Research Article Enhanced Oxidative Stress and Other

Research ArticleEnhanced Oxidative Stress and Other PotentialBiomarkers for Retinopathy in Type 2 DiabeticsBeneficial Effects of the Nutraceutic Supplements

Mariacutea J Roig-Revert1 Antonio Lleoacute-Peacuterez1 Vicente Zanoacuten-Moreno12

Baacuterbara Vivar-Llopis1 Juan Mariacuten-Montiel3 Rosa Dolz-Marco1245

Luis Alonso-Muntildeoz16 Mara Albert-Fort17 Mariacutea I Loacutepez-Gaacutelvez58

David Galarreta-Mira58 Mariacutea F Garciacutea-Esparza6 Carmen Galbis-Estrada125

Carla Marco-Ramirez12 Kian Shoaie-Nia12 Silvia M Sanz-Gonzaacutelez12

Vicente Vila-Bou12 Elena Bendala-Tufanisco129 Joseacute J Garciacutea-Medina1210

Carlo Nucci11 Roberto Gallego-Pinazo1245 J Fernando Areacutevalo12

Maria D Pinazo-Duraacuten125 and Valencia Study on Diabetic Retinopathy (VSDR)

1 Ophthalmic Research Unit ldquoSantiago Grisolıardquo Fundacion Investigacion Biomedica y Sanitaria (FISABIO)90 Gaspar Aguilar Avenida 46017 Valencia Spain

2 Ophthalmology Research Unit Department of Surgery Faculty of Medicine and Odontology University of Valencia15 Blasco Ibanez Avenida 46010 Valencia Spain

3 Ophthalmology Department University Hospital ldquoArnau de Vilanovardquo 12 San Clemente Street 46015 Valencia Spain4 Ophthalmology Department University and Polytechnic Hospital ldquoLa Ferdquo 106 Fernando Abril Martorell Avenida46026 Valencia Spain

5 Spanish Net of Ophthalmic Pathology OFTARED Institute of Health Carlos III 4 Sinesio Delgado Street 28029 Madrid Spain6 Ophthalmology Clinic and Research Department Clinica Oftalmologica Rahhal 52 Cirilo Amoros Street 46004 Valencia Spain7 Ophthalmology Department Hospital Vinaroz 1 Gil de Atrocillo Avenida Vinaroz 12500 Castellon Spain8 Ophthalmology Department University Clinic Hospital 1 Ramon y Cajal Street 47005 Valladolid Spain9 Physiology Department Faculty of Medicine University Cardenal Herrera 1 Seminario Avenida Moncada 46113 Valencia Spain10Ophthalmology Department University Hospital Reina Sofıa 1 Intendente Jorge Palacios Avenida 30003 Murcia Spain11Ophthalmic Unit Department of Experimental Medicine and Surgery University of Rome Tor Vergata Rome Italy12Retina Division The Wilmer Eye Institute The Johns Hopkins University School of Medicine Baltimore MD USA

Correspondence should be addressed to Maria D Pinazo-Duran dolorespinazouves

Received 14 June 2015 Revised 28 August 2015 Accepted 31 August 2015

Academic Editor Kota V Ramana

Copyright copy 2015 Marıa J Roig-Revert 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

We have studied the global risk of retinopathy in a Mediterranean population of type 2 diabetes mellitus (T2DM) patientsaccording to clinical biochemical and lifestyle biomarkersThe effects of the oral supplementation containing antioxidantsomega3 fatty acids (A1205963) were also evaluated Suitable participants were distributed into twomain groups (1) T2DMG (with retinopathy(+DR) or without retinopathy (minusDR)) and (2) controls (CG) Participants were randomly assigned (+A1205963) or not (minusA1205963)to the oral supplementation with a daily pill of Nutrof Omega (R) for 18 months Data collected including demographicsanthropometrics characteristicslifestyle ophthalmic examination (best corrected visual acuity ocular fundus photographsand retinal thickness as assessed by optical coherence tomography) and blood parameters (glucose glycosylated hemoglobintriglycerides malondialdehyde and total antioxidant capacity) were registered integrated and statistically processed by the SPSS150 program Finally 208 participants (130 diabetics (68 +DR62 minusDR) and 78 controls) completed the follow-up Blood analyses

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 408180 12 pageshttpdxdoiorg1011552015408180

2 BioMed Research International

confirmed that the T2DMG+DRpatients had significantly higher oxidative stress (119901 lt 005) inflammatory (119901 lt 005) and vascular(119901 lt 0001) risk markers than the T2DMGminusDR and the CG Furthermore the A1205963 oral supplementation positively changedthe baseline parameters presumptively by inducing metabolic activation and ameliorating the ocular health after 18 months ofsupplementation

1 Introduction

Diabetes mellitus (DM) with a prevalence of logistic growthrises pandemic proportions It has been estimated that over170 million people worldwide are currently affected by DMand it seems that these numbers will augment to over 360million by 2030 [1] A report on the incidence and prevalenceof type 2 diabetes mellitus (T2DM) in 11 European countriesshowed that the age-adjustedcountry-adjusted prevalence in2004 was 102 in men and 85 in women and that theseinequalities were highly related to the bodymass index (BMI)[2] In Spain DM represents a big health problem due toits prevalence of about 10 in the global population aged30ndash89 years [3] Number of diabetics among people aged 45years or older in USA during 2012 was 246 million Amongthem there were 21 million more diabetic men than womenThe total (directindirect) estimated costs of DM in the sametimeframe were $245 billion and after adjusting for agesexthese data concluded that medical expenditures (in general)were double among diabetics compared with the no diabetics[4] Other reports including large population collections alsoconcluded that men had a higher T2DM prevalence thanwomen [5 6] However a more recent estimation of the riskfactors for cardiovascular diseases described that diabeticwomen who smoke or are overweight develop cardiovascularconditions more frequently than men with the same riskfactors [7 8]

Major diabetic complications are the result of a closeinteraction between genes and a wide variety of environ-mental factors Diabetic retinopathy (DR) the microvascularcomplication of DM is responsible for the decreased visionand blindness in young and middle-aged individuals It hasbeen estimated that about 80of the type 2 diabetics (T2DM)and half of patients with type 1 DM (T1DM) develop DRin some point between the diagnosis and 15th year or moreof the disease course [9 10] In this framework it is wellacknowledged that DR appearmore frequently in T1DM thanin T2DM patients and that DR prevalence end points posi-tively correlate with the length of time afflicted byDM as wellas by the glycosilated hemoglobin (HbA1c) and hypertensionblood pressure (HBP) levels [11 12]

The retina is particularly sensitive to hyperglycemia It hasbeen shown that prolonged blood glucose elevation inducesstriking changes in oscillatory potentials in the electroretino-gram (ERG) increased implicit times in the multifocal ERG(mtERG) and dyschromatopsia whereas short-term hyper-glycemia results in an overall decrease in the implicit timesand increase in the amplitudes of the mtERG [13] Chronichyperglycemia induces retinal damage through differentmechanisms such as activation of protein kinase C polyoland hexosamine pathways andor generation of advancedglycation end products (AGEs) Nevertheless hyperglycemiadamages the endothelial cells and pericytes and increases

vascular permeability with rupture of the blood-retinal bar-rier with the subsequent appearance of the retinal hypo-xia processes which in turn induces vascular endothelialgrowth factor (VEGF) expression among other proangio-genic effectors [14 15] In this situation independently of thepresence or not of DR main objectives for implementing eyecare in diabetics have to include maximum nutritional andmetabolic control

Glycemic dysfunction has been widely associated withincreased generation of reactive oxygen (O

2) and nitrogen

species (ROS RNS) [16 17]These are continuously producedin cells during metabolic processes Eventually the electronsare transferred to molecular O

2 by reducing this to H

2O in

a reaction catalyzed by the cytochrome c oxidase accountingfor almost 98 of the molecular O

2used by our body

O2+ 4eminus + 4H+ 997888rarr 2H

2O (1)

It is known that 1-2 of the O2undergoes an incom-

plete reduction by the mitochondria generating the reactiveoxygen species (ROS) very unstable forms such as thesuperoxide anion (O

2

∙minus) formed when O2is reduced by

an electron However this molecule rapidly dismutates toform the hydrogen peroxide (H

2O2) that can itself undergo

dismutation by a reaction that is catalyzed by the manganesesuperoxide dismutase The rate of production of O

2H2O2

depends on the Ca2+ availability Importantly the presence oftransition metals mainly iron can induce H

2O2to generate

the highly reactive hydroxyl radical OH∙minusO2+ eminus = O

2

minus+H+ = HO∙ +O

2997888rarr H

2O2

(2)

