**Dietary and Lifestyle Factors Modulate the Activity of the Endogenous Antioxidant System in Patients with Age-Related Macular Degeneration: Correlations with Disease Severity**

#### **Zofia Ula ´nczyk <sup>1</sup> , Aleksandra Grabowicz <sup>2</sup> , El˙zbieta Cecerska-Hery´c <sup>3</sup> , Daria Sleboda-Taront ´ <sup>3</sup> , El˙zbieta Krytkowska <sup>2</sup> , Katarzyna Mozolewska-Piotrowska <sup>2</sup> , Krzysztof Safranow <sup>4</sup> , Miłosz Piotr Kawa <sup>1</sup> , Barbara Doł˛egowska <sup>3</sup> and Anna Machali ´nska 2,\***


Received: 17 August 2020; Accepted: 2 October 2020; Published: 5 October 2020

**Abstract:** Age-related macular degeneration (AMD) is a common cause of blindness in the elderly population, but the pathogenesis of this disease remains largely unknown. Since oxidative stress is suggested to play a major role in AMD, we aimed to assess the activity levels of components of the antioxidant system in patients with AMD. We also investigated whether lifestyle and dietary factors modulate the activity of these endogenous antioxidants and clinical parameters of disease severity. We recruited 330 patients with AMD (39 with early, 100 with intermediate and 191 with late form of AMD) and 121 controls in this study. At enrolment, patients' dietary habits and physical activity were assessed, and each study participant underwent a thorough ophthalmologic examination. The activity of several components of the antioxidant system were measured in red blood cells and platelets using both kinetic and spectrophotometric methods. Patients with AMD consumed much lower levels of fatty fish and eggs than the control group (*p* = 0.008 and *p* = 0.04, respectively). In the nAMD group, visual acuity (VA) correlated positively with green vegetable consumption (Rs = +0.24, *p* = 0.004) and omega-3-rich oil intake (Rs = +0.17, *p* = 0.03). In the AMD group, the total physical activity MET score correlated positively with VA (Rs = +0.17, *p* = 0.003) and correlated negatively with the severity of AMD (Rs = −0.14, *p* = 0.01). A multivariate analysis of patients and controls adjusted for age, sex, and smoking status (pack-years) revealed that AMD was an independent variable associated with a lower RBC catalase (β = −0.37, *p* < 0.001) and higher PLT catalase (β = +0.25, *p* < 0.001), RBC GPx (β = +0.26, *p* < 0.001), PLT GPx (β = +0.16, *p* = 0.001), RBC R-GSSG (β = +0.13, *p* = 0.009), PLT R-GSSG (β = +0.12, *p* = 0.02) and RBC GSH transferase (β = +0.23, *p* < 0.001) activity. The activities of components of the antioxidant system were associated with disease severity and depended on dietary habits. The observed substantial increase in the activity of many critical endogenous antioxidants in patients with AMD further indicates that the required equilibrium in the antioxidant system is disturbed throughout the course of the disease. Our findings explicitly show that a diet rich in green vegetables, fish and omega-3-rich oils, supplemented by physical exercise, is beneficial for patients with AMD, as it might delay disease progression and help retain better visual function.

#### **1. Introduction**

Age-related macular degeneration (AMD) is an incurable ocular condition of the outer retina that affects approximately 8–10% of the elderly population worldwide [1]. AMD is characterised by a progressive visual impairment and remains the leading cause of visual disability in developed countries [2]. Because of the increasing life expectancy, the number of individuals with AMD is estimated to reach 288 million in 2040 [3,4]. This increasing prevalence of AMD represents a major financial challenge for healthcare systems and is expected to exert increasing socio-economic effects [5].

