Comparative Analysis of Oxidative Stress Biomarkers in 50 Primary Open-Angle Glaucoma Patients

Introduction

Glaucoma is a major global cause of visual deterioration and permanent blindness [1]. It is usually linked to increased intraocular pressure (IOP), results in optic nerve damage, and progresses insidiously, potentially causing severe damage before noticeable symptoms arise [2]. Open-angle glaucoma is an important condition marked by an open angle in the anterior chamber, alterations in the optic nerve head, and a gradual deterioration of peripheral vision that can ultimately lead to loss of the central visual field [3]. Disturbingly, it frequently goes unnoticed until it reaches advanced stages. This highlights the critical importance of early detection and proactive management to safeguard vision and prevent irreversible damage. It was estimated that in 2020, approximately 80 million people worldwide had glaucoma, 74% of whom had open-angle glaucoma [2]. Primary open-angle glaucoma (POAG) is the most prevalent type, marked by heightened resistance to drainage in the trabecular meshwork [3]. POAG is frequently asymptomatic in its initial stages; a comprehensive history and eye examination are crucial for early disease detection [3]. Initial alterations in POAG result in a reduction of peripheral vision, which patients usually do not recognize until 40% of their nerve fibers are affected; it is only at this point that they start to perceive symptoms of “tunnel vision”. Visual field defects typically occur subsequent to asymptomatic thinning of the retinal nerve fiber layer and cupping of the optic nerve. Unlike maculopathies and cataracts, which result in diminished visual acuity, patients do not typically recognize the signs and symptoms of glaucoma. POAG can be clinically evaluated through various diagnostic tools, but the following triad is fundamental to diagnosis: changes in the optic disc or retinal nerve fiber layer, alterations in visual fields, and elevated IOP [3]. The aim of treating POAG is to stop the progression of changes in the optic nerve head and to prevent visual field decline. The idea of target IOP was presented to accomplish this goal. It is estimated that when IOP stays below a specific upper limit, the visual field, optic nerve head, and parameters of the retinal nerve fiber layer (RNFL) will remain stable. This contributes to enhancement of a patient’s quality of life. Topical drops are commonly used in treatment to either decrease aqueous humor production or enhance drainage [4].

The pathophysiology of POAG involves a complex process of progressive degeneration of the optic nerve, ultimately resulting in blindness [5]. Oxidative stress is considered to be a critical factor in this process [6].

Oxidative stress is cellular damage caused by excess reactive oxygen species (ROS). This condition can harm cellular components, including lipid peroxidation of cell membranes, protein damage, and DNA degradation [7]. ROS include various free radicals, including singlet oxygen, superoxide anion (O2•−), and hydroxyl radical. They also encompass non-radical species that arise from the partial reduction of oxygen, particularly hydrogen peroxide (H2O2). ROS are significantly more reactive than molecular oxygen (O2), allowing them to trigger essential biological processes [8]. O2•− is produced during oxidative metabolism through the one-electron reduction of molecular O2. Increased ROS levels and impairments in antioxidant defense mechanisms have been observed in glaucoma patients [9,10].

The action of antioxidant enzymes plays a critical role in regulating oxidative stress. In addition to non-enzymatic molecules like reduced glutathione (GSH), enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione reductase, and glutathione peroxidase are essential for ROS scavenging and maintaining cellular homeostasis [11].

SOD is a crucial enzyme that converts O2•− into H2O2, playing a vital role in the body’s antioxidant defense mechanisms [12]. Lower serum SOD levels may suggest weakened antioxidant capacity, making ocular tissues more vulnerable to oxidative damage [13]. Research assessing serum SOD levels in patients with POAG has produced inconsistent results [14–18].

CAT collaborates with SOD to counteract oxidative species [13], suggesting an increased vulnerability to oxidative damage in ocular tissues. Different results were found in studies evaluating aqueous humor and serum catalase enzyme activities in POAG patients [15,19–21].

Lower serum GSH levels correlate with diminished antioxidant capacity and may indicate increased vulnerability of the optic nerve head [22,23]. The levels of GSH might influence vascular endothelial function, which is essential for optic nerve perfusion. Studies have found reduced GSH levels in samples from POAG patients [24,25].

Malondialdehyde (MDA), a byproduct of lipid peroxidation, is an indicator of oxidative stress in cells [6], and increased oxidative stress is indicated by elevated serum MDA levels. Elevated MDA levels can be associated with damage to retinal ganglion cells, degeneration of the optic nerve, and dysfunction of the trabecular meshwork, all of which are key characteristics of glaucoma [13].

Studies on patients with POAG have found high MDA levels in serum and aqueous humor samples [18,20,26,27].

The present study evaluated oxidative stress parameters that are considered contributors to the pathogenesis of POAG, a disease with a complex etiology, comparing serum levels of MDA, SOD, CAT, and GSH in 50 patients with POAG and 52 healthy controls.

Material and Methods

PATIENT SELECTION:

The study adhered to the principles of the Declaration of Helsinki, and the methodology received approval (No. 04, 5/5/2020) from the local ethics committee. All participants received detailed information necessary about the study, and written informed consent was obtained prior to its commencement. The study included 50 POAG patients (POAG group) and 52 age- and sex-matched controls (Control group).