In fact ROSRNS originated from mitochondrial ornonmitochondrial sources have been related to DM such asxanthine oxidase NADPH oxidase cyclooxygenase lipoxy-genase cytochrome P450 and nitric oxide synthase Tocounteract the effects of ROSRNS our body depends on theendogenous and exogenous antioxidants (AOX) includingthe enzymes superoxide dismutase catalase glutathioneperoxidase and glutathione reductase [18]

Nevertheless ROS overproduction or its deficientremoval leads to vascular disorders that result in protein frag-mentation amino acids aggregation membrane lipids alter-ation andor nucleic acids injury These described changesmay trigger cell damage and death From the describedoxidative background important biomarkers can emergeand obviously these molecules can be determined in DMpatients especially in those suffering chronic diabetes-relatedcomplications [19 20]

The retina is continuously exposed to lightTherefore reti-nal cells live under chronic oxidative stress conditions alsoinduced by the high O

2requirements and the high O

2partial

pressure from the underlying vessels of the choriocapillarisIt has also to be considered that the photoreceptor outer seg-ments are very rich in polyunsaturated fatty acids (PUFAs)

BioMed Research International 3

Table 1 Inclusion and exclusion criteria for the study participants Criteria for eligibility in the Valencia Study on Diabetic Retinopathy(VSDR)

Inclusion

Males and females aged gt25 years and lt80 years with type 2 diabetes for 12 months (at least)Insulin naıveHealthy individuals as controlsNo ocular or systemic diseases or aggressive treatments and no ocular surgery or laser for 12 months (at least)no other oral supplements with antioxidants andor omega 3 fatty acidsProvided written informed consent before any related activities commenceParticipants able to attend the visits and to follow the study guidelines

Exclusion

Males and females aged lt25 years and gt80 yearsInsulin dependent patientsOcular or systemic diseases or aggressive treatments andor previous ocular surgery or laserAntioxidant andor omega 3 fatty acids supplementsNo acceptance for the study participation andor not signing the informed consentParticipants unable to attend the visits or to follow the study guidelines

mainly the omega 3 docosahexaenoic acids (DHA) whichare extremely susceptible to lipid peroxidation and proteinmodifications that may be induced by the lipid peroxidationby-products [21] Current knowledge on the physiologicaland pathological mechanisms of oxidative stress in DR hasbeen collected from a wide spectrum of both the animalmodels research and the epidemiological studies [22 23]However clinical trials have provided ambiguous results onthe effects of the antioxidant [24 25] and PUFAs [26 27]supplements on the development and progression of DR

Chronically elevated glucoseHbA1c levels altogetherwith a series of endogenous and exogenous risk factors leadto the retinopathy The primary goal of the present studyis to investigate the risk of retinopathy in a Mediterraneanpopulation of T2DMpatients according to clinical biochem-ical and lifestyle biomarkers the oxidative stress status beingamong them Secondary goal was to evaluate the effects ofthe oral administration of supplements with antioxidants andomega 3 fatty acids (A1205963) in the oxidative stress outcomesand the inflammation and vascular risk biomarkers to bettermanage eye care and vision among diabetics

2 Material and Methods

21 Community-Based Study Design The study was done inagreementwith theDeclaration ofHelsinki for human studies(as revised in Edinburgh 2000) and all records were treatedaccording to the Spanish law for protecting personal data(LOPD Organic Law 151999 de 13 Dic) Our study wasvalidated by the Ethics Committee and Clinical Research ofthe University Hospital Dr Peset of Valencia (Spain) themain center of the Research Project (number 1233) andby the Ethics Committees of all participating institutionsMoreover the study received the permission of the AgenciaEspanola deMedicamentos y Productos Sanitarios (AEMPS)

We conducted a prospective multicenter study lasting for18 months (Jan 2013ndashJune 2014) that was carried out in 10centers by 32 investigators with the main goal of delineatingthe natural history of DM and eye disease as well as the riskfactors for DR in T2DM patients and to evaluate whetherA1205963 supplements could increase compliance with the healthand vision care among diabetics

The participant investigators were selected to obtainstratified ophthalmologists frommain hospitals pertaining tothe National Health System in the Valencia Community andophthalmology clinics as well as basic researchers involvedin ophthalmology and vision sciences

A total of 360 persons (25ndash80 years old) of bothsexes were preinterviewed for eligibility for the study thatwas determined by the main investigator according tothe inclusionexclusion criteria listed in Table 1 First pointfor recruitment was the diagnosis of T2DM and the durationof disease since diagnosis In parallel healthy individualswere also recruited Therefore from the initial sample 265suitable participants were enrolled signed the informedconsent and were appointed to the first visit of this inter-ventional study Then office visits were programmed everysix months including periodical telephone calls from theinvestigators throughout the study Total study duration was18 months and the characteristics of recruitment are enclosedin Figure 1

22 Patients and Methods

221 Interview Participants who completed the first enroll-ment interview (119899 = 265) were enclosed for demographicspatient characteristics and lifestyle The following vari-ables were recorded age sex ethnicity DM duration BMI(height weight) familial background current medicationsmokingdrinking habits and physical exercise In the mid-twentieth century Professor Ancel Keys observed the nutri-tional habits of people living in the European Mediter-ranean countries leading to the conclusion that a signifi-cant reduction in the incidence of chronic diseases and ahigher lifespan were main characteristics of this area (forfurther reading Keys et al The diet and 15-year death rateinthe seven countries study Am J Epidemiol 1986 124903)Major details of this type of diet are (1) the elevated consump-tion of fruits vegetables legumes olive oil bread and cereals(2) the moderate-to-high ingestion of fish and chicken meat(3) the moderate intake of cheese yogurt and wine and(4) the low levels of consumption of red meat The UNESCOraised the Mediterranean diet in 2013 as a lifestyle in itselfand a cultural heritage for the humanity We used a 14-item


T2 diabetics T2DMG (n = 210) Controls CG (n = 150)

Interview personal and familial characteristics and lifestyle

Ophthalmological examinationBest corrected visual acuityOcular fundus examination and photographsOptical coherence tomography

Classification degree of diabetic retinopathy

CG

18 months of follow-up (n = 208)

Eligibility decision accepting to participate informed consent (n = 265)

Persons attending the department of ophthalmology of the study centers (n = 360)

Subclassification according to antioxidantsomega 3 fatty acids

T2DR+DR

T2DRminusDR

T2DR+DR+A1205963T2DR+DRminusA1205963T2DRminusDR+A1205963T2DRminusDRminusA1205963CG+A1205963CGminusA1205963

Figure 1 Flowchart of the challenges for the recruitment and screening methods of the study participants characteristics of recruitmentsample size and study design

questionnaire to assess the adherence of the sample partic-ipants with the Mediterranean diet (AMD) The Mediter-ranean diet scores indicating compliance to the Mediter-ranean diet distinguished between the participants with ahigh intake of cereals legumes fruits vegetables olive oilfish bread and red wine that were scored positive (1) whilethose with a low intake of these meals were scored negative(0) The results of this questionnaire were reflected as par-ticipants having either good adherence to the Mediterraneandiet (GAMD) or poor adherence to the Mediterranean diet(PAMD)

The T2DMG (119899 = 165) was subclassified according tohaving or not having DR Furthermore each of these sub-groups was homogeneously subdivided into those partici-pants that were randomly assigned to take or not one pillper day of the A1205963 supplementation (T2DMG+DR+A1205963T2DMG+DRminusA1205963 and T2DMGndashDR+A1205963T2DMGndashDRminusA1205963) The CG (119899 = 100) was also randomly assignedto those taking (CG+A1205963) or not (CGminusA1205963) the A1205963supplements A flow chart with the number of patients per-taining to the groups and subgroups is enclosed in Fig-ure 1The nutraceutic formulation contains docosahexaenoicacid (DHA) vitamins (E C B1 B2 B3 B6 B9 and B12)lutein zeaxanthin glutathione hydroxytyrosol and traceelements (Se Mn Zn and Cu) commercialized as NutrofOmega commercialized by Laboratorios Thea SA (Barce-lona Spain) that gently provided all the oral supplementationneeded for the study participants The study-related visitsand the nutraceutic supplements (Nutrof Omega) were givento our participating volunteers (randomly chosen) withoutany charge

222 Ophthalmic Examination A systematized ophthalmo-logical examination was performed to our 265 participantsincluding best corrected visual acuity (BCVA) with eacheye ocular fundus examination and retinographs (ImageNetTopcon Barcelona Spain) and optic coherence tomogra-phy (OCT) examination (Spectral Domain SD-OCT ZeissMadrid Spain)

