Research Article Association of Age-Related ... - Hindawi

Research ArticleAssociation of Age-Related Macular Degeneration withErythrocyte Antioxidant Enzymes Activity and Serum TotalAntioxidant Status

Ivna Plestina-Borjan1 Damir Katusic2 Maria Medvidovic-Grubisic3 Daniela Supe-Domic4

Kajo Bucan1 Leida Tandara4 and Veljko Rogosic1

1Department of Ophthalmology University of Split School of Medicine Spinciceva 1 21000 Split Croatia2Department of Ophthalmology University of Zagreb School of Medicine Salata 3 10000 Zagreb Croatia3Department of Ophthalmology Institute of Navy Medicine Soltanska 1 21000 Split Croatia4Department of Medical Laboratory Diagnostics University of Split School of Medicine Spinciceva 1 21000 Split Croatia

Correspondence should be addressed to Ivna Plestina-Borjan ivnaplestinagmailcom

Received 22 November 2014 Revised 30 January 2015 Accepted 1 February 2015

Academic Editor Ron Kohen

Copyright copy 2015 Ivna Plestina-Borjan 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

The aim was to estimate association of the oxidative stress with the occurrence of age-related macular degeneration (AMD) Theactivities of erythrocyte antioxidant enzymes superoxide dismutase (SOD) glutathione peroxidase (GPx) and catalase (CAT) andadditionally serum total antioxidant status (TAS) were used as indicators of the oxidative stress level 57 AMDpatients (32 early and25 late AMD) and 50 healthy age and gender matched controls were included GPx activity (119875 lt 0001) and serumTAS (119875 = 0015)were significantly lower in AMD patients The difference was not significant for SOD or CAT activities Significant interactionbetween GPx and SOD was detected (119875 = 0003) At high levels of SOD activity (over 75th percentile) one standard deviationdecrease in GPx increases the odds for AMD for six times (OR = 622 119875 lt 0001) ROC analysis revealed that combined valuesof GPx activity and TAS are significant determinants of AMD status Accuracy sensitivity specificity and positive and negativepredictive values were 75 95 52 69 and 90 respectively The study showed that low GPx activity and TAS are associatedwith AMD SOD modulates the association of GPx and AMD The results suggest that erythrocyte antioxidant enzymes activityand serum TAS could be promising markers for the prediction of AMD

1 Introduction

Age-related macular degeneration (AMD) is the leadingcause of legal blindness among people over 55 years in theWestern countries and the third cause of blindness globally[1 2] It is a progressive binocular disorder that affects nearly20of the population between 65 and 75 years of age and 35over the age of 75 [3 4] According to the latest data from theWorldHealthOrganization (WHO) 14million peopleworld-wide are blind or severely visually impaired due to AMD [1]These numbers are especially alarming given the increasingproportion of elderly people in the population

Despite the severity of the problem the etiology andpathogenesis of AMD are poorly understood and todayrsquos

treatment possibilities are not satisfactory Current therapypartially limits the damage only when it has already occurredbut only in 5 of all the cases [5] There are no any availabletreatments for dry form which accounts 90 of AMD cases

It is generally believed that AMD is caused by numerousbiochemical immunogenic and environmental factors [6ndash8] The most recent studies point to the key role of oxidativestress in the pathogenesis of AMD [4 6ndash10] Since oxidativestress involves almost all other assumptive pathogeneses andalmost all risk factors for AMD it could be crucial for theinitiation andprogression of the disease Excessive generationof free radicals and other reactive oxygen species (ROS) andimbalance between their generation and the possibility oftheir degradation by the antioxidant defense system seem to

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2015 Article ID 804054 8 pageshttpdxdoiorg1011552015804054

2 Oxidative Medicine and Cellular Longevity

be the most responsible factor in the development of AMD[10 11]

ROS are generated continuously as a part of normal aer-obic life as a byproduct of normal cellular metabolism (mito-chondrial transport chain) [11] and additionally in the retinaas the product of photochemical reaction between light andoxygen [12ndash14]

The retina particularly macula is the ideal environmentfor the generation of ROS due to the high oxygen consum-mation (because of its high metabolic activity) [15] lifelongexposure to light irradiation [16] high concentration ofpolyunsaturated fatty acids (PUFAs) [10] and abundance ofphotosensitizers [17 18] in photoreceptors and RPE cells

The consequences of oxidative damage on photoreceptorsand RPE cells are severe because they are nonreplicating(postmitotic) cells and must survive a lifetime of oxidativeinsults [9]

The disorder occurs when the antioxidant system canno longer compensate the cumulative oxidative damage Theretina possesses a substantial number of antioxidants in thephotoreceptor and RPE cells (especially in the area of themacula) [10] Antioxidant defense includes enzymes super-oxide dismutase (SOD) glutathione peroxidase (GPx) andcatalase (CAT) nonenzymatic antioxidants (as glutathioneuric acid albumin and bilirubin) and the antioxidantmicro-nutrients (vitamin C vitamin E and carotenoids) [11 19]Antioxidant enzymes which are of endogenous origin andconstitute the first line of antioxidant defense provide amore objective antioxidant state [10 11 19] than antioxidantmicronutrientswhich depends on the current intake and doesnot indicate the real condition of the long-term defenseagainst oxidative stress [19] Antioxidant enzymes (SODCAT and GPx) play the vital role in protecting the photore-ceptors and RPE cells from oxidative damage [10 20]

Hypothesis of oxidative stress induced AMD is supportedby numerous animal tissue cultures or the donors (post-mortem) retinas experiments [20ndash22] but not by clinical andepidemiological studies which are less frequent and oftencontradictory [8 23] Direct estimation of blood oxidantlevels is difficult because of very short free radicals half-lifeHowever oxidative stress can be estimated by measuring theantioxidant enzymes blood levels or activity The greatestchallenge is the development of the blood test that wouldidentify individuals most at risk of developing AMD beforeany signs of the disease become apparent

