The purpose of the survey was to gauge the degrees of superoxide dismutase-1 in AMD patients and analyze the function of Oxidative emphasis, smoke, high blood pressure and other familial factors involved in the pathogenesis of AMD.
Methods: 115 AMD patients and 61 healthy controls were recruited for this survey after informed consent. Serum SOD1 degrees were determined by ELISA and after standardization to entire serum protein these were correlated to assorted hazard factors. Logistic arrested development theoretical account genuineness by sing SOD1 as independent variable have been developed along with ROC curve.
Consequences: The SOD1 degrees were significantly higher in AMD patients as compared to controls. The difference was non important for moisture and dry AMD. However, the difference was important between wet AMD subtypes. Non-significance of Hosmer-Lemeshow goodness of fit statistic ( Chi-square=10.516, df=8, p=0.231 ) indicates the rightness of logistic arrested development theoretical account to foretell AMD.
Decision: Oxidative emphasis in AMD patients may mount compensatory response ensuing in increased degrees of SOD1 in AMD patients. To foretell the hazard of AMD on the footing of SOD1, a logistic arrested development theoretical account show genuineness of 78 % and country under the ROC curve ( 0.827, p=.0001 ) with less standard mistake of 0.033 and near 95 % assurance intervals ( 0.762-0.891 ) further validates the theoretical account.
Age-related macular devolution ( AMD ) is the taking cause of sightlessness in worlds that is characterized by progressive devolution of sunspot taking to severe irreversible loss in vision ( 1-5 ) .The vision loss consequences from either from retinal devolution, ( called prohibitionist or nonexudative AMD ) , or from the choroidal neovascularization ( called CNV ; moisture or exudative AMD ) . The clinical manifestation of AMD includes drusen, hyperplasia of the retinal pigment epithelial tissue ( RPE ) , geographic wasting, and angiogenesis of choroidal vass ( CNVs ) ( 6 ) .
Smoke, intoxicant, oxidative emphasis and familial factors are implicated in the pathogenesis of AMD ( 7 ) but the exact cause of AMD still eludes us. It has been reported that aging is associated with pathological and biochemical alterations in the oculus. In general, aging and AMD is believed to ensue from cumulative and increased oxidative harm. ( 8 ) Oxidative emphasis can exercise cellular or molecular harm mediated by reactive O species ( ROS ) which has been before shown to be implicated with diseases of ageing ( 9,10 ) . The elevated degree of endogenously synthesized ROS are known to be regulated by assorted anti-oxidant, enzymatic and non-enzymatic protective biochemical mechanism like Glutathion peroxiase ( GPx ) , superoxide dismutase ( SOD ) , and catalase ( CAT ) ( 11 ) . ROS which includes free groups, nascent O, H peroxide and the byproducts of O metamorphosis are hurtful for oculus pathophysiology. Due to really high ingestion of O, the high concentration of polyunsaturated fatty acid and direct exposure of light renders retina susceptible to oxidative emphasis ( 12 ) . Assorted factors are responsible for oxidative emphasis generated from aging ; these include reduced degrees of vitamin C and vitamin Tocopherol in plasma ( 13, 14 ) , glutathione degrees lessening, and oxidized glutathione degrees increased in plasma with the age ( 15 ) . Increased Lipid peroxidation is besides reported in aging ( 16, 17 ) and the effects of these unbalanced biochemical alterations lead to increased susceptibleness of retinal pigment epithelial tissue cells ( RPE ) to oxidative harm with the ripening. For illustration, vitamin E degrees and catalase activity have been reported to diminish with aging in RPE cells. ( 18,19 ) . There are several pathological characteristics which have been reported to attach to ripening, for illustration, the increased volume of lipofuscin contents ( Increased Lipid and protein contents ) which enhance the oxidative harm susceptibleness, decreased optical denseness of macular pigment ( 20 ) ensuing in RPE cells confronted by membrane blebbing, a phenomenon observed in aging and AMD eyes ( 21 ) .
We hypothesized that oxidation-reduction change in the oculus might ensue from deranged SOD1 degrees. We therefore analysed the look of ace oxide dismutase 1 ( SOD1 ) in patients of AMD as compared to controls.