TheDR diagnosis was done from ocular fundus examina-tion and photographs which were carried out in each eye ofthe T2DG (119899 = 165) based on the Early Treatment of Dia-betic Retinopathy Study (ETDRS) and the DR severity wasgraded as described before [28] The presence and numberof microaneurysms hemorrhages venous beading andorintraretinal microvascular abnormalities were considered themost relevant factors for monitoring DR progression Infact retinopathy worsened basically by overall augment incounts of microaneurysms and haemorrhages The ETDRSretinopathy severity scale divides the process into 13 levelsranging from absence of retinopathy to severe vitreoushemorrhage

The OCT parameters were measured with the Cirrus SD-OCT direct cross-sectional imaging device of Zeiss Meditec(Dublin CA USA) in a total of 265 eyes with the clinicaldiagnosis of nonproliferative diabetic retinopathy (T2DG(119899 = 165) with or without DR andor clinically significantmacular edema) and in the healthy participants (CG 119899 =100) Prior to the OCT examination each participant wasadministered a drop of tropicamide in both eyes to dilate thepupils Each ophthalmologist visualized the representative A-scan and manually moved the measurement cursors (first atthe signal that represents the internal limitingmembrane and


second at the signal that denotes the retinal pigment epithe-lium) Then the most profound zone of the foveal pit wastaken as the center Six consecutive scans were performed foreach eye Cross-sectional retinal images were optimized foreach scan to obtain the highest intensitydefinition thereforethese scans with signal strengths ge 7 and without artifactswere included in this study Briefly acquisition protocolswere macular cube 200 times 200 HD 5 line raster5 rasterline and HD one line The raw OCT datasets were exportedto a personal computer for analysis The SD-OCT systemanalyzed retinal thickness creating a topographic map andgraphs for quantitatively and qualitatively documenting anychanges in retinal thickness and edema Main exclusioncriteria for this technique were any retinal pathology otherthan DR or opaquemedia such as in cataracts and inability toundergo the OCT examination due to mobility or cognitivedifficulties

223 Sample Processing At baseline and every 6 months ofthe follow-up all participants were visited in the correspond-ing centers and the peripheral blood was collected from theantecubital vein under fasting conditions (between 800 and900 am) Part of the obtained blood tubes was analyzed bycomputerized systems to determine laboratory blood drawsand analysis glucose glycosylated hemoglobin (HbA1c)cholesterol (HDL LDL) and triglycerides Two more EDTAtubes were obtained and transported in optimal conditionsfor preservation to the ophthalmic research centers Eachblood sample was centrifuged at 3000 rpm for 10min andthe plasma and erythrocytes were separated into Eppendorfslabeled and registered to be stored at minus80∘C until processingas described below

(1) Determination of Malondialdehyde- (MDA-) Thiobarbi-turic Acid Reactive Substances (TBARS) Thiobarbituric acidreactive substances (TBARS) includingMDAwere measuredin plasma samples as lipid peroxidation by-products Brieflyat high temperature (90ndash100∘C) under acidic conditionsMDA-TBA complex was formed by the reaction betweenthese molecules and extracted with butanol according toprevious descriptions [28 29] The fluorescence of thesecomplexes was measured in duplicate at 544 nm excitationand 590 nm emission deep relative to the fluorescence of thestandard samples and then the concentration of the sampleswas calculated by extrapolating the results in the standardcurve

(2) Total Antioxidant Activity (TAA) The antioxidant activitywas measured in the plasma samples by means of theantioxidant assay (Cayman Chemical Company Ann ArborMI) commercial kit The kit is based on the antioxidantcapacity of plasma to inhibit the oxidation of ABTS (221015840-azino-di-[3-ethylbenzthiazoline sulphonate]) to ABTS+ bythe metmyoglobin as previously reported [30 31]

23 Statistical Procedures For statistical analysis the excelprogram and the SPSS 150 (SPSS Inc Chicago IL USA)were used All the programmed experiments were performedin duplicate for each sample The continuous variables were

Table 2 Sociodemographic characteristics of diabetics withwith-out retinopathy

T2DM+DR T2DMminusDR 119901lowast

Age (years) 651 plusmn 86 623 plusmn 101 0094Gender ( of women) 527 486 0645DM duration (years) 192 plusmn 68 104 plusmn 71 lt0001lowast

Physical exercise () 273 457 0035lowast

Smoking () 436 157 0002lowast

Alcohol drinking () 382 86 lt0001lowast

BMI (kgm2) 288 plusmn 46 280 plusmn 40 0345GAMD () 182 443 0002lowast

T2DM+DR type 2 diabetes mellitus with diabetic retinopathy T2DMminusDRtype 2 diabetes mellitus without diabetic retinopathy DM diabetes mellitusBMI bodymass index GAMD good adherence toMediterranean diet Dataare shown as mean plusmn standard deviationlowastStatistically significant (119901 lt 005)

expressed as mean plusmn standard deviation of the mean (SD)Categorical variables were expressed as percentages The sta-tistical test performedutilized the twotailed test at 5 level ofsignificance Appropriate analysis of covariance models wasalso performed

3 Results

From the initial suitable participants (119899 = 265) a total of 208participants (130T2DMpatients (62+DRversus 68minusDR) and78 healthy controls) continued and completed altogether the18 months of the study

Demographics patient characteristics and lifestyledetails of the study participants are listed in Table 2 It hasto be emphasized that the HBP was twice frequent in theT2DMG+DR than in the diabetics minusDR (119901 lt 0001)

The BCVA (each eye in separate) was lower in theT2DMG than in the CG [(CG at baseline RE 095 plusmn 010 andLE 096 plusmn 008 119901 lt 0001CG at 18 months RE 095 plusmn 011and LE 095 plusmn 010 119901 lt 0001) (T2DMG at baseline RE081 plusmn 030 and LE 083 plusmn 022 119901 lt 0001T2DMG at 18months RE 078 plusmn 022 and LE 076 plusmn 022 119901 lt 0001)]

The baseline ocular fundus examination revealed that62 patients of the T2DMG displayed DR with the followingdegrees of severity 636 mild DR 291 moderate DRand 73 severe DR From baseline to the end of study 11patients remained without signs of DR progression but therest progressed as follows 291 displayed mild DR 364moderate DR and 145 severe DR Furthermore the 68T2DMG patients that did not have DR showed progressionof disease at the end of study in the following proportions143 of the patients without DR progressed to mild DR71 of the patients without DR progressed to moderate DRand no progression to severe degree of DR was detectedthrough the follow-up in any of these patients In our studythe T2DMGdisplayedDME (clinically significant) at baseline56 DME in the RE versus the 112 in the LE (119901 =0037 119901 = 0003 resp) Moreover DME was more frequentin the T2DMG+DR than in the T2DMGminusDR and the CG


+A1205963 +A1205963 minusA1205963minusA1205963

+onsetprogression of DRminusonsetprogression of DR

Onsetprogression of retinopathy in type-2diabetic patients after 18 months of follow-up

61

39

92

8 9

91

35

65

T2DM+DR T2DMminusDR

Figure 2 Percentages of development or progression of DR in theparticipants during the study course according to being assignedor not to the oral supplementation +A1205963 oral supplementationwith antioxidants and omega 3 fatty acids ndashA1205963 without taking theoral supplementation T2DM+DR type 2 diabetics with retinopathyT2DMminusDR type 2 diabetics without retinopathy

(119901 = 7036119864minus005 for the RE and 119901 = 2214119864minus006 for the LE)At the end of study it was seen that DME was significantlymore frequent in patients with severe DR (50) than in thosewith mild or moderate DME (119901 = 003) A summary of theprogression characteristics based on the ocular fundus andSD-OCT examination of the study participants is reflected inFigure 2

All the results obtained from the ophthalmic examinationof the study participants are enclosed in Table 3

Classical and oxidative stress hematologic parametersfrom the T2DM participants are enclosed in Table 4 Para-meters related to oxidative stress (lipid peroxidation(MDATBARS) total antioxidant activity (TAA)) showedthat the plasmatic MDATBARS levels were significantlyhigher and the TAC was significantly lower in diabetics ascompared to the CG (Table 4) It was also observed that theMDATBARS displayed significantly higher concentrationsand the TAA showed significantly lower activities in theT2DMG+DR with respect to those diabetics without DR andthe CG (Table 4) Data from the oxidative stress parametersaccording to the degree of severity of DR (ETDRS inter-national scale) are reflected in Table 5 The MDATBARSlevels augmented with DR progression but the TAA signifi-cantly decreased with DR progression