We hypothesized that low values of erythrocyte antiox-idant enzymes activity and serum total antioxidant status(TAS) are associated with AMD Our second hypothesis wasthat the interaction between antioxidant enzymes activities(SOD and GPX andor SOD and CAT) could be of the same(or even larger) importance for AMD occurrence as lowenzymes activity values are because the combined actionof the three enzymes forms one metabolic pathway for theprotection against the oxidative damage [11] Based on thesehypotheses we predicted that the evaluation of erythrocyteantioxidant enzymes activity and their interaction and serumTAS level measurement could be useful markers in theidentification and selection of AMDpredisposed individualsBecause the estimation of antioxidant enzymes in retinal cells

in vivo is not possible estimating their activity in peripheralblood would be a considerable advantage in the prediction ofAMD occurrence assuming that the obtained values corre-spond to the levels in the retinal cells

2 Methods

The study was reviewed and approved by the Ethics Com-mittee of the School of Medicine University of Split Croatiaand performed in accordance with the ethical standards inthe Declaration of Helsinki [24] A written informed consentwas obtained from each participant in the study

In total the study included 107 subjects aged 60 yearsor older 57 of them with AMD (25 with late and 32 withearly AMD) and 50 age and gender matched controls withoutapparent AMD-related fundus

The participants in the study were recruited from the reg-ular outpatients of the Eye Clinic (Retinal Department) at theUniversity Hospital in Split during the period from Novem-ber 2012 to December 2013 Subjects with diabetes verifiedcardiovascular disease patients cancer patients smokersalcoholics and patients with advanced cataract or otherdisturbances at the anterior segment of the eye that prevents adetailed fundus examination as well as patients with glau-coma were excluded Patients under or after anti-VEGF treat-ment were not included in the study Patients taking vitaminswith antioxidant effect were asked to stop taking them for2 months Later on they were included in the study if theymet the criteria All respondents taking medications that canlower the activity of antioxidant enzymes (eg large doses ofparacetamol and some cytostatics) were excluded from theresearch

A comprehensive ophthalmologic examination of allsubjects was doneThe best corrected visual acuity (althoughit was not included in the classification of disease) was deter-mined according to the Snellen optotypes A detailed generalophthalmologic examination (anterior segment slit lampexamination and intraocular pressure measurement) wasdone AMDwas diagnosed by slit lamp biomicroscopy of thefundus with the Maistner WF contact lens In all AMDpatients the color fundus photography was taken and fluo-rescein angiography (FA) was done on digital fundus cameraZeiss FF 450 plus IR In the control group only color fundusphotographs were taken and FA was not done as well as inthe AMD patients with allergy history All participants alsounderwent optical coherence tomography (OCT) on CirrusHD-OCTModel 400 Carl Zeiss Meditec

Presence and severity of AMD were determined fromfundus photographs and FA and graded according to theAMD International Classification and Grading System [25]The patients were classified according to the findings in themore affected eye in the case of bilateral affection Earlyform of AMD includes small and large soft drusen (ge63 120583m)as well as confluent drusen and areas of hyperpigmentationandhypopigmentationwith no visible choroidal blood vesselswithin 3000 120583m of foveola Late form of AMD includes wet(neovascular) and dry (geographic atrophy) form in the areawithin 3000 120583m from foveola Neovascular form is defined bythe presence of serous or hemorrhagic RPE or sensory retinal

Oxidative Medicine and Cellular Longevity 3

detachment subretinal neovascularization and fibrovascularscar Geographic atrophy implies sharply limited areas ofdepigmentation greater than 175120583m with visible choroidalvascularization

21 Blood Samples and Enzyme Assays A sample of venousblood (4mL) was taken from all the subjects between 8 and9 am after 12 to 14 hours of overnight fasting in order todetermine the antioxidant erythrocyte enzymes (SOD GPxand CAT) activity and TAS in serum The blood sample forSOD and CAT was drawn into vacuum tubes (Becton Dick-inson) with the addition of ethylenediaminetetraacetic acid(K3-EDTA) as anticoagulant For GPx vacuum tubes (BectonDickinson) were used with the addition of lithium heparinWithin 1 hour of blood samples collection enzymes activitywas measured in erythrocyte lysate spectroscopically at thetemperature of 37∘C For assessing the TAS blood was takenin siliconized vacuum tubes (Becton Dickinson) withoutanticoagulant

The commercial RANSOD assay (Randox LaboratoriesUK) was used to quantify the SOD activity in erythrocytelysate After isolating them from the whole blood the ery-throcytes were lysed following the manufacturerrsquos instruc-tions (procedure applied by Winterbourn et al [26] withminormodifications)Thewhole fresh blood samplewas cen-trifuged at 3000 rpm for 10 minutes Immediately after thisprocedure plasma with leukocytes and platelets was carefullyremoved Erythrocytes were washed 4 times with 09 salineNaCl and centrifuged 10 minutes at 3000 rpm after eachwash Subsequently they were mixed with cold distilled waterand left for 15 minutes at 4∘C to complete the process ofhemolysis Lysate was then diluted with 001molL phosphatebuffer pH 70 (005mL lysate + 1200mL phosphate buffer or50120583L lysate with 12mL of phosphate buffer) SOD activitywas determined spectrophotometrically at a wavelength of505 nm and the temperature of 37∘C in the diluted lysate andexpressed in UgHb

Hemoglobin was measured in the whole blood sample bystandard laboratory method

GPx activity was measured in the sample of whole bloodhemolysate by the commercial RANSEL assay (Randox Labo-ratories Crumlin UK) according to the Paglia andValentinemethod [27] and following the manufacturerrsquos instructionsHeparinized blood (005mL) was diluted with 1mL of thediluting agent (R 3 included in kit) and incubated for 5 min-utes After that 1mL Drabkinrsquos reagent was added and mixedthoroughly Within 20 minutes GPx activity was assessedat 340 nm and 37∘C and expressed in UgHb