As the pathogenesis of AMD is still poorly understood, the treatment options remain limited and are available only for the advanced, neovascular form of AMD (nAMD) [6]. The antiangiogenic treatment targets the main pathophysiological feature of this subtype of AMD: the formation of new, largely malformed vessels (choroidal neovascularization, CNV) [7]. At present, no proven treatment is available for the earlier stages of AMD nor for GA, which are characterised by the accumulation of drusen and retinal atrophy [8]. Therefore, research has focused on the prevention and/or slowing the progression of AMD to its late stages by manipulating modifiable risk factors, among which nutrition and dietary habits are listed as the most important risk factors [9]. Based on research evidence, changes in a patient's dietary habits and the addition of supplements represent a simple and cost-effective method for modifying the risk of developing and progression of AMD [10]. Several observational and experimental studies have been conducted in humans to investigate associations between dietary antioxidants, the consumption of certain foods and AMD [11], including Age Related Eye Disease Study 1 (AREDS1) [12]. Two of the most notable studies, AREDS1 and most recently AREDS2, have contributed to the supplementation strategy currently used in clinical practice [13]. These studies have noted the effects of dietary factors such as omega-3s, carotenoids, lutein/zeaxanthin, vitamins A and D on eye health and suggested that they might affect the course of AMD. These findings resulted in the development of recommendations and clinical practice guidelines that have been used as a decision-making tool in clinical settings [14]. In general, the most cost-effective and seemingly achievable strategy for the prevention of progression of AMD to its later stages appears to be a general healthy lifestyle that is achieved by a healthy diet and exercise [15,16].

The pathogenesis of AMD has been attributed to both modifiable and unmodifiable factors, including age, genetics and active smoking [17]. The most well-known genetic factors associated with an increased risk of AMD are polymorphisms in the *CFH* Y402H (complement factor H) [18,19] and *ARMS2* (age-related maculopathy susceptibility 2) genes [20]. Recently, our team identified another variant associated with an increased risk for AMD that is located in the peripherin-2 (*PRPH2*) gene, which encodes a photoreceptor-specific protein vital for rod and cone cell formation and stability [21]. The detrimental effect of the major modifiable risk factor for AMD, cigarette smoking, which increases the risk of AMD 2–4 times [22–24], has been attributed to the induction of angiogenesis, impairments in choroidal circulation, activation of the immune system and generation of oxidative damage [22,25,26]. In fact, risk factors other than smoking that contribute to AMD development, such as light exposure, diet, and vitamin D levels, among others, also exert well-documented effects on oxidative stress, which corresponds to cellular damage caused by reactive oxygen species (ROS) [27].

At the molecular level, the retinal environment is particularly susceptible to oxidative stress, as it is constantly exposed to light and is characterised by increased oxygen consumption and a high proportion of polyunsaturated fatty acids [27,28]. ROS directly damage DNA (particularly in mitochondria) and lipids in the photoreceptors, leading to the deterioration of the retinal pigment epithelium (RPE) [29]. These oxidatively damaged molecules then accumulate in the macular area and become a continuous source of chronic oxidative stress [30]. The retina protects itself from oxidative damage by producing a considerable number of antioxidants in the photoreceptor and RPE cells, including the enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), glutathione transferase (GST) and glutathione reductase (R-GSSG) and nonenzymatic antioxidants glutathione (GSH), carotenoids, uric acid, albumin and many others [31]. Disturbances in the tight balance of antioxidant system might contribute to AMD pathogenesis [32].

In the present study, we aimed to assess the activity levels of several components of the antioxidant system in patients with AMD and controls and to explore the role of dietary habits in AMD development. In particular, we wanted to investigate whether lifestyle factors modulate the concentrations of these endogenous antioxidants and clinical parameters of disease severity. We also focused on possible associations of antioxidant activity with genetic risk factors for AMD.