The adherence to theMediterranean diet nutritional factsof the study participants were reflected in the 14-item ques-tionnaire Specifically average values for the 14-item scorewere significantly higher in the CG (98 plusmn 20) versus theT2DMG (64 plusmn 1) (119901 lt 005) The comparison of the pro-oxidant and antioxidant markers analyzed in the presentstudy in the T2DMG with poor and good adherence to theMediterranean diet (PAMDGAMD) is reflected in Table 6

When assessing the oxidative stress status at the endof the eighteen months of the follow-up it was seen thatplasmatic MDATBARS levels significantly decreased in theT2DM+A1205963 subgroup with respect to the T2DMminusA1205963

Table 3 Ophthalmic examination records of the study participants

T2DM

BCVA REBaseline 081 plusmn 029618 months 078 plusmn 0220119901 value 0176

BCVA LEBaseline 083 plusmn 02218 months 078 plusmn 022119901 value 0388

IOP RE (mmHg)Baseline 152 plusmn 2818 months 156 plusmn 25119901 value 0099

IOP LE (mmHg)Baseline 156 plusmn 2918 months 161plusmn 25119901 value 0048lowast

RNFLT RE (120583m)Baseline 25165 plusmn 227918 months 25414 plusmn 3160119901 value 0001lowast

RNFLT LE (120583m)Baseline 25853 plusmn 5502918 months 26222 plusmn 638119901 value 0011lowast

MT RE (119899) Baseline 56 (13)18 months 72 (9)

MT LE (119899) Baseline 112 (14)18 months 104 (13)

DM minus R diabetes mellitus without retinopathy DM + R diabetes mellituswith retinopathy BCVA best corrected visual acuity RE right eye LE lefteye IOP intraocular pressure RNFL retinal nerve fiber layer thicknessMTmacular thickness Data are shown as mean plusmn standard deviationlowastStatistically significant (119901 lt 005)

patients (Figure 3(a)) Furthermore the TAC significantlyincreased in the T2DM+A1205963 subgroup while the nonsup-plemented participants did not show any noticeable change(Figure 3(b))

4 Discussion

The retinopathy is the major cause of blindness among adultdiabetics (aged 20ndash75 years) in the developed world [1ndash6] In this population-based multicenter study carried outin 265 participants from the Mediterranean area of Spain(T2DMG 119899 = 165 versus CG 119899 = 100) ldquoThe ValenciaStudy on Diabetic Retinopathy (VSDR)rdquo we have identifiedsignificant risk factors of DR such as theHBP duration of theDM physical exercise the BMI the smokingdrinking habitsthe dyslipidemia and the oxidative stress In fact plasmaticoxidative stress biomarkers increased and antioxidant activitydecreased in the diabetics with DR with respect to thosewithout DR and the healthy controls data consistent withprevious reports fromour group andother authors [18 19 22ndash25] More precise information about each of these risk factorsis provided below

The HBP was present in 764 of our T2DM+DR versus586 of the T2DMminusDR and 93 of the CG (119901 lt 0001)Emdin et al [32] studying patients with T2DM emphasized


Table 4 Haematologic parameters in diabetics withwithout retinopathy at baseline and at 18 months of follow-up

T2DMminusDR119901

T2DM+DR119901

Baseline End of study Baseline End of studyMDA (120583mL) 237 plusmn 139 243 plusmn 098 0724 363 plusmn 130 383 plusmn 181 0482TAS (120583M) 264 plusmn 152 287 plusmn 131 0203 196 plusmn 132 209 plusmn 139 0020lowast

HbA1c () 712 plusmn 155 699 plusmn 130 0307 787 plusmn 148 872 plusmn 145 0046lowast

Total-c (mgmL) 1868 plusmn 394 1843 plusmn 376 0669 1788 plusmn 326 1813 plusmn 338 0522HDL-c (mgmL) 504 plusmn 131 516 plusmn 142 0479 515 plusmn 130 514 plusmn 130 0959LDL-c (mgmL) 1043 plusmn 286 1023 plusmn 352 0687 999 plusmn 294 1026 plusmn 354 0587DM minus R diabetes mellitus without retinopathy DM + R diabetes mellitus with retinopathy MDA malondialdehyde TAS total antioxidant status HbA1cglycosilated hemoglobin total-c total-cholesterol HDL-c higher density lipoprotein-cholesterol LDL-c lower density lipoprotein-cholesterol Data are shownas mean plusmn standard deviationlowastStatistically significant (119901 lt 005)

Malondialdehyde (120583mL)

Baseline End of study

⋆

454

353

252

151

050

T2DR+A1205963T2DRminusA1205963

(a)


454

353

252

151

050

Total antioxidant activity (120583M)

⋆

+A1205963minusA1205963

(b)

Figure 3 Oxidative and antioxidant status in the type 2 diabetics during the 18months of follow-up according to being assigned or not to theoral supplementation of A1205963 supplements T2DR+A1205963 type 2 diabetics with retinopathy taking the oral supplementation T2DRminusA1205963type 2 diabetics with retinopathy not taking the oral supplementation +A1205963 assigned to the oral supplementation minusA1205963 not assigned tothe oral supplementation

Table 5 Oxidative status in diabetics according to the degree ofretinopathy (ETDRS International scale)

T2DMG MDATBARS (120583molL) TAA (120583molL)minusDR 119 plusmn 053 384 plusmn 124+DR

Mild DR 300 plusmn 174 238 plusmn 130Moderate DR 494 plusmn 129 129 plusmn 059Severe DR 536 plusmn 051 110 plusmn 034119901 valuelowast 2997119864 minus 006

lowastlowast4857119864 minus 005

lowastlowast

minusDR diabetes mellitus without retinopathy +DR diabetes mellitus withretinopathy MDA malondialdehyde TAA total antioxidant activity Dataare shown as mean plusmn standard deviationlowast119901 value obtained from ANOVA analysislowastlowastStatistically significant (119901 lt 005)

that appropriated HBP lowering was related to improvedmortality and other positive clinical changes in the affectedpatients Barrios and Escobar [33] reported that DM and

HBP are closely related disorders creating an optimumbackground for atherosclerosis In fact the strict controlof both processes is pivotal to ameliorate macroangiopa-thymicroangiopathy prognosis in the affected patients

The lead time between the diagnosis of the disease and thefirst appointment for the present study was considered as theduration of DM Average DM durability in our participantswas 142 plusmn 82 years Furthermore in the T2DMG+DR itwas 192 plusmn 68 years while in those without retinopathythe duration of DM was 104 plusmn 71 years In our work thisparameter was also related to the degree of severity of DRas shown in Table 5 We demonstrated that the duration ofDM was strongly associated with a higher risk of retinopathy(OR = 118 119901 lt 0001) Other authors also reported astrong relationship between DM duration and retinopathyneuropathy nephropathy and peripheral vasculopathy in asample of 1157 individuals of Northwest India [34]

Healthy participants performing physical exerciseaccounted for almost double than the diabetics (CG 68versus T2DMG 376 119901 lt 0001) Regarding the presence


Table 6 Comparison of the plasmatic prooxidant and antioxidant markers found in the diabetes mellitus patients according to havingpoorgood adherence to Mediterranean diet

T2DMminusDR T2DM+DR119901lowast

PAMD GAMD PAMD GAMDMDA1 (120583mL) 230 plusmn 114 247 plusmn 167 377 plusmn 126 301 plusmn 132 lt0001ab

MDA2 (120583mL) 232 plusmn 085 258 plusmn 111 412 plusmn 178 249 plusmn 131 lt0001abc

TAA1 (120583M) 257 plusmn 150 271 plusmn 158 186 plusmn 110 239 plusmn 206 0051TAA2 (120583M) 311 plusmn 150 258 plusmn 099 191 plusmn 138 288 plusmn 117 0001a

T2DMminusRD type 2 Diabetes mellitus patients without diabetic retinopathy T2DM+DR type 2 diabetes mellitus patients with diabetic retinopathy PAMDpoor adherence to Mediterranean diet GAMD good adherence to Mediterranean diet MDA1 malondialdehyde-baseline MDA2 malondialdehyde-end ofstudy TAA1 total antioxidant activity-baseline TAA2 total antioxidant activity-end of study Data are shown as mean plusmn standard deviationlowast119901 value obtained from ANOVA analysis

a significant differences between DM minus DR PAMD and DM + DR PAMD b significant differences between DM minus DR GAMD and DM + DR PAMD csignificant differences between DM + DR PAMD and DM + DR GAMD

of retinopathy significantly higher percentage of subjectsperforming physical exercise was observed in T2DMGminusDRthan in the T2DMG+DR (45 versus 27 119901 = 0035) Ourresults coincide with those reporting that physical exercise isbeneficial with respect to both the glycemic control and thediabetes-related comorbidities including the vitreoretinaldisorders [35]