SOD and GPx measurements were done on automaticanalyzer Architect C8000 (Abbott Diagnostic USA)

Catalase activity was also determined in erythrocytelysate using the OxiSelect catalase activity assay (Cell BiolabsSan Diego CA USA)The assay was performed following themanufacturerrsquos assay procedure The CAT degrades H

2O2to

water and molecular oxygen and the amount of degradedH2O2is proportional to the enzyme activityThe color change

of the reactionmixturewasmeasured spectrophotometricallyat 520 nm on the BioTek analyzer (MTX Lab Systems LLC

Virginia USA) CAT activity was calculated using a calibra-tion curve and expressed as UgHb

TAS was determined in serum using the commerciallyavailable TAS kit (Randox Laboratories Crumlin UK)following the manufacturerrsquos instruction by the methodreported byMiller et al [28] ABTS [221015840-azinobis(3-ethylben-zothiazoline-6-sulfonate)] incubated with peroxidase (met-myoglobin) and hydrogen peroxide (H

2O2) produces free

radical cation ABTS∙+ green in color (detected on 600 nm)Antioxidants present in the added serum sample suppressthe formation of the color The concentration of antioxidantsis inversely proportional to the development of the colorConcentration of TAS is expressed as mmolL of Troloxequivalents TAS was also measured on automatic analyzerArchitect C8000

22 Statistical Procedures The level of statistical significancewas set to 119875 lt 005 and all confidence intervals were givenat 95 level In all instances two-tailed tests of statisticalsignificance were used The univariate analysis of differencesin median values of TAS GPx SOD and CAT betweenAMD and control group was done by Mann-Whitney 119880 testwith Monte Carlo two-tailed statistical significance based on10000 sampled tables The distributions were described bymedians and interquartile ranges The absolute and relativedifferences between medians in AMD and in control groupwere reported The confidence intervals (CI) of the differ-ence of two medians were calculated based on the methodproposed by Bonett and Price [29] The multivariate logisticregression analysis was used to estimate enzymes activity asindependent predictors of the occurrence of AMD and theinteraction between GPx and SOD activities All markerswere standardized before the analysis and expressed as 119911 val-ues The standardization was done by subtracting arithmeticmeans from all data and dividing the differences by standarddeviation of each marker The moderating effect of SOD onassociation of GPx activity and AMD was analyzed by ldquoPro-cessrdquo release 212 Andrew F Hayes theOhio State University2014 SOD value defining the region of statistically significantassociation of GPx and AMD was assessed by Johnson-Neyman technique as implemented in the ldquoProcessrdquo Theprobabilities of AMD were calculated from odds as followsprobability = odds ratio(1 + odds ratio) The optimal cut-off points of TAS and GPx for the prediction of AMD weredetermined by Receiver Operating Characteristic (ROC)analysis andYouden index J Twomarkers were dichotomizedand combined into the composite predictor with two values(1) at least one of two indices bellow the cut-off value and (2)both TAS and GPx above the cut-off values The diagnosticaccuracy of the combined predictor was accessed by the areaunder the curve sensitivity specificity positive and negativepredictive values and likelihood ratios Data analysis wasdone by R Development Core Team (2008) (R a languageand environment for statistical computing) R Foundation forStatistical Computing Vienna Austria ISBN 3-900051-07-0 URL httpwwwR-projectorg ROC curve analysis wasdone by MedCalc Statistical Software version 133 (MedCalcSoftware bvba Ostend Belgium httpwwwmedcalcorg2014)


Table 1 Difference of median erythrocyte SOD GPx and CAT activities and serum TAS between AMD patients and healthy control groupassociation of decrease in markers activity with AMD

AMD Control119889abs (95 CI) 119889rel 119875

Multivariatelowast

(119899 = 57) (119899 = 50) OR (95 CI) 119875

TAS mmolL 147 (133ndash171) 164 (147ndash177) minus017 (minus028ndashminus005) 10 0015 234 (142ndash386) 0001GPx UgHb 39 (33ndash48) 54 (43ndash62) minus15 (minus21ndashminus9) 28 lt0001 410 (219ndash770) lt0001SOD UgHb 1389 (1185ndash1601) 1413 (1221ndash1575) minus25 (minus166ndash116) 2 0984 110 (068ndash179) 0701CAT UgHb 52 (48ndash58) 54 (47ndash60) minus2 (minus6ndash2) 4 0426 070 (041ndash120) 0191Values are presented as median (interquartile range)AMD = age-related macular degeneration TAS = serum total antioxidant status index GPx = glutathione peroxidase SOD = superoxide dismutase CAT =catalase119875 = Mann-Whitney 119880 test with Monte Carlo two-tailed statistical significance based on 10000 sampled tables 119889abs = absolute difference between medians119889rel = relative difference between medians 95 CI = 95 confidence interval of difference between medians OR = odds ratio for AMD at unit decrease ofstandardized markersrsquo values multivariate (adjusted) binary logistic regressionlowastValues of TAS GPx SOD and CAT were standardized before the multivariate logistic regression analysis arithmetic mean was subtracted and the differencewas divided by standard deviation

Table 2 Conditional effect of decrease in GPx activity on AMD at different levels of SOD

SODlowast (119911 values) Effect of decrease of GPx119861 (95 CI) 119875 OR Probability of AMD

10th percentile (minus131) minus002 (minus079ndash074) 0953 098 04925th percentile (minus073) 055 (000ndash109) 0049 173 06350th percentile (minus006) 120 (063ndash176) lt0001 331 07775th percentile (059) 183 (098ndash267) lt0001 622 08690th percentile (128) 250 (127ndash373) lt0001 1216 092GPx = glutathione peroxidase SOD = superoxide dismutase 119861 = regression coefficient 95 CI = 95 confidence intervals 119875 = level of statistical significanceOR = odds ratio for AMD at unit decrease in standardized GPx values probability = probability of AMD predicted by logistic regression of decrease of GPx 119911valueslowastValues of GPx SOD and CAT were standardized before the logistic regression analysis and expressed as 119911 values arithmetic mean was subtracted and thedifference was divided by standard deviation