#### **2. Materials and Methods**

#### *2.1. Study Groups and Initial Management*

For this study we recruited 330 patients with AMD (39 with early, 100 with intermediate and 191 with late form of AMD) from the outpatient population of the First Department of Ophthalmology of Pomeranian Medical University in Szczecin, Poland (ethical code is KB-0012/141/13). For the control group, we enrolled 121 age-matched participants with no symptoms or signs of macular degeneration (absence of drusen, neovascularization or pigmentary abnormalities). We excluded patients with significant chronic systemic conditions (diabetes mellitus, renal failure, collagen/neoplastic disease, hepatic dysfunction, etc.) or ongoing retinal disease except AMD (in AMD groups), i.e., glaucoma or intraocular inflammatory diseases from the participation in the study. A consent form was signed by all patients before enrolment in the trial, in accordance with the tenets of the Declaration of Helsinki.

Demographic characteristics (age, gender, time of symptom onset, time to presentation) and the following vascular risk factors present at the time of the enrolment were recorded: hypertension, hyperlipidaemia, diabetes mellitus, atrial fibrillation, ischaemic heart disease, cardiomyopathy, prior cerebrovascular events or renal artery stenosis (atherosclerosis). The following anthropometric and nutritional parameters were also assessed in all patients: waist circumference [cm], waist/hip ratio (WHR), and body mass index (BMI) [weight (kg)/height (m)<sup>2</sup> ]. Cumulative pack-years of smoking were determined from the recorded average number of smoked cigarettes per day and smoking years. The actual blood pressure (BP) was determined in all subjects prior to the ophthalmic examination.

#### *2.2. Dietary Habits and Physical Activity Assessment*

At the enrolment, each patient completed Food Frequency Questionnaire (FFQ) and International Physical Activity Questionnaire (IPAQ) with the help of the member of the research team.

We modified a quantitative Food Frequency Questionnaire (FFQ) to assess the intake of the following food groups rich in nutrients considered important in the AMD aetiology and oxidative processes to evaluate the participants' dietary habits: fatty fish, eggs, green vegetables, fruit and fruit juice, omega-3-rich oils, simple and complex carbohydrates, as recommended previously [11,13,33,34]. Three different frequencies in terms of portions per week were available for selection for each food type and alcoholic drink as follows: fatty fish: <1, 2–4, and >4; eggs: <1, 2–4, and >4; green vegetables: <2, 2–7, and >7; fruit and fruit juice: <2, 2–7, and >7; omega-3-rich oils: <2, 2–7, and >7; simple carbohydrates: <2, 2–7, and >7; complex carbohydrates: <2, 2–7, and >7; beer: 0, ≤1, and 2–7; wine: 0, <2, and 2–7; and vodka: 0, ≤1, and 2–3.

Each participant completed the International Physical Activity Questionnaire (IPAQ) with the assistance of the member of the research team due to participants' vision impairment, which comprises 7 questions regarding all types of physical activity associated with daily life, work and leisure performed in the last seven days. The duration of each activity included in the final data was 10 min or longer with no interruptions at any moment. The physical activity score was presented in MET-min per week units and was calculated by multiplying a factor specific for each activity by several days spent performing the activity and time in min spent on the activity per day. Weekly activity was measured by adding scores of each of the activities.

#### *2.3. Ophthalmologic Examination*

The patients were examined by an ophthalmologist with a comprehensive ophthalmologic evaluation, including best-corrected visual acuity using the Early Treatment Diabetic Retinopathy Study (ETDRS) chart, intraocular pressure measurement, fundus photography, autofluorescence imaging, spectral-domain OCT, and fluorescein or indocyanine green angiography (Spectralis, Heidelberg Engineering, Carlsbad, CA, USA). The severity of AMD was classified according to Ferris et al. [35]: patients with medium drusen (63–125 m) and without pigmentary abnormalities were classified as early AMD group, patients with large drusen or with pigmentary abnormalities associated with at least medium drusen were classified as intermediate AMD group, and patients with lesions associated with neovascular AMD or geographic atrophy were classified to have late AMD. The examinations were carried out in a blinded manner.

