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115 AMD patients and 61 healthy controls were recruited for this study after informed consent. Serum SOD1 levels were determined by ELISA and after normalization to totalserum protein these were correlated to various risk factors. Logistic regression model authenticity by considering SOD1 as independent variable have been developed along with ROC curve.
Results: The SOD1 levels were significantly higher in AMDpatients as compared to controls. The difference was not significant for wet and dry AMD. However, the difference was significantbetween wet AMD subtypes. Non-significance of Hosmer-Lemeshow goodness of fit statistic (Chi-square=10.516, df=8, p=0.231) indicates the appropriateness of logistic regression model to predict AMD.
Conclusion: Oxidative stress in AMD patients may mount compensatory response resulting in increased levels of SOD1 in AMD patients. To predict the risk of AMD on the basis of SOD1, a logistic regression model show authenticity of 78% and area under the ROC curve (0.827, p=.0001) with less standard error of 0.033 and close 95% confidence intervals (0.762-0.891) further validates the model.
AMD is the leadingcause of blindness in humans that is characterized by progressive degeneration of macula leading to severe irreversible loss in vision (1-5).The vision loss results from either from retinal degeneration, (nonexudative or dry AMD), or from the choroidal neovascularization (wet or exudative AMD). The clinical manifestation of AMD includes drusen, geographic atrophy, hyperplasia of the retinal pigment epithelium (RPE) and angiogenesis of choroidal vessels (CNVs) (6).
Smoking, alcohol, oxidative stress and genetic factors are occupied 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 changes in the eye. In general, aging and AMD is believed to result from cumulative and increased oxidative damage. (8) Oxidative stress can exert molecular or cellular damage mediated by reactive oxygen species (ROS) which has been earlier shown to be implicated with diseases of ageing (9,10). The elevated level of endogenously synthesized ROS are known to be regulated by various anti-oxidant, enzymatic and non-enzymatic protective biochemical mechanism like Glutathion peroxiase (GPx), superoxide dismutase (SOD), and catalase (CAT) (11). ROS which includes free radicals, nascent oxygen, hydrogen peroxide and the by-products of oxygen metabolism are deleterious for eye pathophysiology. Due to high consumption of O2, the high concentration of polyunsaturated fatty acid and direct exposure of light renders retina susceptible to oxidative stress (12). Various factors are responsible for oxidative stress generated from aging; these include decreased levels of vitamin C and vitamin E in plasma (13, 14), oxidized glutathione levels increased in plasma and glutathione levels decrease with the age (15). Increased Lipid peroxidation is also reported in aging (16, 17) and the consequences of these imbalanced biochemical changes lead to increased susceptibility of retinal pigment epithelium cells (RPE) to oxidative damage with the aging. For example, catalase activity and vitamin E levels have been reported to decrease with aging in RPE cells. (18,19). There are several pathological features which have been reported to accompany aging, for example, the increased volume of lipofuscin contents (Increased Lipid and protein contents) which enhance the oxidative damage susceptibility, decreased optical density of macular pigment (20) resulting in RPE cells confronted by membrane blebbing, a phenomenon observed in AMD eyes and aging (21).
We hypothesized that oxidoreduction alteration in the eye might result from deranged SOD1 levels. We therefore analysed the expression of super oxide dismutase 1 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 oxygen and hydrogen peroxide (H2O2 ) (22). SOD is an antioxidant enzyme in the defense system against ROS. Super oxide dismutase catalyzes the dismutation reaction of O2- (superoxide radical anion) to H2O2, which is then catalyzed to H2O and O2 by catalase and glutathione peroxidase. There are three major families of super oxide dismutase, depending on metal co-factors: Cu-Zn SOD (SOD1), present in cytosol, Mn (Fe)- SOD (SOD2) present in mitochondrial matrix, and the extracellular SOD (SOD3) interstitium of the tissues as a secretory form. (23)
The amount and activity of the Cu-Zn SOD (SOD1) is highest amongthe three isoenzymes in human retina (23) so it seems reasonable to screen SOD1 for possible role in accelerating age linked changes in the retina.
Currently there is no study examining the role of SOD 1 in Indian AMD patients and this investigations will likely provide the substrate for future therapies in AMD
This study was been approved by Institute Ethics Committee of Post-Graduate Institute of Medical Education and Research, Chandigarh, India vide letter No. Micro/10/1411. Patients and controls were first informed about the study 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 criteria was strictly followed. Briefly, all AMD patients underwent ophthalmic examination by a retina specialist for best corrected visual acuity, dilated fundus examination and slit lamp biomicroscopy of anterior segment. All AMD patients were subjected to fluorescein fundus angiography (FFA) and optical coherence tomography (OCT). AMD diagnosis was based on FFA and ophthalmoscopic findings.