3 Results

A total of 107 subjects were enrolled in this study 57 patientsin AMD group (25 with late and 32 with an early form ofAMD) and 50 in control group The groups were properlymatched by age and gender In the AMD group of patientstherewere 36 (614) female and 21 (386)male while in thecontrol group there were 31 (62) female and 19 (38) maleIn the AMD group of patients themedian of age was 77 years(interquartile range 71ndash82 years) while the median of age inthe control group was 775 years (interquartile range 73ndash81)

GPx activitywas significantly lower in subjectswithAMDcompared to controls by 15UgHb (119875 lt 0001) (Table 1)Likewise median of TAS value was 017mmolL lower inAMD group than in the control group indicating statisticallysignificant difference between these two groups (119875 = 0015)When adjusted for the effects of all four antioxidant indica-tors decrease of GPx and TAS has shown significant asso-ciation with AMD No statistically significant differences inactivity of erythrocyte SOD (119875 = 0984) andCAT (119875 = 0426)were observed between AMD patients and controls

Although SOD activity has not been significantly asso-ciated with AMD it had statistically significant moderatingeffect on the association of GPx and AMD (119875 = 0003) AtSOD activity value lower than 1st quartile that is lower thanminus073 standard deviations (corresponding to 1209UgHb)

differences in GPx activity were not significantly associatedwith AMD (Table 2) Above 75th percentile of SOD valueswhich is higher than 059 standard deviations (correspondingto 1581UgHb) one standard deviation decrease in GPxincreases the odds for AMD for six times (OR = 622 119875 lt0001) The higher the value of SOD activity is the higherthe association of decrease in GPx activity with AMD is(Figure 1)

Interactions between SOD andCAT (119875 = 0810) andGPxand CAT (119875 = 0679) were not statistically significant

The ROC analysis was performed in order to investigatethe potential of combined GPx activity and TAS levels asclinical tool for the prediction of AMD status (healthy versusdiseased) and to identify the threshold levels of GPx activityand TAS that discriminate well patients from the healthysubjects with the maximized sensitivity Pretest probabilityof disease or the expected prevalence was set to 25 as theaverage prevalence in the population ge65 years of age [2 4]Optimal cut-off values determined by Youden index 119869 were144mmolL for TAS and 4897UgHb for GPx

Twomarkers were combined into the final predictor withtwo values (1) at least one of two indices bellow the cut-offvalue and (2) both TAS and GPx above the cut-off valuesTotal accuracy measured by area under the curve (AUC) ofthe classification of patients into AMD or control group wasAUC = 75 (95 CI 66ndash79 119875 lt 0001) with sensitivity


minus15 minus10 minus05

GPx (z values)

00

00 05 10 15

25th percentile minus072575th percentile 059190th percentile 128

Median minus0057

SOD values)(z

02

04

06

08

10Pr

obab

ility

of A

MD

10th percentile minus131

Figure 1 Probability of age-related macular degeneration (AMD)predicted by glutathione peroxidase (GPx) activity values at differentlevels of superoxide dismutase (SOD) activity all values are stan-dardized and expressed as 119911 values arithmetic mean was subtractedand the difference was divided by standard deviations

of 95 and specificity of 52 (Table 3) Positive likelihoodratio for AMD was 197 (95 CI 152ndash226) Negative like-lihood ratio was 010 (95 CI 003ndash031) Number needed todiagnose was 21 (95 CI 182ndash337)

4 Discussion

Thegoal of the present study was to investigate the hypothesisthat erythrocyte antioxidant enzymes activity (SOD GPXand CAT) and the serum TAS are associated with thepathogenesis of AMD

No significant difference was observed in erythrocyteSOD and CAT activity between AMD patients and controlsin our study while the GPx activity and TAS level weresignificantly lower in the patients with AMD compared to thecontrols Moreover significant interaction between GPx andSOD was detected At very low SOD activity values decreasein GPx activity was not associated with AMD but at highSOD values decrease in GPx significantly increased the oddsfor AMD (more than six times)

Previously published research data based mainly onexamining the levels or the activity of the antioxidantenzymes in donor retinal tissues [30] tissue cultures [31] oranimal models [22 32] as well as the results of few clinicalstudies [8 33ndash35] seem to be in contradiction It has beenproposed that antioxidant enzymes are lower in AMD

Table 3 Prediction of AMD based on the combined erythrocyteGPx activity and serum TAS value

AMD(119899 = 57)

Control(119899 = 50) Predictive value

At least onebellow thecut-offlowast

54 (947) 24 (480) Positive069 (063ndash072)

Both TAS andGPx above thecut-off

3 (53) 26 (520) Negative090 (074ndash097)

Total 57 (1000) 50 (1000)Sensitivity

095(087ndash099)

Specificity052

(043ndash056)Values are presented as 119899 ()AMD = age-related macular degeneration TAS = serum total antioxidantstatus index GPx = glutathione peroxidaselowastCut-off values were set up based on the Youden index 119869 le144 for TASle4897 for GPx

patients contributing to the development of the disease Weassume that also higher SOD accompanied by reduced GPxand reduced or unchanged CAT activity (as shown in ourstudy) contributes significantly to the formation of the toxiclevel of hydrogen peroxide (H

2O2) and to an increased oxida-

tive damage Removal of excess of superoxide anions by SODis an important antioxidant defense mechanism althoughtoo much SOD (in relation to the activities of H

