unit)

SLE patients Healthy controls1198772

119875 value 1198772

119875 valueHemoglobin (gdL) minus02 01 minus02 02

RBC (times1000000mL) minus01 05 minus01 05

WBC (times1000mL) minus01 04 006 07

Platelets (times1000mL) 008 05 004 08

Hematocrit () minus01 04 minus014 03

ESR (mmh) 05 lt00001 02 009

4 Discussion

Systemic lupus erythematosus is an autoimmune disorderthat affects many different tissues and organs in the body[1] The exact cause of the disease is not fully known butenvironmental factors and genetic and hormonal disordersare involved in development of SLE [4] Some studies revealedthat the inability of the antioxidant defense system to copewith oxidative stress plays an important role in the patho-genesis of SLE [17 18] The increased oxidative stress inpatients with SLE can lead to the oxidative modifications ofproteins lipids and DNA in cells [19] The main objectiveof the present study was therefore to evaluate prooxidant-antioxidant balance (PAB) in patients with systemic lupuserythematosus (SLE) and its relationship with laboratoryfindings and clinical manifestations In this study we showedthat an increase in PAB value among SLE patients in com-parison to the control group and PAB was correlated withESRMoreover increased PABwas found in SLE patients withalopecia discoid rash oral ulcers arthritis and nephritisZhang et al [20] and Morgan et al [21] demonstrated thatthe protein oxidation markers are associated with chronicinjuries in SLE patients These studies support strongly therole of oxidative stress in pathogenesis of SLE In addi-tion Shah et al [22] showed that malondialdehyde level as

200

150

100

50

PAB

valu

e (H

K un

it)

SLE patients Controls

30

P lt 00001

Figure 1 Comparison of PAB values between SLE patients and con-trol group

a marker of lipid peroxidation was significantly higher in SLEpatients compared to control group In a recent study theantioxidant activities of some enzymes namely superoxidedismutase catalase and glutathione peroxidase and the levelof glutathione molecules were significantly lower in SLEpatients than in controls Likewise in another study authorsrevealed a significant relationship between malondialdehydelevel and ESR in patients with SLE [23] Similarly the presentstudy showed a significant positive correlation between thePAB value and ESR in SLE patients In a study urinaryF2-isoprostane excretion was evaluated as a biomarker foroxidative stress and the findings showed that there wasno significant correlation between this biomarker and somemajor symptoms such as inflammation [24] Researchers else-where illustrated that antibody levels against hydroxynonenaland malondialdehyde in sera obtained from SLE patientshad a significant positive correlation with disease activity sothat the patients with score higher than 6 determined bySLE disease activity index (SLEDAI) had higher levels of thementioned antibodies [25] However it appears that patientswith inactive disease also have high levels of lipoperoxidationand protein oxidation [26] Furthermore it was revealed thatthe balance between oxidants and antioxidants is impairedin lupus patients and the consumption of antioxidants suchas vitamin E eicosapentaenoic acid and docosahexaenoicacid can be effective in reducing the symptoms of the disease[27] It was also observed that the use of vitamins C andE leads to a considerable decrease in lipid peroxidationbut not in other evaluated oxidative stress markers [28]In the current study the PAB value evaluation showedthat the prooxidant-antioxidant balance in SLE patients wassignificantly higher than in healthy controls Therefore thefindings of this study showed that the PAB method can beused as a new method representing the intensity of oxidativestress in patients with SLE Accordingly at the final pointwe suggest strongly that antioxidants as a complementarytherapy should be always added to medications of SLEpatients In conclusion increased PAB values were observed


Table 3 Comparison of PAB values between SLE patients with and without different manifestation

SLE manifestations PAB (UK units)119875 value

SLE patients with SLE patients withoutPhotosensitivity 152 plusmn 37 140 plusmn 49 03Alopecia 164 plusmn 42 132 plusmn 36 0002Malar rash 154 plusmn 46 140 plusmn 33 01Discoid rash 176 plusmn 42 140 plusmn 39 0005Oral ulcers 158 plusmn 42 132 plusmn 38 002Anemia 150 plusmn 38 144 plusmn 47 06Arthritis 158 plusmn 39 112 plusmn 29 00001Nephritis 182 plusmn 29 134 plusmn 40 00002Serositis 154 plusmn 58 147 plusmn 41 07Neurological involvement 151 plusmn 36 146 plusmn 44 07ANA 147 plusmn 42 147 plusmn 48 1Anti-dsDNA 144 plusmn 44 154 plusmn 38 04

in SLE patients compared to controls Higher PAB was foundin SLE patients with alopecia In addition PAB was correlatedwith ESR These findings suggest that the measurement ofPAB may be useful to show oxidative stress condition in SLEpatients