The major antioxidant system in the retina consists of three superoxide dismutase ( SOD ) isoenzymes that catalyse dismutation of superoxide into O and H peroxide ( H2O2 ) ( 22 ) . Superoxide dismutase is an antioxidant enzyme involved in the defence system against reactive O species ( ROS ) . SOD catalyzes the dismutation reaction of superoxide extremist anion ( O2- ) to hydrogen peroxide, which is so catalyzed to innocuous O2 and H2O by glutathione peroxidase and catalase. There are three major households of SOD, depending on metal co-factors: Cu-Zn SOD ( SOD1 ) , present in cytosol, Mn ( Fe ) – Turf ( SOD2 ) nowadays in mitochondrial matrix, and the extracellular SOD ( SOD3 ) interstitium of the tissues as a secretory signifier. ( 23 )
The activity and sum of the Cu-Zn SOD ( SOD1 ) is highest among the three isoenzymes in the human retina ( 23 ) so it seems sensible to test SOD1 for possible function in speed uping age related alterations in the retina.
Presently there is no survey analyzing the function of SOD 1 in Indian AMD patients and this probes will probably supply the substrate for future therapies in AMD
This survey was been approved by Institute Ethics Committee of Post-Graduate Institute of Medical Education and Research, Chandigarh, India vide missive No. Micro/10/1411. Patients and controls were foremost informed about the survey and henceforth enrolled in patient/control group after obtaining written performa from all participants. All enrolled participants were recruited from Department of Ophthalmology, PGIMER, Chandigarh ( India ) in which phenotypic standards was purely followed. Briefly, all AMD patients underwent ophthalmic scrutiny by a retina specializer for best corrected ocular sharp-sightedness, slit lamp biomicroscopy of anterior section and dilated fundus scrutiny. All AMD patients were subjected to fluorescein fundus angiography ( FFA ) and optical coherency imaging ( OCT ) . The diagnosing of AMD was based on ophthalmoscopic and FFA findings.
We included 176 case-control samples dwelling of 115 AMD patients from Eye Centre, PGIMER, Chandigarh, India with 61 genetically unrelated healthy controls as per inclusion and exclusion standards. However, some demographic item was non available for some topics.
Inclusion and exclusion standards
The inclusion standards for patients in both groups included the age of 50 old ages or older with the diagnosing of dry AMD with drusen more than five in at least one oculus and/or choroidal neovascularization.. The controls representing the survey included those that were of age 50 old ages or older and had no drusen with absence of other diagnostic standards for AMD.
The exclusion standards besides included the retinal diseases affecting the photoreceptors and/or outer retinal beds other than AMD loss such as high nearsightedness, retinal dystrophies, cardinal serous retinopathy, vein occlusion, diabetic retinopathy, uveitis or similar outer retinal diseases that have been present prior to the age of 50 and opacities of the optic media, restrictions of papillose dilation or other jobs sufficient to prevent equal stereo fundus picture taking. These conditions include occluded students due to synechiae, cataracts and opacities due to optic diseases.
Collection of Blood and Serum separation: About 4.0 milliliters of blood was collected in serum centrifuge tubing ( BD Biosciences, USA ) from both AMD and controls and left for 30 proceedingss at 370c to let it to coagulate harmonizing to the criterion processs. Serum was later separated by centrifugation at 3000 revolutions per minute for 30 proceedingss. The detached serum was frozen at -800 until analysis.
Entire protein appraisal
Bradford check was used to gauge the entire serum proteins for standardization to SOD1 degrees from ELISA. The process was carried out harmonizing to maker ‘s recommendations. Serum samples were diluted 1500 times in dual distilled H2O. The standard curve was generated by utilizing protein Bovine Serum Albumin ( BSA ) as criterion. Diluted samples and BSA standard protein were assorted with coomassie superb blue G – 250 dye ( Bradford reagent ) in 4:1 ratio followed by incubation at room temperature for 10 mins – 15 mins on shaker. The optical density was read at 595nm in 680XR theoretical account of Microplate reader ( Biorad, Hercules, CA, USA ) . The standard curve of BSA was estimated with quadratic tantrum or additive theoretical accounts.