2O2remov-

ing enzymes GPx and CAT) may be deleterious H2O2is

itself ROS and can be converted into amore reactive anddam-aging hydroxyl radical (OH∙) (via Fenton reaction) whichreadily initiates lipid peroxidation chain reaction In ouropinion lipid peroxidation is the main oxidative mechanismfor damaging photoreceptor outer segment lipid membranes(rich with PUFAs) RPE cell and organelles membranes(especially mitochondrial) [11 36] leading to the develop-ment of AMD Therefore SOD acts in cooperation withGPx and CAT which convert H

2O2to nontoxic products

water and molecular oxygen protecting the photoreceptorsand RPE cells from oxidative damage GPx is more effectivethan CAT in detoxification of H

2O2because of its kinetic

properties [37] Other than on H2O2 GPx acts on lipid

hydroperoxides and on lipid peroxidation-derived toxic alde-hydes reducing them to corresponding alcohols [11]

The decrease inGPx activity can result from the increasedenzyme consumption caused by a higher level of oxidativestress or from the deficiency of essential metal cofactors(selenium) On the other hand it can be a consequence of thedecreased expression of genes that regulate enzymes produc-tion or the formation of inactive enzyme complexes (becauseof genetic variants of enzymeswith variable activities) [11 37]

Animal studies support our results indicating that lowvalues ofGPx contribute to the development ofAMD[22 38]Contrary to our results the PathologiesOculaires Liees a lrsquoAge(POLA) study [8] found that higher levels of plasmaGPxwereassociated with a ninefold increase in late AMD prevalenceIn POLA study GPx plasma levels were measured notactivities Measuring the levels of antioxidant enzymes is less


reliable than the determination of their activities Higher lev-els do not always mean higher activities due to genetic-basedcreation of inactive enzyme complex [11] There is also noinformation about the interaction between GPx and otherantioxidant enzymes in POLA study

To our knowledge our study is the first to examine theinteraction between the antioxidant enzymes activity ratio inAMD patients compared to healthy controls

The study indicates that increased SOD activity at lowGPx activity (as well as lack between SOD and CAT interac-tion) significantly increases the probability for AMD It couldbe crucial in explaining the impact of antioxidant enzymeactivity in the occurrence of AMD in our patients (stressingthe impact of lipid peroxidation)

Several experimental studies have provided a solid back-ground for our research [11 39] The antioxidant defenseoperates as a balanced and coordinated system (SOD andGPx and SOD and CAT) Previous clinical studies on theimpact of antioxidant enzymes on AMD differently con-ceived from our study provide contradictory data becausethey did not examine interaction of enzyme activities in asso-ciation with AMD as we did [8 33 34] Some of them mea-sured serumor erythrocytes level (not activity) of antioxidantenzymes [8 34] and others measured only particular antiox-idant enzymes (not all three) with [35 40] or without TASlevels [33 34 37]

In the present study the level of serum TAS (representingalso serum nonenzymatic antioxidants) was found to be sig-nificantly lower in patients with AMD compared to controlsThese results are consistent with other studies [35 40] that allassociate lower serum TAS level with a higher risk of AMDdevelopment

The method of measuring serum TAS levels by Randoxcommercial TAS kit as well as other methods (Ferric-reduc-ing antioxidant power assay oxygen radical absorbing capac-ity etc) [11] has certain limits One of themain disadvantagesof this method is that sample diluting might result in falsepositive TAS values but that was not required in our researchbecause in none of the samples TAS values were above25mmolL [41] In addition control serum was assayed ineach batch of samples for the estimation of analytical impre-cision

We included serumTAS in our study because of synergis-tic and compensatory effect between the intracellular antiox-idant enzymes and extracellular nonenzymatic antioxidantsin minimizing oxidative damage [11] Our results indicatelack of compensatory TAS effect in lowering tissue damage inAMDpatients contrary to the findings in the control group ofsubjects

We have also tried to predict AMD occurrence basedon the combined optimal cut-off values of erythrocyte GPxactivity and serum TAS level determined by ROC analysiswith themaximized test sensitivityTheROC analysis showedthat combined values of these two markers could providepromising predictive model for AMD (with 75 of AUC and95 of the test sensitivity)

The main limitation of this study was a relatively smallsample size In addition our predictive model has not beenevaluated on the independent sample (that is our next step)

but we think that this limitation did not affect our resultssignificantly

In conclusion we can say it is likely that low peripheralblood antioxidant enzymes activity well reflects retinal cellsantioxidant status and could contribute to the occurrence ofAMDAssessment of antioxidant erythrocyte enzyme activityand its interaction combined with serum TAS measurementcould be a promising marker in predicting the occurrence ofAMD

The antioxidantmechanism is not completely clearThereare stillmany open questions to be answered for example canthe overexpression of antioxidant enzymes which generallyprovides protection against oxidative stress in cell culturemodels be applied to the whole organism or to what extentdoes the overexpression of antioxidant enzymes provideprotection or how to reestablish antioxidant enzymes activitybalance Our results demonstrate the need for future geneticbiochemical and biomolecular research

Conflict of Interests

None of the authors has any conflict of interests to disclose

Authorsrsquo Contribution

The contribution of each author was as follows Ivna Plestina-Borjan and Damir Katusic provided conception and designand undertook the overall management of the study and ana-lyzed and interpreted all data Daniela Supe-Domic and LeidaTandara did all laboratory tests Maria Medvidovic-Grubisicand Kajo Bucan contributed to the statistical analysis anddrafting of the paper Veljko Rogosic contributed to the finalversion of the paper All authors read and approved the finalpaper

Acknowledgments

The authors thank Professor Ana Marusic from the Depart-ment of Research in Biomedicine and Health University ofSplit School ofMedicine past President of theWorld Associ-ation of Medical Editors and Council of Science Editors andthe currentVice President of EuropeanAssociation of ScienceEditors and Professor Predrag Sikiric from theDepartment ofPharmacology University of Zagreb School of Medicine forthe critical reading of the paper