The importance of data regarding the BMI from ourparticipants was notorious Significant differences betweenthe CG and the T2DMG (138 + 38 kgm2 versus 284 +43 kgm2 119901 lt 0001) were detected When the presence ofretinopathy was analyzed it was seen that the differences ofBMI between the T2DMG+DR and T2DMGminusDR were notsignificant (288 + 47 kgm2 versus 280 + 40 kgm2 119901 =0345) In agreement with these data it has been reportedthat BMI in correlation with HbA1c HBP and cholesterolseems to be related to DR progression in T2DM patients[36] Furthermore it has been suggested that BMI may beconsidered as a predictive marker for the development andprogression of DR [36 37] In addition to the carbohydraterestriction dietary recommendations on this topic includesaturated fat restriction (lt7 of daily caloric intake) andcholesterol restriction (lt200mgdL) among diabetics

Tobacco use is a major cause of disease disability andpremature death worldwide Our results showed a higherfrequency of smokers among the T2DMG than in the CG(344 versus 20 resp 119901 = 003) Diabetics also displayedhigher alcohol consumption than the healthy participants(248 versus 173) but the difference was not statisticallysignificant (119901 = 0217) Furthermore the T2DMG+DRshowed significant differences regarding tobacco and alcoholconsumption with respect to the T2DMGminusDR (119901 = 0001and 119901 lt 0001 resp) In fact it has been described thatdrinking in excess has to be avoided because it inducesketoacidosis and hypertriglyceridemia Moreover it has to beemphasized that alcohol taken outside the meals can causehypoglycemia crisis and a relevant increase of a host of healthproblems [38 39]

Oxidative stress affecting the cell and tissue morphologyand functions as well as leading to cell death is amajor playerin the pathogenesis of a wide variety of retinal diseases This

fact called our attention regarding DR because knowledgeon the precise molecular mechanisms of DR developmentand progression is far from complete Up to date there isno definitive treatment for the disease In the MediterraneanSpanish region of Valencia 500000 diabetics (from a totalof 5004844 inhabitants) have been recorded in the last yearBlood samples from our diabetics were analyzed for theoxidative and antioxidant activities as well as for determiningthe glycemia HbA1 HDLLDL cholesterol and triglyceridesDespite the classical parameters that were significantly higherin diabetics (glycemia HbA1C) as expected it was quitesurprising that the triglycerides levels were significantlylower in the CG than in the T2DMG at baseline (975 plusmn598mgdL versus 1496 plusmn 966mgdL 119901 lt 0001) and after18 months (1147 plusmn 693mgdL versus 1426 plusmn 809mgdL119901 = 0014) However this data was not statistically significantbetween the diabetics withwithout DR at baseline (13363 plusmn6516mgdL versus 15686 plusmn 11485mgdL 119901 = 0385)neither after 18 months (1340 plusmn 659mgdL versus 1413 plusmn910mgdL 119901 = 0812) Other authors concluded that nosignificant association between DR triglycerides HDL andtotal cholesterol was detected in a sample of Danish diabetics[40] Miljanovic et al did not find associations betweenlipid profile and DR progression [41] In contrast the globalcase-control study in 13 countries concluded that DR wasassociated with triglycerides and high-density lipoproteincholesterol in matched analysis [42]

It has been widely recognized that MDATBARS areassociated with the production of AGEs implicated in the DRdevelopment This mechanism is mainly due to AGEs accu-mulation in the ocular tissues by increasing cell permeabilityangiogenesis and disruption of the internal blood-retinalbarrier [43] PlasmaticMDATBARS levels were significantlyhigher and the TAA levels were significantly lower in theT2DMG than in the CG Furthermore the oxidative statuswas significantly higher and the antioxidant activity wassignificantly lower in diabetics +RD with respect to dia-betics minusRD data similar to previous reports demonstratingenhanced oxidative stress in T2DM patients [16ndash19 23 28]Interesting results came from the relationship between theDR degree of severity and the plasmatic oxidative stress


markers pointing to the fact that the prooxidants signifi-cantly increased and the antioxidants significantly decreasedin plasma samples of the T2DMG according to the ETDRSseverity scale as shown in Table 5 Data strongly suggeststhat the hyperglycemia created a particular oxidative stressbackground in the retina of the participant diabetics that inturns influences the onset and progression ofDR as reflectedthroughout the 18 months of the follow-up

We also observed that a nutraceutic formulation con-taining A1205963 is capable of counteracting the oxidative stressdetected in this diabetic Mediterranean population asreflected in Figure 2 and Tables 4ndash6 In addition to the anti-oxidant enzymes the robust antioxidant capacities of ahigh variety of nonenzymatic antioxidantsmdashamong themare vitamins (ascorbic acid (vit C) 120572-tocopherol (vit E))transition metals (selenium (Se) zinc (Zn) copper (Cu)and manganese (Mn)) glutathione carotenoids (120573-carotenelutein and lycopene) hormones (melatonin oestrogen)phenols (catechin quercetin) and so forthmdashand the addedbenefits of the antioxidant and anti-inflammatory propertiesof the PUFAs are well defined in the literature [21 2224ndash27] Therefore possible protecting mechanisms againstoxidative stress of the oral supplementation with a combi-nation of A1205963 in our diabetics may include a mixture ofthe strong antioxidant activity from vitamins C and E thecarotenoids lutein and zeaxanthin the free radical scavengingglutathione and the trace elements Se Zn Cu and Mnas well as the DHA and DHA-metabolic by-products allof which are participating in scavenging excessive oxidantspreventing free radical generation recycling other antiox-idants and probably ameliorating the mitochondrial func-tions (oxidative phosphorylation) In this framework it hasbeen reported that systemic interventions are highly requiredfor controlling DR patients Among these early detectionof hyperglycemia and intensive glycemic control togetherwith hypertension and dyslipidemia therapy have to beprogrammed As a consequence of the results raised duringthis study course strategies for avoidingDRdevelopment andprogression should therefore aim to prevent specifically theeffects of glycation but also the oxidative stress consequencesTherefore it is reasonable to consider that biotherapies withmultiple targets can be more useful than unique drugs in DRtreatment [43]

However it is notorious that studies with oral supple-ments on DR have yielded conflicting findings Among thestudies on the role of A1205963 in the prevention of DR theSan Luis Valley Diabetes Study did not find protective effectin DR and vitamin E on DR of the b-carotene vitaminC and vitamin E [44] The Third National Health andNutrition Examination study described that 120572-tocopheroland vitamin C serum levels were not associated with DR[45]Moreover theDiabetesControl andComplicationsTrialdescribed that patients assigned to a low-fat diet decreasedthe rate of DR progression as compared with patients notfollowing the low-fat diet [46] In addition antioxidant andantiangiogenic properties of edible berries were shown to beprotective for DR [47] Garcıa-Medina et al [48] designeda follow-up study in the Mediterranean area of Spain inT2DM patients with nonproliferative diabetic retinopathy

evaluating the effect of A1205963 supplementation over a 5-yearfollow-up The DR stage showed a retardation of progressionin the subgroup with supplementation but worsened inthe nonsupplemented subgroup It was also described thatthe oral supplements maintained the antioxidant defensesactivity as shown by the high plasmatic TAA levels whichwas related to the decreased oxidative plasma activity Inseveral experimental studies using different strains it wasshown that increasing the diversity of antioxidants providessignificantly more protection than using a single antioxidantfor the supplementation Moreover when antioxidants wereadministered to diabetic rats with the main goal of assessingthe ability of these agents to inhibit the development ofDR the authors concluded that long-term administrationof antioxidants could inhibit the development of the earlyphases of DR [49] Hence the oral supplementation withA1205963 represents an achievable adjunct to help preserve visionin diabetics

It is recognized that proliferative diabetic retinopathy(PDR) is the most usual sight-threatening vitreoretinal dam-age in T1DM patients while diabetic macular edema (DME)is considered the main cause of visual impairment andblindness in the T2DM patients [50 51] In our study theT2DMG displayed DME (clinically significant) at baseline56 DME in the RE versus the 112 in the LE (119901 = 0037119901 = 0003 resp) Moreover DME was more frequent inthe T2DMG+DR than in the T2DMGminusDR and the CG (119901 =7036119864minus005 for the RE and 119901 = 2214119864minus006 for the LE) Among

the recognized risk factors are the hyperglycemia HBP dys-lipidemia DMduration and oxidative stress status Howeverour study supports the benefits of the A1205963 supplementsas well as the important modifiable component for the riskfactors profile for better managing the diabetic eyes andvision