#### *2.4. Blood Sample Collection, RBC and PLT Preparation*

Peripheral venous blood (approx. 7.5 mL) was collected from the AMD group and controls into two types of tubes containing ethylenediaminetetraacetic acid (EDTA) or sodium citrate as an anticoagulant. The blood sample in the EDTA tube was centrifuged at 3000× *g* for 10 min to separate the plasma and buffy coat from red blood cells (RBCs). The plasma and buffy coat were removed from RBCs and 3 mL of deionised water were added to induce haemolysis. The sample was then centrifuged at 13,500× *g* for 5 min to separate the haemolysate from the pellet of red blood cell membranes. Haemoglobin levels were assayed using Drabkin's method. All results obtained for the activity of antioxidant enzymes were calculated per 1 g of haemoglobin in RBCs.

Platelets were obtained from venous blood collected in a tube containing 109 mM sodium citrate (3.2%, 9:1; *v*/*v*). Blood was centrifuged (10 min; 20 ◦C; 10,000 rpm) to obtain platelet-rich plasma (PRP), which was transferred to a new tube and centrifuged again (10 min; 20 ◦C; 3824 rpm). The resulting platelet-poor plasma (PPP) was placed in a fresh tube and stored at −80 ◦C until further analysis. The platelet pellet was washed twice with Tyrode's solution (pH 7.4), suspended in 1 mL of Tyrode's solution and the number of platelets was determined using spectrophotometry. The suspension was stored at −80 ◦C until further analysis. The platelet suspension was thawed (37 ◦C) and frozen (−80 ◦C) twice, and the obtained platelet lysate was centrifuged (10 min; 4 ◦C; 3824 rpm). All results obtained for the activity of antioxidant enzymes were calculated per 1 g of platelet lysate protein. Protein levels were assayed using the Lowry protein assay.

We used automated methods and commercially available assays to measure fasting glucose and lipid levels (including triglycerides, total cholesterol and high (HDL) and low-density (LDL) lipoproteins) in all patients with AMD and controls.

#### *2.5. The Activity of Antioxidant Enzymes*

A spectrophotometric method was used to establish the concentrations of reduced glutathione (GSH). The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione transferase (GST), glutathione reductase (R-GSSG) in red blood cells (RBCs) and platelets (PLT) were obtained using kinetic methods. The measurements in RBCs and PLT were performed using a UV/VIS Lambda 650 spectrophotometer (Perkin-Elmer, Waltham, MA, USA) and similar protocols (specified below). The extracellular haemoglobin concentration in plasma samples was determined using a spectrophotometric method [36,37] with the same spectrophotometer.

#### 2.5.1. GSH Concentration

The (haemo)lysate was diluted, mixed with a precipitation solution (1.67 g of metaphosphoric acid, 0.2 g of EDTA-Na2, 30 g of NaCl and 100 mL of H2O; Sigma-Aldrich, St. Louis, MO, USA), incubated

(5 min, 4 ◦C) and centrifuged (550× *g* for 10 min). The supernatant was diluted with phosphoric buffer (pH 7.9), DTNB (5,5'-dithiobis-(2-nitrobenzoic acid), Sigma-Aldrich, St. Louis, MO, USA) was added, and then the mixture was incubated for 15 min at 25 ◦C. The detection wavelength was λ 412 nm. The GSH concentration was calculated using the molar absorption coefficient (e = 13,600 M−<sup>1</sup> cm−<sup>1</sup> ).

### 2.5.2. SOD Activity

In a test tube, a mixture of (haemo)lysate, chloroform:ethanol (3:5; *v*/*v*) solution, and distilled water was combined. The mixtures were subsequently vortexed and centrifuged (5 min; 4 ◦C; 3824× *g*). The study material (Na2CO3/NaHCO<sup>3</sup> buffer, SOD extract and adrenaline solution; Sigma-Aldrich, St. Louis, MO, USA) was incubated for 3 min at 37 ◦C. The absorbance of the study material was recorded in 5 min at a wavelength of 320 nm (in 30 ◦C).