References

[1] World Health Organization ldquoVision 2020 The right to sightGlobal initiative for the elimination of avoidable blindnessMagnitude and causes of visual impairmentrdquo Fact Sheet No282 2004 httpwhqlibdocwhointfact sheet2004FS 282pdf

[2] World Health Organization Vision 2020 The Right to SightGlobal Initiative for the Elimination of Avoidable BlindnessActions Plan 2006ndash2011WorldHealthOrganization 2013 httpwwwwhointblindnessVision2020 reportpdf

[3] M A Zarbin ldquoCurrent concepts in the pathogenesis of age-related macular degenerationrdquo Archives of Ophthalmology vol122 no 4 pp 598ndash614 2004


[4] H R Coleman C-C Chan F L Ferris III andE Y Chew ldquoAge-relatedmacular degenerationrdquoTheLancet vol 372 no 9652 pp1835ndash1845 2008

[5] A Lux H Llacer F M A Heussen and A M Joussen ldquoNon-responders to bevacizumab (Avastin) therapy of choroidal neo-vascular lesionsrdquo British Journal of Ophthalmology vol 91 no10 pp 1318ndash1322 2007

[6] X Ding M Patel and C-C Chan ldquoMolecular pathology ofage-related macular degenerationrdquo Progress in Retinal and EyeResearch vol 28 no 1 pp 1ndash18 2009

[7] M A Brantley Jr M P Osborn B J Sanders et al ldquoPlasmabiomarkers of oxidative stress and genetic variants in age-related macular degenerationrdquo American Journal of Ophthal-mology vol 153 no 3 pp 460ndash467 2012

[8] CDelcourt J-P Cristol C L Leger BDescomps and L PapozldquoAssociations of antioxidant enzymes with cataract and age-relatedmacular degeneration the POLA studyrdquoOphthalmologyvol 106 no 2 pp 215ndash222 1999

[9] S Khandhadia A Cree and A Lotery ldquoOxidative damage andmacular degenerationrdquo in Systems Biology of Free Radicals andAntioxidants I Lather Ed pp 3625ndash3653 Springer BerlinGermany 2014

[10] P Tokarz K Kaarniranta and J Blasiak ldquoRole of antiox-idant enzymes and small molecular weight antioxidants inthe pathogenesis of age-related macular degeneration (AMD)rdquoBiogerontology vol 14 no 5 pp 461ndash482 2013

[11] B Halliwell and J M C Gutteridge Free Radicals in BiologyandMedicine OxfordUniversity Press NewYork NYUSA 3rdedition 1999

[12] I Plestina-Borjan and M Klinger-Lasic ldquoLong-term exposureto solar ultraviolet radiation as a risk factor for age-relatedmacular degenerationrdquo Collegium Antropologicum vol 31 sup-plement 1 pp 33ndash38 2007

[13] R W Young ldquoSolar radiation and age-related macular degen-erationrdquo Survey of Ophthalmology vol 32 no 4 pp 252ndash2691988

[14] G-Y Sui G-C Liu G-Y Liu et al ldquoIs sunlight exposure arisk factor for age-related macular degeneration A systematicreview and meta-analysisrdquo British Journal of Ophthalmologyvol 97 no 4 pp 389ndash394 2013

[15] D-Y Yu and S J Cringle ldquoOxygen distribution and consump-tion within the retina in vascularised and avascular retinas andin animal models of retinal diseaserdquo Progress in Retinal and EyeResearch vol 20 no 2 pp 175ndash208 2001

[16] P N Youssef N Sheibani and D M Albert ldquoRetinal lighttoxicityrdquo Eye vol 25 no 1 pp 1ndash14 2011

[17] J J Hunter J I W Morgan W H Merigan D H Sliney J RSparrow and D R Williams ldquoThe susceptibility of the retinato photochemical damage from visible lightrdquo Progress in Retinaland Eye Research vol 31 no 1 pp 28ndash42 2012

[18] J R Sparrow E Gregory-Roberts K Yamamoto et al ldquoThebisretinoids of retinal pigment epitheliumrdquo Progress in Retinaland Eye Research vol 31 no 2 pp 121ndash135 2012

[19] J Curran-Celentano J Hammond BR T A Ciulla D ACooper L M Pratt and R B Danis ldquoRelation between dietaryintake serum concentrations and retinal concentrations oflutein and zeaxanthin in adults in aMidwest populationrdquoAmer-ican Journal of Clinical Nutrition vol 74 no 6 pp 796ndash8022001

[20] R N Frank R H Amin and J E Puklin ldquoAntioxidantenzymes in themacular retinal pigment epithelium of eyes with

neovascular age-related macular degenerationrdquo The AmericanJournal of Ophthalmology vol 127 no 6 pp 694ndash709 1999

[21] E Kaemmerer F Schutt T U Krohne F G Holz and J KopitzldquoEffects of lipid peroxidation-related protein modifications onRPE lysosomal functions and POS phagocytosisrdquo InvestigativeOphthalmology and Visual Science vol 48 no 3 pp 1342ndash13472007

[22] M G Nicolas K Fujiki K Murayama et al ldquoStudies on themechanism of early onset macular degeneration in cynomolgusmonkeys II Suppression of metallothionein synthesis in theretina in oxidative stressrdquoExperimental Eye Research vol 62 no4 pp 399ndash408 1996

[23] A E Fletcher ldquoFree radicals antioxidants and eye diseases evi-dence from epidemiological studies on cataract and age-relatedmacular degenerationrdquo Ophthalmic Research vol 44 no 3 pp191ndash198 2010

[24] World Medical Association Declaration of Helsinki ldquoEthicalprinciples for medical research involving human subjectsrdquoBulletin of theWorldHealth Organization vol 79 no 4 pp 373ndash374 2001

