Age-related macular degeneration is the leading cause of blindness in humans that is characterized by progressive degeneration of macula, and consequences of this degeneration leading to severe irreversible loss in vision (1-5).The resulted vision loss subjected to either from retinal degeneration, (called dry or nonexudative AMD), or from the choroidal neovascularization (called CNV; wet or exudative AMD). The clinical manifestation of AMD includes drusen, hyperplasia of the retinal pigment epithelium (RPE), geographic atrophy, and angiogenesis of choroidal vessels (CNVs). (6).
Smoking, alcohol, oxidative stress and genetic factors are implicated in the pathogenesis of AMD (7) but at present exact cause of AMD is not known. It has been reported that aging is associated with pathological and biochemical changes in the eye. In general, aging and AMD is resulted from cumulative and increased oxidative damage. (8) Oxidative stress, refers to cellular or molecular damage that is caused by reactive oxygen species (ROS), has been implicated to any age related diseases and aging.(9,10) and this eleveted level of endogenously synnthesize ROS can be regulated by differents anti-oxidants enzymatic and non-enzymatic protective biochemical mechanism like Glutathion peroxiase (GPx), superoxide dismutase (SOD), and catalase (CAT). (11) ROS including, free radicals, nascent oxygen, hydrogen peroxide and the by-products of oxygen metabolism. Due to high consumption of oxygen which directly took from environment, high concentration of polyunsaturated fatty acid and direct exposure of light, make the retina to susceptible for oxidative stress. (12) Cumulatively lots of factors are responsible for oxidative generated aging like decreased levels of vitamin C and vitamin E in plasma (13, 14), glutathione levels decrease, and oxidized glutathione levels increased in plasma 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, vitamin E levels and catalase activity decreased with aging in RPE cells. (18,19). There is lots of pathological features have been reported in aging like, increased volume of lipofuscin contents (Increased Lipid and proteins contents), which enhance the oxidative damage susceptibility, decreased optical density of macular pigment,(20) and RPE cells that facing the phototoxicity exhibit membrane blebbing, phenomenon observed in aging and AMD eyes.(21).
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We hypothesized that oxidoreduction disturbance in eye results in alteration of SOD1 levels. Our concern of this research paper was to find out the expression of super oxide dismutase 1 (SOD1) in age related macular degeneration patients as compare to controls.
Main antioxidant system in the retina is comprised of three superoxide dismutase (SOD) isoenzymes that catalyse dismutation of superoxide into oxygen and hydrogen peroxide (H2O2 ) (22) .
Superoxide dismutase (SOD) is an antioxidant enzyme involved in the defense system against reactive oxygen species (ROS). SOD catalyzes the dismutation reaction of superoxide radical anion (O2-) to hydrogen peroxide, which is then catalyzed to innocuous O2 and H2O by glutathione peroxidase and catalase. There are three major families of SOD, depending on metal co-factors: Cu-Zn SOD (SOD1), present in cytosol, Mn (Fe)- SOD (SOD2) exist in mitochondrial matrix, and the extracellular SOD (SOD3) interstitium of the tissues as a secretory form. (23)
The activity and amount of the Cu-Zn SOD (SOD1) is highest among the three isoenzymes in the human retina (23) so it seems reasonable to predicts and analyze that SOD1 would accelerate age related changes in the retina.
Currently there is no study on SOD 1 in Indian AMD patients therefore; we thought to check the SOD 1 levels in North Indian population.
Study Participants:- This study has 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 were enrolled in patient/control group after obtaining written performa from all participants. All enrolled participants were referred from Department of Ophthalmology, PGIMER, Chandigarh (India) in which phenotypic criteria had strictly followed. In briefly, all AMD patients underwent for ophthalmic examination by a retina specialist for best corrected visual acuity, slit lamp biomicroscopy of anterior segment and dilated fundus examination. All AMD patients were subjected to fluorescein fundus angiography (FFA) and optical coherence tomography (OCT). The diagnosis of AMD was based on ophthalmoscopic and FFA findings.
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We included 176 case-control samples composed of 115 AMD patients from Eye Centre, PGIMER, Chandigarh, India with 61 genetically unrelated healthy controls as per inclusion and exclusion criteria along. However, some demographic detail was not available for some subjects.
Inclusion and exclusion criteria
The inclusion criteria for patients in both groups included the age of 50 years or older with the diagnosis of advanced AMD defined by geographic atrophy and/or choroidal neovascularization with drusen more than five in at least one eye. 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 include the retinal diseases involving the photoreceptors and/or outer retinal layers other than AMD loss such as high myopia, retinal dystrophies, central 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 ocular media, limitations of papillary dilation or other problems sufficient to preclude adequate stereo fundus photography. These conditions include occluded pupils due to synechiae, cataracts and opacities due to ocular diseases.