Disclosure

This paper was extracted from the MS thesis (registrationnumber 2160) at Zahedan University of Medical Sciences

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgment

The authors thank Zahedan Deputy of Research Affairs forfunding this project

References

[1] J C Crispın S-N C Liossis K Kis-Toth et al ldquoPathogenesis ofhuman systemic lupus erythematosus recent advancesrdquo Trendsin Molecular Medicine vol 16 no 2 pp 47ndash57 2010

[2] N Agmon-Levin M Mosca M Petri and Y ShoenfeldldquoSystemic lupus erythematosus one disease or manyrdquo Autoim-munity Reviews vol 11 no 8 pp 593ndash595 2012

[3] A P Grammatikos and G C Tsokos ldquoImmunodeficiency andautoimmunity lessons from systemic lupus erythematosusrdquoTrends in Molecular Medicine vol 18 no 2 pp 101ndash108 2012

[4] G J Pons-Estel D Wojdyla G McGwin et al ldquoThe AmericanCollege of Rheumatology and the Systemic Lupus InternationalCollaborating Clinics Classification criteria for systemic lupuserythematosus in two multiethnic cohorts a commentaryrdquoLupus vol 23 no 1 pp 3ndash9 2014

[5] G C Tsokos and G M Kammer ldquoMolecular aberrationsin human systemic lupus erythematosusrdquo Molecular MedicineToday vol 6 no 11 pp 418ndash424 2000

[6] A Perl ldquoOxidative stress in the pathology and treatment ofsystemic lupus erythematosusrdquo Nature Reviews Rheumatologyvol 9 no 11 pp 674ndash686 2013

[7] V Lobo A Patil A Phatak and N Chandra ldquoFree radicalsantioxidants and functional foods impact on human healthrdquoPharmacognosy Reviews vol 4 no 8 pp 118ndash126 2010

[8] A Bhatnagar and A Aggarwal ldquoOxidative stressmdasha majorplayer in the pathophysiology of systemic lupus erythematosusrdquoin Systems Biology of Free Radicals and Antioxidants pp 2539ndash2559 Springer Berlin Germany 2014

[9] D Shah NMahajan S Sah S K Nath and B Paudyal ldquoOxida-tive stress and its biomarkers in systemic lupus erythematosusrdquoJournal of Biomedical Science vol 21 article 23 2014

[10] J Fujii T Kurahashi T Konno THomma andY Iuchi ldquoOxida-tive stress as a potential causal factor for autoimmune hemolyticanemia and systemic lupus erythematosusrdquo World Journal ofNephrology vol 4 no 2 pp 213ndash222 2015

[11] S H Zaieni Z Derakhshan and R Sariri ldquoAlternations ofsalivary antioxidant enzymes in systemic lupus erythematosusrdquoLupus vol 24 no 13 pp 1400ndash1405 2015

[12] M Horoz C Bolukbas F F Bolukbas et al ldquoMeasurementof the total antioxidant response using a novel automatedmethod in subjects with nonalcoholic steatohepatitisrdquo BMCGastroenterology vol 5 article 35 2005

[13] 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

[14] K I Berker K Gulu B Demirata and R Apak ldquoA novel anti-oxidant assay of ferric reducing capacity measurement usingferrozine as the colour forming complexation reagentrdquo Analyt-ical Methods vol 2 no 11 pp 1770ndash1778 2010

[15] D H Alamdari K Paletas T Pegiou M Sarigianni C Befaniand G Koliakos ldquoA novel assay for the evaluation of the pro-oxidant-antioxidant balance before and after antioxidant vita-min administration in type II diabetes patientsrdquo Clinical Bio-chemistry vol 40 no 3-4 pp 248ndash254 2007

[16] 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


[17] Y G Perez L C G Perez Rita de Cassia M Netto D S N deLima and E S Lima ldquoMalondialdehyde and sulfhydryl groupsas biomarkers of oxidative stress in patients with systemic lupuserythematosusrdquo Revista Brasileira de Reumatologia vol 52 no4 pp 658ndash660 2012