Serum from AMD patients and controls was used to find the quantitative sensing of SOD1 utilizing commercially available enzyme linked immunosorbant check ( AB Frontier ; Catalog # LF-EK0101 ) as per maker ‘s protocol and optical density was read at 450 nanometers utilizing 680XR theoretical account of Microplate reader ( Biorad, Hercules, USA ) . Sample checks were performed in extra. The process to analyze the SOD1 degrees was provided by maker of the kit. This check recognizes native and recombinant human SOD1 with sensing more than 12.5pg/ml. The standard curve was generated by additive arrested development analysis for SOD1 appraisal in both patients and controls. All the values were normalized to entire serum protein.
A trained interviewer collected the information on demographic features, medical history, and lifestyle hazard factors like smoke, intoxicant etc utilizing a criterion hazard factor questionnaire [ 60,61 ] . Smokers were defined as those holding smoked at least three coffin nails per twenty-four hours or 54 boxes for at least 6 months and were segregated farther into tobacco users and ne’er tobacco users. Non vegetarian patients were defined as those holding chicken, meat or fish for at least 6 months and alcoholc consumer patients were defined as those holding whisky, rum, vino or place made intoxicant for at least 6 months. Hypertension was defined as systolic blood force per unit area less than140 millimeter Hg, diastolic blood force per unit area than 90 mm Hg at scrutiny, or diagnosed by a doctor antecedently, self-reported by the participant ‘s responses to whether a doctor had of all time informed them of this diagnosing and whether they had of all time taken medicines for this status. Similar protocols have been used earlier in old surveies ( 46 ) . Subjects were besides asked to describe any anterior diagnosing of shot, usage of antihypertensive medicines, megrim, diabetes or history of bosom diseases.
All statistical computations were carried out by statistical merchandise and service solutions SPSS ( IBM SPSS Statistics 20.0, Chicago, Illinois, USA ) package. The premise of normalcy was tested with the aid of Normal Quantile secret plan ( Q-Q secret plan ) and it was observed that informations were non usually distributed. Therefore, Mann-Whitney U-test was applied for comparing two groups. For comparing more than two groups, Kruskal Wallis one-way analysis of discrepancy ( ANOVA ) followed by post-hoc for multiple comparings was applied. The P ?0.05 was considered important. The step R2 ( coefficient of finding ) was used to find the goodness of standard curve tantrum for ELISA and entire protein. The additive and quadratic arrested developments with R2 & A ; gt ; 0.80 were considered to be a good tantrum. In order to place the hazard factors associated with AMD, a logistic arrested development was carried out and adjusted odds ratios were besides obtained. ROC curve for predicted modal was drawn.
Drumhead statistics of all of import variables are reported in Table 1. The serum SOD1 degree was found to be significantly higher in AMD patients as compared to controls ( Figure 1, Table 2, p=0.0001 ) . However, there is no important difference between the degrees of prohibitionist and wet AMD ( Table 2, p=0.117 ) . Furthermore, in the moisture AMD bomber groups there was a important difference. The degrees of SOD1 in preponderantly authoritative ( p=0.022 ) and supernatural ( p=0.023 ) were significantly higher as compared to minimal authoritative ( Figure 2 A ) . An independent analysis was carried out while seting the hazard factors to AMD. Important hazard factors like smoke, intoxicant, nutrient wonts, gender, high blood pressure, and bosom diseases were analyzed to analyze their association with SOD1. The SOD1 degrees were found to be higher among hypertensive ( Figure 1B, Table 2, p=0.015 ) , those with bosom disease ( Figure 1C, Table 2, p=0.002 ) and male AMD patients ( Figure 1D, Table 2, p=0.035 ) as compared to ne’er hypertensive, those without bosom disease and female AMD patients severally. However, there was no important difference between AMD tobacco users versus AMD ne’er tobacco users, intoxicant consumers versus ne’er alcohol consumers and vegetarian versus non-vegetarian ( Table 2 ) . The degrees were non found to be important when compared between Avastin treated AMD patients versus not treated AMD patients ( Table 2 ) . It has been observed that there was important association of degrees of SOD1 with AMD subtypes ( Chi-square= 6.326, p=.042 ) , gender ( Chi-square= 6.860, p=.032 ) , and smoke ( Chi-square= 6.291, p=.043 ) . The anticipation equation for AMD, by sing SOD1 as independent variable, shows that 78 % of the instances have been right classified ( exemplary genuineness 78 % ) with close assurance intervals for ROC curve. The country under ROC is 0.827 ( p=.0001 ) with standard mistake of 0.033 and assurance interval ( 0.762 – 0.891 ) ( Figure 3 ) .