We included 176 case-control samples consisting of 115 AMD patients from Eye Centre, PGIMER, Chandigarh, India with 61 genetically unrelated healthy controls as per inclusion and exclusion criteria. However, some demographic detail was not available for some subjects.
Inclusion and exclusion criteria
The inclusion criteria for AMD patients included the age of 50 years or older with the diagnosis of dry AMD with drusen more than five in at least one eye and/or choroidal neovascularization.. The controls constituting the study included those that were of age 50 years or older and had no drusen with absence of other diagnostic criteria for AMD.
The exclusion criteria also included the retinal diseases involving the photoreceptors and/or outer retinal layers other than AMD loss such as high myopia, central serous retinopathy, diabetic retinopathy, retinal dystrophies, vein occlusion, uveitis or similar outer retinal diseases that have been present earlier to the age of 50 and opacitiesof the ocular media, limitations of papillary dilation or other problems enough to preclude satisfactory stereo fundus photography. These conditions includeoccluded pupils due to cataracts, synechiae and opacities due to ocular diseases.
Collection of Blood and Serum separation: About 4.0 ml of blood was collected in serum separator tube (BD Biosciences, USA) from both AMD and controls and left for 30 minutes at 370c to allow it to clot according to the standard procedures. Serum was subsequently separated by centrifugation for 30 minutes at 3000 rpm. The separated serum was frozen at -800 until analysis.
Total protein estimation
Bradford assay was used to estimate the total serum proteins for normalization to SOD1 levels from ELISA. The procedure was carried out according to manufacturer's recommendations. Serum samples were diluted 1500 times in double distilled water. The standard curve was generated by using protein Bovine Serum Albumin (BSA). Diluted samples and BSA standard protein were mixedwith coomassie brilliant blue G - 250 dye (Bradford reagent) in 4:1 ratio followed by incubation atroom temperature for 10 mins - 15 mins on shaker. The absorbance was read at 595nm in 680XR model of Microplate reader (Biorad, Hercules, CA, USA). The standard curve of BSA was estimated with quadratic fit or linear models.
Serum from AMD patients and controls was used to determine the quantitative detection of SOD1 using commercially available enzyme linked immunosorbant assay (AB Frontier; Catalog # LF-EK0101) as per manufacturer's protocol and absorbance was read at 450 nm using 680XR model of Microplate reader (Biorad, Hercules, USA). Sample assays were performed in duplicate. The procedure to analyse the SOD1 levels was provided by manufacturer of the kit. This assay recognizes native and recombinant human SOD1 with detection more than 12.5pg/ml. The standard curve was generated by linear regression analysis for SOD1 estimation in both controls and patients. All the values were normalized to totalserum protein.
A trained interviewer collected the information on medical history, demographic characteristics and lifestyle risk factors like smoking, alcohol etc using a standard risk factor questionnaire [60,61]. Smokers were defined as those having smoked at least three cigarettes per day or 54 boxes for at least 6 months and were segregated further into smokers and never smokers. Non vegetarian patients were defined as those having chicken, meat or fish for at least 6 months and alcoholc consumer patients were defined as those having whiskey, rum, wine or home made alcohol for at least 6 months. Hypertension was defined as diastolic blood pressure ≥90 mm Hg, systolic blood pressure ≥140 mm Hg at examination, or diagnosed by a physician previously, self-reported by the participant's responses to whether a physician had ever informed them of this diagnosis and whether they had ever taken medications for this condition. Similar protocols have been used earlier in previous studies (46). Subjects were also asked to report any prior diagnosis of migraine, use of antihypertensive medications, stroke, diabetes or history of heart diseases.
All statistical calculations were carried out by statistical product and service solutions SPSS (IBM SPSS Statistics 20.0, Chicago, Illinois, USA ) software. The assumption of normality was tested with the help of Normal Quantile plot (Q-Q plot) and it was observed that data were not normally distributed. Therefore, Mann-Whitney U-test was applied for comparing two groups. For comparing more than two groups, Kruskal Wallis one-way analysis of variance (ANOVA) followed by post-hoc for multiple comparisons was applied. The p ≤0.05 was considered significant. The measure R2 (coefficient of determination) was used to determine the goodness of standard curve fit for ELISA and total protein. The linear and quadratic regressions with R2 >0.80 were considered to be a good fit. In order to identify the risk factors associated with AMD, a logistic regression was carried out and adjusted odds ratios were also obtained. ROC curve for predicted modal was drawn.