[18] Y Li G Gorelik F M Strickland and B C RichardsonldquoOxidative stress T cell DNAmethylation and lupusrdquo Arthritisand Rheumatology vol 66 no 6 pp 1574ndash1582 2014

[19] M A B Lozovoy A N C Simao S R Oliveira et al ldquoRela-tionship between iron metabolism oxidative stress and insulinresistance in patients with systemic lupus erythematosusrdquoScandinavian Journal of Rheumatology vol 42 no 4 pp 303ndash310 2013

[20] Q Zhang DQ Ye G P Chen andY Zheng ldquoOxidative proteindamage and antioxidant status in systemic lupus erythemato-susrdquo Clinical and Experimental Dermatology vol 35 no 3 pp287ndash294 2010

[21] P E Morgan A D Sturgess and M J Davies ldquoIncreased levelsof serum protein oxidation and correlation with disease activityin systemic lupus erythematosusrdquo Arthritis and Rheumatismvol 52 no 7 pp 2069ndash2079 2005

[22] D Shah R Kiran A Wanchu and A Bhatnagar ldquoOxidativestress in systemic lupus erythematosus relationship to Th1cytokine and disease activityrdquo Immunology Letters vol 129 no1 pp 7ndash12 2010

[23] S Z Hassan T A Gheita S A Kenawy A T Fahim I M El-Sorougy and M S Abdou ldquoOxidative stress in systemic lupuserythematosus and rheumatoid arthritis patients relationshipto disease manifestations and activityrdquo International Journal ofRheumatic Diseases vol 14 no 4 pp 325ndash331 2011

[24] I Avalos C P Chung A Oeser et al ldquoOxidative stress insystemic lupus erythematosus relationship to disease activityand symptomsrdquo Lupus vol 16 no 3 pp 195ndash200 2007

[25] G Wang S S Pierangeli E Papalardo G A S Ansari and MF Khan ldquoMarkers of oxidative and nitrosative stress in systemiclupus erythematosus correlation with disease activityrdquoArthritisand Rheumatism vol 62 no 7 pp 2064ndash2072 2010

[26] M Lozovoy A Simao C Panis et al ldquoOxidative stress is associ-ated with liver damage inflammatory status and corticosteroidtherapy in patients with systemic lupus erythematosusrdquo Lupusvol 20 no 12 pp 1250ndash1259 2011

[27] I K Mohan and U N Das ldquoOxidant stress anti-oxidantsand essential fatty acids in systemic lupus erythematosusrdquoProstaglandins Leukotrienes and Essential Fatty Acids vol 56no 3 pp 193ndash198 1997

[28] L S Tam E K Li V Y F Leung et al ldquoEffects of vitaminsC and E on oxidative stress markers and endothelial functionin patients with systemic lupus erythematosus a double blindplacebo controlled pilot studyrdquo The Journal of Rheumatologyvol 32 no 2 pp 275ndash282 2005

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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EndocrinologyInternational Journal of



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BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


and numerous nonenzymatic antioxidants [7] Oxidativestress is essentially an imbalance between productions ofreactive oxygen species (ROS) and antioxidant defensemech-anism [8] The increased production of free radicals disruptsredox status and can increase the expression of a wide varietyof inflammatory molecules that may be important causesof the inflammation and tissue damage [9] Several studiessuggested that oxidative stress could be a risk factor forautoimmune diseases such as SLE [8ndash10] Impaired antioxi-dant status has been reported in the saliva of SLE patients too[11]

The oxidative stress is evaluated by various methods inwhich oxidants and antioxidants aremeasured separately [12ndash14] Accordingly the PAB (prooxidant-antioxidant balance)method was developed to provide a rapid and reliabledetection of oxidant and antioxidant balance simultaneously[15] To the best of our knowledge there is no published reportabout the PAB assay in SLE patients therefore current studywas aimed at evaluating prooxidant-antioxidant balance inpatientswith SLE and its relationshipwith laboratory findingsand clinical manifestations

2 Materials and Methods

21 Participants This study was confirmed by the UniversityResearch Ethics Committee (UREC) of the Zahedan Univer-sity of Medical Science In this case-control study 60 patientswith SLE (54 women and 6 men) with a mean age of 3057 plusmn774 years as a test group and 60 healthy individuals (54women and 6 men) with a mean age of 3107 plusmn 75 years asa control group were enrolled All participants completed theinformed consent form Patients in this study were selectedin accordance with the American College of Rheumatology(ACR) criteria for SLE Moreover the participants in thecontrol group had no systemic diseases and their antinuclearantibody (ANA) test was negative