One additional interesting aspect of our work is thatcompliance with the Mediterranean diet was assessed by abrief 14-item validated questionnaire which is faster and lesscomplicated to the participants than other questionnairesbeing considered very useful by the investigators [52 53]The Mediterranean diet is a nutritional and lifestyle patternbased on the traditional dietary habits of Greece Italy andSpain [52] The term Mediterranean diet was set by AncelKeys in 1960 In our study course we have detected thatthe average values of the 14-item Mediterranean diet scoreswere significantly higher in the CG versus the T2DMG(119901 lt 005) Moreover comparison of the prooxidant andantioxidant markers in the T2DMG with GAMD or PAMDalso reported significant differences (Table 6) These resultspoint to those people with GAMD with the above describeddietary habits who had a lesser risk of DR which agree withprevious reports regarding the lesser risk of chronic andcardiovascular diseases among the Mediterranean countriesinhabitants [50ndash52] At this point we may suggest that thistype of questionnaire is useful for evaluating the role of theMediterranean diet in the ocular diseases

A limitation of this study was the relatively small samplesize of 265 participants at baseline (T2DMG 119899 = 165 versusCG 119899 = 100) and the important patients lost to follow-up(a total of 208 participantsmdashamong them are 130 patients


from the T2DMG (62 +DR versus 68 minusDR) and 78 healthyindividuals from the CGmdashcontinued and completed alto-gether the 18 months of the study) For this reason thesefindings are helpful enough to understand the characteristicsof the studied population but we are trying to control theseimportant variables for further research to better generalizeour results to the broader community

5 Conclusions

Healthy living includes everything we have to plan forimproving our wellbeing Specifically diabetics have to beable to handle lifersquos challenges including exercise being awareof smoking and drinking in excess andmaintaining the bodyweight All of these have to be considered key milestonesfor eye care in diabetics The DM induced an oxidativebackground in T2DM participants of the Mediterraneanstudy area that was enhanced in patients +DR The A1205963formulation utilized herein provided the appropriate vita-mins minerals and PUFAs resulting in decreased plasmaticoxidative level and increased antioxidant activity in diabeticsAntioxidant supplementation may be useful to help managediabetics at risk of retinopathy and vision loss

Disclosure

Maria D Pinazo-Duran is Coordinator of the VSDR RosaDolz-Marco Maria I Lopez-Galvez David Galarreta-MiraCarmen Galbis-Estrada Jose J Garcıa-Medina Roberto Gal-lego-Pinazo and Maria D Pinazo-Duran are OFTARED(Spanish Net of Ophthalmic Pathology) researchers

Conflict of Interests

All authors of this work have disclosed that they have nosignificant financial relationships or financial interests in thecommercial companies that are related to this study or paper

Authorsrsquo Contribution

Maria J Roig-Revert Antonio Lleo-Perez Vicente Zanon-Moreno and Barbara Vivar-Llopis collaborated equally in thepresent work sharing the first place

Acknowledgments

A detailed list of all the study investigators is presentedherein B Vivar-Llopis V Zanon-Moreno A LLeo-Perez MJ Roig-Revert J Marın-Montiel L Alonso Munoz R Dolz-Marco M Andreu-Fenoll K Shoaie Nia C Marco-RamirezC Galbis-Estrada O Alvarez-Barrachina M Perez-RamosG Rogla Navarro M Albert-Fort M F Garcıa-Esparza MSaleh Rahhal V Vila-Bou M Villalonga M L Redon ALlorca-Cardenosa R Gallego-Pinazo M Lopez-Galvez DGalarreta E Bendala-Tufanisco S Sanz-Gonzalez J J Gar-cıa-Medina F Santander-Trentini V Chaques C NucciJ F Arevalo and Maria D Pinazo-Duran Barbara Vivar-Llopis received a research fellowship from LaboratoriosTheaSA Barcelona (Spain) during the study course The authorswould like to thank Professor Dolores Corella (University ofValencia Spain) for helping with the 14-item questionnaireon the Mediterranean diet

References

[1] S Wild G Roglic A Green R Sicree and H King ldquoGlobalprevalence of diabetes estimates for the year 2000 and projec-tions for 2030rdquoDiabetes Care vol 27 no 5 pp 1047ndash1053 2004

[2] A Espelt C Borrell L Palencia et al ldquoSocioeconomic inequal-ities in the incidence and prevalence of type 2 diabetes mellitusin Europerdquo Gaceta Sanitaria vol 27 no 6 pp 494ndash501 2013

[3] J Lopez-Bastida M Boronat J Oliva-Moreno andW SchurerldquoCosts outcomes and challenges for diabetes care in SpainrdquoGlobalization and Health vol 9 article 17 9 pages 2013

[4] Centers for Disease Control and Prevention National DiabetesStatistics Report Estimates of Diabetes and Its Burden in theUnited States US Department of Health and Human ServicesAtlanta Ga USA 2014

[5] IM Stratton A I AdlerHAWNeil et al ldquoAssociation of gly-caemia withmacrovascular andmicrovascular complications oftype 2 diabetes (UKPDS 35) prospective observational studyrdquoBritish Medical Journal vol 321 no 7258 pp 405ndash412 2000

[6] WYang J Lu JWeng et al ldquoPrevalence of diabetes amongmenand women in Chinardquo The New England Journal of Medicinevol 362 no 12 pp 1090ndash1101 2010

[7] A Tschaftary and S Oertelt-Prigione ldquoSex and gender dif-ferences in cardiovascular preventionrdquo Deutsche MedizinischeWochenschrift vol 139 no 49 pp 2541ndash2545 2014

[8] G A Lutty ldquoEffects of diabetes on the eyerdquo Investigative Oph-thalmology amp Visual Science vol 54 no 14 pp ORSF81ndashORSF87 2013

[9] R Klein B E K Klein and S E Moss ldquoThe Wisconsin Epi-demiologic Study of Diabetic Retinopathy III Prevalence andrisk of diabetic retinopathy when age at diagnosis is 30 or moreyearsrdquo Archives of Ophthalmology vol 102 no 4 pp 527ndash5321984

[10] T W Gardner S F Abcouwer A J Barber and G R JacksonldquoAn integrated approach to diabetic retinopathy researchrdquoArchives of Ophthalmology vol 129 no 2 pp 230ndash235 2011

[11] J W Yau S L Rogers R Kawasaki et al ldquoGlobal prevalenceand major risk factors of diabetic retinopathyrdquo Diabetes Carevol 35 no 3 pp 556ndash564 2012

[12] G Bertelsen T Peto H Lindekleiv et al ldquoTromsoslash eye studyprevalence and risk factors of diabetic retinopathyrdquo Acta Oph-thalmologica vol 91 no 8 pp 716ndash721 2013

[13] K KlempM Larsen B Sander A Vaag P B Brockhoff andHLund-Andersen ldquoEffect of short-term hyperglycemia on multi-focal electroretinogram in diabetic patients without retinopa-thyrdquo Investigative Ophthalmology amp Visual Science vol 45 no10 pp 3812ndash3819 2004

[14] A S De Vriese T J Verbeuren J Van De Voorde N HLameire and P M Vanhoutte ldquoEndothelial dysfunction indiabetesrdquo British Journal of Pharmacology vol 130 no 5 pp963ndash974 2000

[15] N Ferrara and H-P Gerber ldquoThe role of vascular endothelialgrowth factor in angiogenesisrdquoActaHaematologica vol 106 no4 pp 148ndash156 2001

[16] G B Arden and S Sivaprasad ldquoHypoxia and oxidative stress inthe causation of diabetic retinopathyrdquoCurrentDiabetes Reviewsvol 7 no 5 pp 291ndash304 2011


[17] LMonnier EMas C Ginet et al ldquoActivation of oxidative stressby acute glucose fluctuations compared with sustained chronichyperglycemia in patients with type 2 diabetesrdquo The Journal ofthe AmericanMedical Association vol 295 no 14 pp 1681ndash16872006

[18] M Valko D Leibfritz J Moncol M T D Cronin M Mazurand J Telser ldquoFree radicals and antioxidants in normal physi-ological functions and human diseaserdquo International Journal ofBiochemistry and Cell Biology vol 39 no 1 pp 44ndash84 2007