The major ground for vision loss in aged population is accounted for by AMD ( 47 ) . Many surveies have attempted to tie in assorted biomarkers and candidate marks in the pathogenesis of AMD with conflicting studies and unverified in non Caucasic populations. Evidence suggests that oxidative emphasis plays an of import function in the pathogenesis of AMD ( 48,49 ) .
This survey was conducted to find whether the serum SOD1 degrees are altered in AMD patients as compared to normal controls. Our consequences indicate that the SOD1 degree increased significantly in AMD as compared to normal controls. To our cognition this non Caucasian survey is foremost to show elevated SOD1 serum degrees in Indian AMD patients. Higher degrees of SOD1 in AMD patients indicate the oxidative harm lending to AMD. During oxidative emphasis, the organic structure uses its defence mechanism to minimise the procedure of lipid peroxidation by utilizing the antioxidant enzymes such as SOD ensuing in increased activity of this enzyme likely due to compensatory response.
Retina is really susceptible for lipid peroxidation [ 19,20 ] which increases with age in macular part [ 19 ] . This is associated with cellular harm which involves largely decreased cellular antioxidants [ 21 ] . In our consequences the high degrees of SOD1 indicates that lipid peroxidation and oxidative emphasis are involved in tissue harm in AMD patients. Whether increased SOD1 degrees in our survey are so due to compensatory ordinance or cause of AMD can be determined by carry oning longitudinal survey base done intermediate or early Age-related macular degeneration patients.
SOD1 degrees of supernatural and preponderantly authoritative AMD patients were found to be higher as compared to minimally authoritative AMD patients. This corresponds to disease badness whether induced by SOD1 or ensuing from disease. Previously, it has been shown that the protein content of SOD1 and SOD2 in RPE homogenates additions in ulterior phases of AMD [ 26 ] .
The fact that SOD1 degrees were found to be higher among hypertensive and bosom disease patients could be ascribed to high oxidative emphasis in these patients. It was shown that the oxidative emphasis could be involved in the cardiovascular diseases and hypertensive patients [ 50,51 ] . It is pertinent to indicate out that although SOD1 is an antioxidant its over look can take to increased oxidative emphasis. Surveies on transgenic animate beings have shown that high degrees of SOD lead to increased hypersensitivity to oxidative emphasis [ 52,53 ] . It has hence frequently been proposed that the negative effects seen with high degrees of SOD are caused by an increased degree of the merchandise of the dismutation reaction, H peroxide [ 54 ] .
Previously we have reported that the VEGFR2 degrees increased significantly in the AMD patients as compared to normal control [ 59 ] . We hypothesized that there is positive correlativity between the increased SOD1 and VEGFR2 degrees because oxidative emphasis has been correlated antecedently with increased production of VEGF under in vitro conditions, and is thought to be involved in the upregulation of VEGF look [ 55,56 ] . In add-on, several surveies affecting animate being and tissue civilization theoretical accounts have indicated that oxidative emphasis is besides a critical go-between in the transduction of the mitogenic effects of VEGF. [ 57,58 ] .
We have attempted to foretell AMD based on SOD1 utilizing logistic arrested development, which showed 78 % theoretical account predictivity and country under curve is 0.827. The high value of AUC may be used to name AMD patients with really less Standard mistake. The association with gender, smoke and AMD types means that the increased degrees of SOD1 are associated with these factors.
Therefore, the oxidative emphasis is considered as of import causative factor for AMD, which can take to induced programmed cell death of RPE and farther consequence in damage of RPE map. ( 28-30 ) .