Summary statistics of all important variables are reported in Table 1. The serum SOD1 level was found to be significantly higher in AMD patients as compared to controls (Figure 1, Table 2, p=0.0001). However, there is no significant difference between the levels of dry and wet AMD (Table 2, p=0.117). Moreover, in the wet AMD sub groups there was a significant difference. The levels of SOD1 in predominantly classic (p=0.022) and occult (p=0.023) were significantly higher as compared to minimal classic (Figure 2 A). An independent analysis was carried outwhile adjusting the risk factors to AMD. Important risk factors like smoking, alcohol, food habits, gender, hypertension, and heart diseases were analyzed to examine their association with SOD1. The SOD1 levels were found to be higher among hypertensive (Figure 1B, Table 2, p=0.015), those with heart disease (Figure 1C, Table 2, p=0.002) and male AMD patients (Figure 1D, Table 2, p=0.035) as compared to never hypertensive, those without heart disease and female AMD patients respectively. However, there was no significant difference between AMD smokers versus AMD never smokers, alcohol consumers versus never alcohol consumers and vegetarian versus non-vegetarian (Table 2). The levels were not found to be significant when compared between Avastin treated AMD patients versus non treated AMD patients (Table 2). It has been observed that there was significant association of levels of SOD1 with AMD subtypes (Chi-square= 6.326, p=.042), gender (Chi-square= 6.860, p=.032), and smoking (Chi-square= 6.291, p=.043). The prediction equation for AMD, by considering SOD1 as independent variable, shows that 78% of the cases have been correctly classified (model authenticity 78%) with close confidence intervals for ROC curve. The area under ROC is 0.827 (p=.0001) with standard error of 0.033 and confidence interval (0.762 - 0.891) (Figure 3).
The major reason for vision loss in elderly population is accounted for by AMD (47). In the pathogenesis of AMD a lot of studies have attempted to correlate various targets and biomarkers with conflicting reports and unverified in non Caucasian populations. Facts suggests that oxidative stress acts major role in the pathogenesis of AMD (48,49).
This study was carried out to determine whether the serum SOD1 levels are altered in AMD patients as compared to normal controls. Our results indicate that the SOD1 level increased significantly in AMD as compared to normal controls. To our knowledge this non Caucasian study is first to demonstrate elevated SOD1 serum levels in Indian AMD patients. Higher levels of SOD1 in AMD patients indicate the oxidative damage contributing to AMD. Body uses its defense mechanism during oxidative stress to minimize the process of lipid peroxidation by using antioxidant enzymes like SOD. It results in increased activity of this enzyme probably due to compensatory response.
Retina is very susceptible for lipid peroxidation [19,20] which increases with age in macular region . This is associated with cellular damage which involves mostly decreased cellular antioxidants . In our results the high levels of SOD1 indicates that lipid peroxidation and oxidative stress are involved in tissue damage in AMD patients. Whether increased SOD1 levels in our study are indeed due to compensatory regulation or cause of AMD can be determined by conducting longitudinal study base done intermediate or early AMD patients.
SOD1 levels of occult and predominantly classic AMD patients were found to be higher as compared to minimally classic AMD patients. This corresponds to disease severity whether induced by SOD1 or resulting from disease. Previously, it has been shown that the protein content of SOD1 and SOD2 in RPE homogenates increases in later stages of AMD .
The fact that SOD1 levels were found to be higher among hypertensive and heart disease patients could be ascribed to high oxidative stress in these patients. It was shown that the oxidative stress could be involved in the cardiovascular diseases and hypertensive patients [50,51]. It is pertinent to point out that although SOD1 is an antioxidant its over expression can lead to increased oxidative stress. Studies on transgenic animals have shown that increased levels of SOD lead to more hypersensitivity to oxidative stress [52,53]. So the negative effects seen withhigh levels of SOD are caused by an increased level of the product ofthe dismutation reaction yield hydrogen peroxide .
Previously we have reported that the VEGFR2 levels increased significantly in the AMD patients as compared to normal control . We hypothesized that there is positive correlation between the increased SOD1 and VEGFR2 levels because under in vitro conditions oxidative stress has been correlated previously with upregulation of VEGF, and is thought to be involved in the increased expression of VEGF [55,56]. In addition, several studies involving tissue culture and animal models have pointed out that oxidative stress is a critical moderator in the transduction of the mitogenic effects of VEGF. [57,58].
We have attempted to predict AMD based on SOD1 using logistic regression, which showed 78% model predictivity and area under curve is 0.827. The high value of AUC may be used to diagnose AMD patients with very less Standard error. The association with gender, smoking and AMD types means that the increased levels of SOD1 are associated with these factors.
Therefore, the oxidative stress is considered as important causative factor for AMD, which can lead to induced apoptosis of RPE and further result in impairment of RPE function. (28-30).