[25] A C Bird N M Bressler S B Bressler et al ldquoAn internationalclassification and grading system for age-related maculopathyand age-related macular degeneration The International ARMEpidemiological Study Grouprdquo Survey of Ophthalmology vol39 no 5 pp 367ndash374 1995

[26] C C Winterbourn R E Hawkins M Brian and R W CarrellldquoThe estimation of red cell superoxide dismutase activityrdquoJournal of Laboratory and Clinical Medicine vol 85 no 2 pp337ndash341 1975

[27] 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

[28] N J Miller C Rice-Evans M J Davies V Gopinathan andA Milner ldquoA novel method for measuring antioxidant capacityand its application to monitoring the antioxidant status inpremature neonatesrdquoClinical Science vol 84 no 4 pp 407ndash4121993

[29] D G Bonett and R M Price ldquoStatistical inference for a linearfunction of medians confidence intervals hypothesis testingand sample size requirementsrdquo PsychologicalMethods vol 7 no3 pp 370ndash383 2002

[30] M R Liles D A Newsome and P D Oliver ldquoAntioxidantenzymes in the aging human retinal pigment epitheliumrdquoArchives of Ophthalmology vol 109 no 9 pp 1285ndash1288 1991

[31] Z Liu L Sun L Zhu et al ldquoHydroxytyrosol protects retinalpigment epithelial cells from acrolein-induced oxidative stressandmitochondrial dysfunctionrdquo Journal of Neurochemistry vol103 no 6 pp 2690ndash2700 2007

[32] V Justilien J-J Pang K Renganathan et al ldquoSOD2 knockdownmouse model of early AMDrdquo Investigative Ophthalmology andVisual Science vol 48 no 10 pp 4407ndash4420 2007

[33] Z Yildirim N I Ucgun and F Yildirim ldquoThe role of oxidativestress and antioxidants in the pathogenesis of age-relatedmacular degenerationrdquo Clinics vol 66 no 5 pp 743ndash746 2011

[34] A Anand N K Sharma A Gupta S Prabhakar S K Sharmaand R Singh ldquoSuperoxide dismutase1 levels in north Indianpopulation with age-related macular degenerationrdquo OxidativeMedicine and Cellular Longevity vol 2013 Article ID 365046 7pages 2013

[35] X L Shen L H Jia P Zhao et al ldquoChanges in blood oxidativeand antioxidant parameters in a group of chinese patients with


age-related macular degenerationrdquo Journal of Nutrition Healthand Aging vol 16 no 3 pp 201ndash204 2012

[36] H Lin H Xu F-Q Liang et al ldquoMitochondrial DNA damageand repair in rpe associated with aging and age-related maculardegenerationrdquo Investigative Ophthalmology and Visual Sciencevol 52 no 6 pp 3521ndash3529 2011

[37] S M Cohen K L Olin W J Feuer L Hjelmeland C L Keenand L S Morse ldquoLow glutathione reductase and peroxidaseactivity in age-related macular degenerationrdquo British Journal ofOphthalmology vol 78 no 10 pp 791ndash794 1994

[38] L Lu B C Oveson Y-J Jo et al ldquoIncreased expression ofglutathione peroxidase 4 strongly protects retina from oxidativedamagerdquo Antioxidants and Redox Signaling vol 11 no 4 pp715ndash724 2009

[39] S Usui B C Oveson T Iwase et al ldquoOverexpression of SOD inretina need for increase in H

2

O2

-detoxifying enzyme in samecellular compartmentrdquo Free Radical Biology and Medicine vol51 no 7 pp 1347ndash1354 2011

[40] Y Totan R Yagci Y Bardak et al ldquoOxidative macromoleculardamage in age-related macular degenerationrdquo Current EyeResearch vol 34 no 12 pp 1089ndash1093 2009

[41] J Lamont J Campbell and P FitzGerald ldquoMeasurement ofindividual vs total antioxidantsrdquo Clinical Chemistry vol 43 no5 pp 852ndash854 1997

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


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

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


be the most responsible factor in the development of AMD[10 11]

ROS are generated continuously as a part of normal aer-obic life as a byproduct of normal cellular metabolism (mito-chondrial transport chain) [11] and additionally in the retinaas the product of photochemical reaction between light andoxygen [12ndash14]

The retina particularly macula is the ideal environmentfor the generation of ROS due to the high oxygen consum-mation (because of its high metabolic activity) [15] lifelongexposure to light irradiation [16] high concentration ofpolyunsaturated fatty acids (PUFAs) [10] and abundance ofphotosensitizers [17 18] in photoreceptors and RPE cells

The consequences of oxidative damage on photoreceptorsand RPE cells are severe because they are nonreplicating(postmitotic) cells and must survive a lifetime of oxidativeinsults [9]

The disorder occurs when the antioxidant system canno longer compensate the cumulative oxidative damage Theretina possesses a substantial number of antioxidants in thephotoreceptor and RPE cells (especially in the area of themacula) [10] Antioxidant defense includes enzymes super-oxide dismutase (SOD) glutathione peroxidase (GPx) andcatalase (CAT) nonenzymatic antioxidants (as glutathioneuric acid albumin and bilirubin) and the antioxidantmicro-nutrients (vitamin C vitamin E and carotenoids) [11 19]Antioxidant enzymes which are of endogenous origin andconstitute the first line of antioxidant defense provide amore objective antioxidant state [10 11 19] than antioxidantmicronutrientswhich depends on the current intake and doesnot indicate the real condition of the long-term defenseagainst oxidative stress [19] Antioxidant enzymes (SODCAT and GPx) play the vital role in protecting the photore-ceptors and RPE cells from oxidative damage [10 20]