[19] R Mancino D di Pierro C Varesi et al ldquoLipid peroxidationand total antioxidant capacity in vitreous aqueous humorand blood samples from patients with diabetic retinopathyrdquoMolecular Vision vol 17 pp 1298ndash1304 2011

[20] M D Pinazo-Duran R Gallego-Pinazo J J Garcıa-Medinaet al ldquoOxidative stress and its downstream signaling in agingeyesrdquo Clinical Interventions in Aging vol 9 pp 637ndash652 2014

[21] P C Calder ldquoPolyunsaturated fatty acids inflammation andimmunityrdquo Lipids vol 36 no 9 pp 1007ndash1024 2001

[22] V Ciddi and D Dodda ldquoTherapeutic potential of resveratrol indiabetic complications in vitro and in vivo studiesrdquo Pharmaco-logical Reports vol 66 no 5 pp 799ndash803 2014

[23] MHe H Pan C Xiao andM Pu ldquoRoles for redox signaling byNADPH oxidase in hyperglycemia-induced heme oxygenase-1expression in the diabetic retinardquo Investigative Ophthalmologyamp Visual Science vol 54 no 6 pp 4092ndash4101 2013

[24] R A Kowluru Q Zhong J M Santos M ThandampallayamD Putt and D L Gierhart ldquoBeneficial effects of the nutritionalsupplements on the development of diabetic retinopathyrdquoNutrition and Metabolism vol 11 no 1 article 8 2014

[25] P KMukherjee V LMarcheselli J C D R VaccariW C Gor-don F E Jackson and N G Bazan ldquoPhotoreceptor outer seg-ment phagocytosis attenuates oxidative stress-induced apopto-sis with concomitant neuroprotectin D1 synthesisrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 104 no 32 pp 13158ndash13163 2007

[26] M D Pinazo-Duran and L Bosca-Gomar ldquoAnti-inflammatoryproperties of polyunsaturated fatty acid omega 3 Indications inophthalmologyrdquoArchivos de la Sociedad Espanola de Oftalmolo-gia vol 87 no 7 pp 203ndash205 2012

[27] A Gębka E Serkies-Minuth and D Raczynska ldquoEffect ofthe administration of alpha-lipoic acid on contrast sensitivityin patients with type 1 and type 2 diabetesrdquo Mediators ofInflammation vol 2014 Article ID 131538 7 pages 2014

[28] M Uchiyama and M Mihara ldquoDetermination of malonalde-hyde precursor in tissues by thiobarbituric acid testrdquo AnalyticalBiochemistry vol 86 no 1 pp 271ndash278 1978

[29] W Wasowicz J Neve and A Peretz ldquoOptimized steps in flu-orometric determination of thiobarbituric acidreactive sub-stances in serum importance of extraction pH and influence ofsample preservation and storagerdquo Clinical Chemistry vol 39no 12 pp 2522ndash2526 1993

[30] Y Kambayashi N T Binh H W Asakura et al ldquoEfficient assayfor total antioxidant capacity in human plasma using a 96-wellmicroplterdquo Journal of Clinical Biochemistry and Nutrition vol44 no 1 pp 46ndash51 2009

[31] A C da Cruz F Petronilho C C V Heluany et al ldquoOxidativestress and aging correlation with clinical parametersrdquo AgingClinical and Experimental Research vol 26 no 1 pp 7ndash12 2014

[32] C A Emdin K Rahimi B Neal T Callender V Perkovic andA Patel ldquoBlood pressure lowering in type 2 diabetes a sys-tematic review and meta-analysisrdquoThe Journal of the AmericanMedical Association vol 313 no 6 pp 603ndash615 2015

[33] V Barrios and C Escobar ldquoDiabetes and hypertension Whatis newrdquoMinerva Cardioangiologica vol 57 no 6 pp 705ndash7222009

[34] R P Agrawal V Ola P Bishnoi S Gothwal P Sirohi and RAgrawal ldquoPrevalence of micro and macrovascular complica-tions and their risk factors in type-2 diabetes mellitusrdquo Journalof Association of Physicians of India vol 62 no 6 pp 504ndash5082014

[35] O Hamdy L J Goodyear and E S Horton ldquoDiet and exercisein type 2 diabetes mellitusrdquo Endocrinology and MetabolismClinics of North America vol 30 no 4 pp 883ndash907 2001

[36] A B Herrera-Rangel C Aranda-Moreno T Mantilla-Ochoa etal ldquoInfluence of the body mass index on the occurrence of fallsin patients with type 2 diabetes mellitusrdquo Obesity Research andClinical Practice 2015

[37] S Kastelan M Tomic A Gverovic Antunica S Ljubic JSalopek Rabatic and M Karabatic ldquoBody mass index a riskfactor for retinopathy in type 2 diabetic patientsrdquo Mediators ofInflammation vol 2013 Article ID 436329 8 pages 2013

[38] B Eliasson ldquoCigarette smoking and diabetesrdquo Progress in Car-diovascular Diseases vol 45 no 5 pp 405ndash413 2003

[39] CC Lee R P Stolk A I Adler et al ldquoAssociation between alco-hol consumption and diabetic retinopathy and visual acuitymdashthe AdRem studyrdquo Diabetic Medicine vol 27 no 10 pp 1130ndash1137 2010

[40] M N Hove J K Kristensen T Lauritzen and T Bek ldquoTheprevalence of retinopathy in an unselected population of type2 diabetes patients from Arhus County Denmarkrdquo Acta Oph-thalmologica Scandinavica vol 82 no 4 pp 443ndash448 2004

[41] B Miljanovic R J Glynn D M Nathan J E Manson and DA Schaumberg ldquoA prospective study of serum lipids and riskof diabetic macular edema in type 1 diabetesrdquo Diabetes vol 53no 11 pp 2883ndash2892 2004

[42] F M Sacks M P Hermans P Fioretto et al ldquoAssociationbetween plasma triglycerides and high-density lipoproteincholesterol and microvascular kidney disease and retinopathyin type 2 diabetes mellitus a global case-control study in 13countriesrdquo Circulation vol 129 no 9 pp 999ndash1008 2014

[43] P Gillery ldquoAdvanced glycation end products (AGEs) freeradicals and diabetesrdquo Journal de la Societe de biologie vol 195no 4 pp 387ndash390 2001

[44] E J Mayer-Davis R A Bell B A Reboussin J Rushing J AMarshall and R F Hamman ldquoAntioxidant nutrient intake anddiabetic retinopathy the San Luis Valley Diabetes Studyrdquo Oph-thalmology vol 105 no 12 pp 2264ndash2270 1998

[45] A E Millen M Gruber R Klein B E K Klein M Palta and JAMares ldquoRelations of serum ascorbic acid and120572-tocopherol todiabetic retinopathy in theThirdNational Health andNutritionExamination Surveyrdquo American Journal of Epidemiology vol158 no 3 pp 225ndash233 2003

[46] The Diabetes control and Complications Trial ldquoInfluence ofintensive diabetes treatment on body weight and compositionof adults with type 1 diabetes in the diabetes control andcomplications trialrdquoDiabetes Care vol 24 no 10 pp 1711ndash17212001


[47] S Zafra-Stone T YasminM Bagchi AChatterjee J AVinsonand D Bagchi ldquoBerry anthocyanins as novel antioxidants inhuman health and disease preventionrdquoMolecular Nutrition andFood Research vol 51 no 6 pp 675ndash683 2007

[48] J J Garcıa-Medina M D Pinazo-Duran M Garcia-MedinaV Zanon-Moreno and S Pons-Vazquez ldquoA 5-year follow-upof antioxidant supplementation in type 2 diabetic retinopathyrdquoEuropean Journal of Ophthalmology vol 21 no 5 pp 637ndash6432011

[49] X Li M Zhang and H Zhou ldquoThe morphological featuresand mitochondrial oxidative stress mechanism of the retinalneurons apoptosis in early diabetic ratsrdquo Journal of DiabetesResearch vol 2014 Article ID 678123 8 pages 2014

[50] J F Arevalo ldquoDiabetic macular edema changing treatmentparadigmsrdquoCurrent opinion in ophthalmology vol 25 no 6 pp502ndash507 2014

[51] R Dolz-Marco R Abreu-Gonzalez M Alonso-Plasencia andR Gallego-Pinazo ldquoTreatment decisions in diabetic macularedemabased onoptical coherence tomography retinal thicknessmap LET classificationrdquoGraefersquos Archive for Clinical and Exper-imental Ophthalmology vol 252 no 10 pp 1687ndash1688 2014