Collection of Blood and Serum separation: Collected 4.0 ml of blood sample in serum separator tube (BD Biosciences, USA) from the AMD as well as from control cases and left for 30 minutes at 370c to allow it to clot according to the standard hospital procedure. Serum was subsequently separated by centrifugation at 3000 rpm for 30 minutes. The separated serum was frozen at -800 until analysis.
Total protein estimation
Bradford assay was used to calculate the Concentration of serum total protein to normalize value of SOD1 calculated from ELISA. The procedure was done according to manufacturer's recommendations. Serum samples were diluted 1500 times in double distilled water. Standard curve was generated by using protein Bovine Serum Albumin (BSA) as standards. Diluted samples and BSA standard protein were mixed with coomassie brilliant blue G - 250 dye (Bradford reagent) in 4:1 ratio followed by incubation at room 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.
The separated serum from the blood of AMD patients and controls had been 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 check the SOD1 levels had followed as the instructions 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 patients and controls. All the values were normalized to total serum protein.
A trained interviewer has collected the information on demographic characteristics, medical history, and lifestyle risk factors like smoking alcohol etc using a standard risk factor questionnaire. Smokers were defined as having smoked at least 1 cigarette per day for at least 6 months and segregated in to smokers and never smokers. Hypertension was defined as systolic blood pressure less than140 mm Hg, diastolic blood pressure than 90 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 stroke, use of antihypertensive medications, migraine, diabetes and history of heart diseases.
All statistical evolutions were carried out by statistical package and service solutions (SPSS; IBM SPSS Statistics 20.0, Chicago, Illinois, USA) 20.0 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. Mann-Whitney U-test was therefore, applied for comparing the two groups. For comparing more than two groups, Kruskal Wallis one-way analysis of variance (ANOVA) followed by post-hoc was applied for multiple comparisons. 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.
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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 compare to controls (Figure 1, Table 2, p=0.0001). But there is no significant difference between the levels of dry and wet AMD (Table 2, p=0.117). However, in the subgroups of wet AMD there was a significant difference. The levels of SOD1 in predominantly classic (p=0.022) and occult (p=0.023) were significantly higher as compare to minimal classic. An independent analysis was carried out while 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 in hypertensive (p=0.015), with heart disease (0.002) and male AMD patients (0.035) as compare to non hypertensive, without heart disease and female AMD patients respectively (Table 2). 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 again significant when compare between avastin treated AMD patients versus non treated AMD patients (Table 2).
Discussion:- Majority of vision loss in elderly population is accounted by AMD (47). Many studies have attempted to associate various biomarkers and candidate targets in the pathogenesis of AMD. Evidences suggest that oxidative stress play an important role in the pathogenesis of AMD (48,49).
To our knowledge this study is first to demonstrate the SOD serum levels in Indian AMD patients. This study was conducted to determine whether the serum SOD levels increase or decrease in AMD patients as compared to normal controls. Our results indicate that the SOD1 level increased significantly in AMD as compared to normal controls. Higher levels of SOD1 in AMD patients showed that the oxidative damage increased in these as compared to normal. During oxidative stress, the body uses its defense mechanism to minimize the process of lipid peroxidation by using the antioxidant enzymes such as SOD, thus, the activity of this enzyme become higher as oxidative stress increases.
Retina is very susceptible for lipid peroxidation [19,20] which increases with age in macular region . Lipid peroxidation is free radical related process which may occur enzymetic and non enzymetic control. This is associated mostly 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 mechanism of tissue damage in AMD patients. The increased SOD1 levels in our study may be a compensatory regulation in response to increased oxidative stress.
Levels of Occult and predominantly classic AMD patients were higher as compared to minimally classic AMD patients. This may be due to the severity of disease. Previously it was shown that the protein content of SOD1 and SOD2 in RPE homogenates increases in later stages of AMD .
SOD1 levels were found higher in hypertensive and heart disease patients, which may be due to high oxidative stress in these patients. It was shown that the oxidative stress is involved in the cardiovascular diseases and hypertensive patients [50,51].
Although SOD1 is an antioxidant however its overexpression can lead to increased oxidative stress. Studies on transgenic animals showed that high levels of SOD lead to increased hypersensitivity to oxidative stress [52,53]. It has therefore often been
proposed that the negative effects seen with high levels of SOD are caused by an increased level of the product of the dismutation reaction, hydrogen peroxide .
The oxidative stress considered as important causative factor for AMD, which can lead to induced apoptosis of RPE and further consequence lead to impairment of RPE function. (28-30).