22 Sampling Two-milliliter fasting blood samples werecollected from all the subjects The blood samples werecentrifuged immediately and their sera were separated andthen stored at minus80∘C until use

23 Chemicals 331015840551015840-Tetramethylbenzidine (TMB) pow-der and chloramine T trihydrate were obtained from Appli-Chem (Darmstadt Germany) peroxidase enzyme and uricacid were obtained from Sigma-Aldrich (St Louis MO) andhydrogen peroxide and DMSO were obtained from MerckCompany (Darmstadt Germany)

24 Prooxidant-Antioxidant Balance (PAB) Assay The PABassay was performed in accordance with the methodexplained in detail by Alamdari et al [16] Briefly the PABmethod was used to determine the prooxidant-antioxidantbalance value

241 Preparation of Solutions 60mg TMB powder wasweighed and 10mL DMSO was added The TMB cation wasprepared as follows 20mL of acetate buffer [005M buffer

pH 45] 400 120583L of TMBDMSO and 70 120583L of fresh chlo-ramine T (100mM) solution were mixed and incubatedat room temperature for 2 hours in dark place 25U ofperoxidase enzyme solution was added into 20mL TMBcation dispensed into 1mL aliquots and frozen at minus20∘C theTMB solution was prepared with 200120583L of TMBDMSO in10mL of acetate buffer [005M buffer pH = 58] in orderto prepare working solution 1mL TMB cation was added in10mL of TMB solution incubated at room temperature for2min in a dark place and used freshly

242 Procedure 10 120583L of 200120583L of working solution in eachwell of a 96-well plate each sample standard or blank wasadded and the mixture was allowed to incubate at 37∘C for12min in a dark place at the end of the incubation time100 120583L of 2N HCl was added to each well the absorbance at450 nm was read against distilled water Hydrogen peroxidewas considered as a standard in different concentrations (0ndash100) After drawing the standard curve the PAB valuewas calculated for each unknown sample according to thestandard curve using OD obtained for each sample in the testand control groups

25 Data Analysis All the statistical analysis was carriedout by SPSS software version 16 Data have been presentedas mean plusmn SD as well as percentage Data in each groupshowed a normal distribution by Kolmogorov-Smirnov (K-S) statistical test Comparison between test and controlgroups was performed using the independent sample 119905-testIn addition Pearsonrsquos correlation coefficient test was used todetermine the correlation among different factors

3 Results

Table 1 shows demographic and laboratory data related to thepatients and control groups The data analysis showed thatthere was statistically a significant difference between meansof RBC hemoglobin WBC platelets hematocrit and ESR intwo independent groups

As shown in Figure 1 the mean of PAB values in SLEpatients (1473 plusmn 42HK) was higher than the healthy group(848 plusmn 322HK) and it was highly significant statistically(119875 lt 00001)

In addition Table 2 shows the correlation between thePAB value and laboratory findings in the patients and controlgroups In SLE patients there was a positive significant corre-lation between the PAB value and erythrocyte sedimentationrate (ESR) (119903 = 0492 119875 lt 0001)

Moreover as has been shown in Table 3 the PAB valueswere significantly different in SLE patients with and withoutsome clinicalmanifestations such as alopecia (164plusmn42 versus132 plusmn 36 119875 = 0002) discoid rash (176 plusmn 42 versus 140 plusmn39 119875 = 0005) oral ulcers (158 plusmn 42 versus 132 plusmn 38 119875 =0002) arthritis (158 plusmn 39 versus 112 plusmn 29 119875 = 00001) andnephritis (182plusmn29 versus 134plusmn40119875 = 00002)Therewere nocorrelations between PAB values and photosensitivity malarrash anemia serositis neurological involvement ANA andanti-dsDNA manifestations





119875 value





119875 value 1198772






ESR (mmh) 05 lt00001 02 009

4 Discussion


200

150

100

50

PAB

valu

e (H

K un

it)


30

P lt 00001








Disclosure




Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















119875 value





119875 value 1198772






ESR (mmh) 05 lt00001 02 009

4 Discussion


200

150

100

50

PAB

valu

e (H

K un

it)


30

P lt 00001








Disclosure




Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















Disclosure




Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Prooxidant-Antioxidant Balance in ...downloads.hindawi.com/journals/ad/2016/4343514.pdfLupus Erythematosus and Its Relationship with Clinical and Laboratory Findings