Hypothesis of oxidative stress induced AMD is supportedby numerous animal tissue cultures or the donors (post-mortem) retinas experiments [20ndash22] but not by clinical andepidemiological studies which are less frequent and oftencontradictory [8 23] Direct estimation of blood oxidantlevels is difficult because of very short free radicals half-lifeHowever oxidative stress can be estimated by measuring theantioxidant enzymes blood levels or activity The greatestchallenge is the development of the blood test that wouldidentify individuals most at risk of developing AMD beforeany signs of the disease become apparent

We hypothesized that low values of erythrocyte antiox-idant enzymes activity and serum total antioxidant status(TAS) are associated with AMD Our second hypothesis wasthat the interaction between antioxidant enzymes activities(SOD and GPX andor SOD and CAT) could be of the same(or even larger) importance for AMD occurrence as lowenzymes activity values are because the combined actionof the three enzymes forms one metabolic pathway for theprotection against the oxidative damage [11] Based on thesehypotheses we predicted that the evaluation of erythrocyteantioxidant enzymes activity and their interaction and serumTAS level measurement could be useful markers in theidentification and selection of AMDpredisposed individualsBecause the estimation of antioxidant enzymes in retinal cells

in vivo is not possible estimating their activity in peripheralblood would be a considerable advantage in the prediction ofAMD occurrence assuming that the obtained values corre-spond to the levels in the retinal cells

2 Methods

The study was reviewed and approved by the Ethics Com-mittee of the School of Medicine University of Split Croatiaand performed in accordance with the ethical standards inthe Declaration of Helsinki [24] A written informed consentwas obtained from each participant in the study

In total the study included 107 subjects aged 60 yearsor older 57 of them with AMD (25 with late and 32 withearly AMD) and 50 age and gender matched controls withoutapparent AMD-related fundus

The participants in the study were recruited from the reg-ular outpatients of the Eye Clinic (Retinal Department) at theUniversity Hospital in Split during the period from Novem-ber 2012 to December 2013 Subjects with diabetes verifiedcardiovascular disease patients cancer patients smokersalcoholics and patients with advanced cataract or otherdisturbances at the anterior segment of the eye that prevents adetailed fundus examination as well as patients with glau-coma were excluded Patients under or after anti-VEGF treat-ment were not included in the study Patients taking vitaminswith antioxidant effect were asked to stop taking them for2 months Later on they were included in the study if theymet the criteria All respondents taking medications that canlower the activity of antioxidant enzymes (eg large doses ofparacetamol and some cytostatics) were excluded from theresearch

A comprehensive ophthalmologic examination of allsubjects was doneThe best corrected visual acuity (althoughit was not included in the classification of disease) was deter-mined according to the Snellen optotypes A detailed generalophthalmologic examination (anterior segment slit lampexamination and intraocular pressure measurement) wasdone AMDwas diagnosed by slit lamp biomicroscopy of thefundus with the Maistner WF contact lens In all AMDpatients the color fundus photography was taken and fluo-rescein angiography (FA) was done on digital fundus cameraZeiss FF 450 plus IR In the control group only color fundusphotographs were taken and FA was not done as well as inthe AMD patients with allergy history All participants alsounderwent optical coherence tomography (OCT) on CirrusHD-OCTModel 400 Carl Zeiss Meditec

Presence and severity of AMD were determined fromfundus photographs and FA and graded according to theAMD International Classification and Grading System [25]The patients were classified according to the findings in themore affected eye in the case of bilateral affection Earlyform of AMD includes small and large soft drusen (ge63 120583m)as well as confluent drusen and areas of hyperpigmentationandhypopigmentationwith no visible choroidal blood vesselswithin 3000 120583m of foveola Late form of AMD includes wet(neovascular) and dry (geographic atrophy) form in the areawithin 3000 120583m from foveola Neovascular form is defined bythe presence of serous or hemorrhagic RPE or sensory retinal









2O2to





2O2) produces free






Multivariatelowast

(119899 = 57) (119899 = 50) OR (95 CI) 119875





3 Results










GPx (z values)

00

00 05 10 15


Median minus0057

SOD values)(z

02

04

06

08

10Pr

obab

ility

of A

MD




4 Discussion





AMD(119899 = 57)



54 (947) 24 (480) Positive069 (063ndash072)


3 (53) 26 (520) Negative090 (074ndash097)


095(087ndash099)

Specificity052





2O2remov-



























Acknowledgments


References












































2

O2









of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























2O2to





2O2) produces free






Multivariatelowast

(119899 = 57) (119899 = 50) OR (95 CI) 119875





3 Results










GPx (z values)

00

00 05 10 15


Median minus0057

SOD values)(z

02

04

06

08

10Pr

obab

ility

of A

MD




4 Discussion





AMD(119899 = 57)



54 (947) 24 (480) Positive069 (063ndash072)


3 (53) 26 (520) Negative090 (074ndash097)


095(087ndash099)

Specificity052





2O2remov-



























Acknowledgments


References












































2

O2









of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Multivariatelowast

(119899 = 57) (119899 = 50) OR (95 CI) 119875





3 Results










GPx (z values)

00

00 05 10 15


Median minus0057

SOD values)(z

02

04

06

08

10Pr

obab

ility

of A

MD




4 Discussion





AMD(119899 = 57)



54 (947) 24 (480) Positive069 (063ndash072)


3 (53) 26 (520) Negative090 (074ndash097)


095(087ndash099)

Specificity052





2O2remov-



























Acknowledgments


References












































2

O2









of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















GPx (z values)

00

00 05 10 15


Median minus0057

SOD values)(z

02

04

06

08

10Pr

obab

ility

of A

MD




4 Discussion





AMD(119899 = 57)



54 (947) 24 (480) Positive069 (063ndash072)


3 (53) 26 (520) Negative090 (074ndash097)


095(087ndash099)

Specificity052





2O2remov-



























Acknowledgments


References












































2

O2









of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































Acknowledgments


References












































2

O2









of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























































2

O2









of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















2

O2









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 Association of Age-Related ... - Hindawi