[52] M A Martınez-Gonzalez A Garcıa-Arellano E Toledo etal ldquoA 14-item mediterranean diet assessment tool and obesityindexes among high-risk subjects the PREDIMED trialrdquo PLoSONE vol 7 no 8 Article ID e43134 2012

[53] R Estruch M A Martınez-Gonzalez D Corella et al ldquoEffectsof a Mediterranean-style diet on cardiovascular risk factors arandomized trialrdquo Annals of Internal Medicine vol 145 no 1pp 1ndash11 2006

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


confirmed that the T2DMG+DRpatients had significantly higher oxidative stress (119901 lt 005) inflammatory (119901 lt 005) and vascular(119901 lt 0001) risk markers than the T2DMGminusDR and the CG Furthermore the A1205963 oral supplementation positively changedthe baseline parameters presumptively by inducing metabolic activation and ameliorating the ocular health after 18 months ofsupplementation

1 Introduction

Diabetes mellitus (DM) with a prevalence of logistic growthrises pandemic proportions It has been estimated that over170 million people worldwide are currently affected by DMand it seems that these numbers will augment to over 360million by 2030 [1] A report on the incidence and prevalenceof type 2 diabetes mellitus (T2DM) in 11 European countriesshowed that the age-adjustedcountry-adjusted prevalence in2004 was 102 in men and 85 in women and that theseinequalities were highly related to the bodymass index (BMI)[2] In Spain DM represents a big health problem due toits prevalence of about 10 in the global population aged30ndash89 years [3] Number of diabetics among people aged 45years or older in USA during 2012 was 246 million Amongthem there were 21 million more diabetic men than womenThe total (directindirect) estimated costs of DM in the sametimeframe were $245 billion and after adjusting for agesexthese data concluded that medical expenditures (in general)were double among diabetics compared with the no diabetics[4] Other reports including large population collections alsoconcluded that men had a higher T2DM prevalence thanwomen [5 6] However a more recent estimation of the riskfactors for cardiovascular diseases described that diabeticwomen who smoke or are overweight develop cardiovascularconditions more frequently than men with the same riskfactors [7 8]

Major diabetic complications are the result of a closeinteraction between genes and a wide variety of environ-mental factors Diabetic retinopathy (DR) the microvascularcomplication of DM is responsible for the decreased visionand blindness in young and middle-aged individuals It hasbeen estimated that about 80of the type 2 diabetics (T2DM)and half of patients with type 1 DM (T1DM) develop DRin some point between the diagnosis and 15th year or moreof the disease course [9 10] In this framework it is wellacknowledged that DR appearmore frequently in T1DM thanin T2DM patients and that DR prevalence end points posi-tively correlate with the length of time afflicted byDM as wellas by the glycosilated hemoglobin (HbA1c) and hypertensionblood pressure (HBP) levels [11 12]

The retina is particularly sensitive to hyperglycemia It hasbeen shown that prolonged blood glucose elevation inducesstriking changes in oscillatory potentials in the electroretino-gram (ERG) increased implicit times in the multifocal ERG(mtERG) and dyschromatopsia whereas short-term hyper-glycemia results in an overall decrease in the implicit timesand increase in the amplitudes of the mtERG [13] Chronichyperglycemia induces retinal damage through differentmechanisms such as activation of protein kinase C polyoland hexosamine pathways andor generation of advancedglycation end products (AGEs) Nevertheless hyperglycemiadamages the endothelial cells and pericytes and increases

vascular permeability with rupture of the blood-retinal bar-rier with the subsequent appearance of the retinal hypo-xia processes which in turn induces vascular endothelialgrowth factor (VEGF) expression among other proangio-genic effectors [14 15] In this situation independently of thepresence or not of DR main objectives for implementing eyecare in diabetics have to include maximum nutritional andmetabolic control

Glycemic dysfunction has been widely associated withincreased generation of reactive oxygen (O

2) and nitrogen

species (ROS RNS) [16 17]These are continuously producedin cells during metabolic processes Eventually the electronsare transferred to molecular O

2 by reducing this to H

2O in

a reaction catalyzed by the cytochrome c oxidase accountingfor almost 98 of the molecular O

2used by our body

O2+ 4eminus + 4H+ 997888rarr 2H

2O (1)

It is known that 1-2 of the O2undergoes an incom-

plete reduction by the mitochondria generating the reactiveoxygen species (ROS) very unstable forms such as thesuperoxide anion (O

2

∙minus) formed when O2is reduced by

an electron However this molecule rapidly dismutates toform the hydrogen peroxide (H

2O2) that can itself undergo

dismutation by a reaction that is catalyzed by the manganesesuperoxide dismutase The rate of production of O

2H2O2

depends on the Ca2+ availability Importantly the presence oftransition metals mainly iron can induce H

2O2to generate

the highly reactive hydroxyl radical OH∙minusO2+ eminus = O

2

minus+H+ = HO∙ +O

2997888rarr H

2O2

(2)

In fact ROSRNS originated from mitochondrial ornonmitochondrial sources have been related to DM such asxanthine oxidase NADPH oxidase cyclooxygenase lipoxy-genase cytochrome P450 and nitric oxide synthase Tocounteract the effects of ROSRNS our body depends on theendogenous and exogenous antioxidants (AOX) includingthe enzymes superoxide dismutase catalase glutathioneperoxidase and glutathione reductase [18]

Nevertheless ROS overproduction or its deficientremoval leads to vascular disorders that result in protein frag-mentation amino acids aggregation membrane lipids alter-ation andor nucleic acids injury These described changesmay trigger cell damage and death From the describedoxidative background important biomarkers can emergeand obviously these molecules can be determined in DMpatients especially in those suffering chronic diabetes-relatedcomplications [19 20]

The retina is continuously exposed to lightTherefore reti-nal cells live under chronic oxidative stress conditions alsoinduced by the high O

2requirements and the high O

2partial

pressure from the underlying vessels of the choriocapillarisIt has also to be considered that the photoreceptor outer seg-ments are very rich in polyunsaturated fatty acids (PUFAs)



Inclusion


Exclusion
















CG





T2DR+DR

T2DRminusDR























3 Results






+A1205963 +A1205963 minusA1205963minusA1205963



61

39

92

8 9

91

35

65

T2DM+DR T2DMminusDR








T2DM











4 Discussion





T2DMminusDR119901

T2DM+DR119901






⋆

454

353

252

151

050


(a)


454

353

252

151

050


⋆

+A1205963minusA1205963

(b)






lowastlowast































5 Conclusions


Disclosure






Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Inclusion


Exclusion
















CG





T2DR+DR

T2DRminusDR























3 Results






+A1205963 +A1205963 minusA1205963minusA1205963



61

39

92

8 9

91

35

65

T2DM+DR T2DMminusDR








T2DM











4 Discussion





T2DMminusDR119901

T2DM+DR119901






⋆

454

353

252

151

050


(a)


454

353

252

151

050


⋆

+A1205963minusA1205963

(b)






lowastlowast































5 Conclusions


Disclosure






Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















CG





T2DR+DR

T2DRminusDR























3 Results






+A1205963 +A1205963 minusA1205963minusA1205963



61

39

92

8 9

91

35

65

T2DM+DR T2DMminusDR








T2DM











4 Discussion





T2DMminusDR119901

T2DM+DR119901






⋆

454

353

252

151

050


(a)


454

353

252

151

050


⋆

+A1205963minusA1205963

(b)






lowastlowast































5 Conclusions


Disclosure






Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































3 Results






+A1205963 +A1205963 minusA1205963minusA1205963



61

39

92

8 9

91

35

65

T2DM+DR T2DMminusDR








T2DM











4 Discussion





T2DMminusDR119901

T2DM+DR119901






⋆

454

353

252

151

050


(a)


454

353

252

151

050


⋆

+A1205963minusA1205963

(b)






lowastlowast































5 Conclusions


Disclosure






Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















+A1205963 +A1205963 minusA1205963minusA1205963



61

39

92

8 9

91

35

65

T2DM+DR T2DMminusDR








T2DM











4 Discussion





T2DMminusDR119901

T2DM+DR119901






⋆

454

353

252

151

050


(a)


454

353

252

151

050


⋆

+A1205963minusA1205963

(b)






lowastlowast































5 Conclusions


Disclosure






Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















T2DMminusDR119901

T2DM+DR119901






⋆

454

353

252

151

050


(a)


454

353

252

151

050


⋆

+A1205963minusA1205963

(b)






lowastlowast































5 Conclusions


Disclosure






Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of









































5 Conclusions


Disclosure






Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























5 Conclusions


Disclosure






Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















5 Conclusions


Disclosure






Acknowledgments


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





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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