DIAGNOSIS:

POAG diagnosis was based on intraocular pressure >21 mmHg, glaucomatous cupping observed in fundus evaluation, open iridocorneal angle in gonioscopy, and reliable visual field defects determined by automated perimetry results using the 24-2 test on the Humphrey visual field analyzer (pattern standard deviation [PSD] <5% or glaucoma hemifield test results outside the normal limits in 99%). Patients with secondary open-angle glaucoma, such as steroid-induced glaucoma, pigmentary glaucoma, pseudo-exfoliation glaucoma, and traumatic glaucoma, were excluded.

Detailed medical histories, including age, sex, systemic diseases, prior ocular surgeries, and glaucoma treatments, were recorded.

INCLUSION CRITERIA:

Participants eligible for the glaucoma group were those aged 40 and above who had been diagnosed with POAG. The control group consisted of individuals who were at least 40 years old and had no ocular or systemic conditions.

EXCLUSION CRITERIA:

In addition to the criteria previously mentioned, the exclusion criteria included (Criteria I for the POAG group and Criteria II–V for both groups): (1) any type of glaucoma other than POAG, such as primary and secondary angle-closure glaucoma, normotensive glaucoma, and secondary open-angle glaucoma; (2) ocular diseases such as corneal pathologies, uveitis, diabetic retinopathy, and retinal vein occlusion; (3) a history of intraocular surgery; (4) systemic diseases that affect antioxidant enzyme levels, such as diabetes mellitus, autoimmune disorders, hepatic and renal conditions, and smoking; and (5) the use of systemic steroids or antioxidant vitamin supplements.

BLOOD SAMPLE COLLECTION:

Three milliliters of venous blood were collected from all patients and control subjects. Samples were centrifuged at 5000 rpm for 10 minutes, and then were carefully separated and stored at −80°C until further analysis.

SOD ASSAY: Serum SOD activity was evaluated using the method proposed by Sun et al [28]. SOD accelerates the dismutation of toxic superoxide radicals (O2−) formed during oxidative energy production to hydrogen peroxide and molecular oxygen. This method is based on the reading of the optical density (OD) at 560 nm wavelength of the blue-colored formazan dye given by superoxide radicals formed by xanthine and xanthine oxidase (XOD) with nitro blue tetrazolium (NBT). SOD in the sample inhibits the formazan reaction by removing superoxide radicals. One unit of SOD is a 50% inhibition of the NBT reduction rate under experimental conditions.

ACTIVITY CALCULATION:

1 unit of SOD: Enzyme activity that inhibits NBT reduction by 50%.

Activity was calculated in U/ml.

CAT ACTIVITY ASSAY:

The activity of CAT in serum was assessed using the methodology outlined by Aebi et al [29].

SOLUTIONS USED:

Two tubes were taken first, 1.4 ml of 30 mM H₂O₂ was added to the blind tube and 0.1 ml of phosphate buffer was added. 1.4 ml of 30 mM H₂O₂ was added to the sample tube. 0.1 ml of enzyme was added and mixed with vortex. Absorbances were read at 240 nm twice at 30-second intervals and the activity was determined. If the absorbance value is greater than 10, the sample can be diluted 1/5 using, for example, 80 μl of 0.9% NaCI solution on 20 μl of serum. After this, 1400 μl of 30 mM H₂O₂ is added to the tube in the same way. The absorbance value is multiplied by 5. If the absorbance value does not fall below 10, the sample can be diluted 1/10 by using 90 μl of 0.9% NaCI solution on 10 μl of serum. After this, 1400 μl of 30 mM H₂O₂ is added to the tube in the same way. The absorbance value is multiplied by 10.

ACTIVITY CALCULATION:

E.U. = (2.3 / Δx) × [(log A1 / log A2)] Activity was calculated in U/L.

Δx = 30 seconds

2,3 = Optical density of 1 μmol H₂O₂ in a 1 cm light path

GSH ASSAY:

Measurement of GSH levels was performed utilizing the method developed by Beutler et al [30].

CALCULATION:

Glutathione concentration was calculated in units of mmol/g protein.

MDA ASSAY:

MDA, a byproduct of lipid peroxidation due to free radical interaction with fatty acids, was quantified by measuring the colored complex formed with thiobarbituric acid [31].

APPLICATION OF THE METHOD:

We placed 200 μl of tissue homogenate into a tube, then 800 μl phosphate buffer, 25 μl BHT solution, and 500 μl 30% TCA were added. The tubes were mixed in a vortex and kept on ice for 2 hours, then centrifuged at 2000 rpm for 15 min. 1 ml of the supernatant was taken and transferred to other tubes. To these, 75 μl EDTA and 25 μl TBA were added. The tubes were mixed in a vortex and kept in a hot water bath for 15 min. Then, they were brought to room temperature and their absorbance was read at 532 nm in a UV/Vis spectrophotometer.

MALONDIALDEHYDE LEVEL CALCULATION:

Level calculation: calculated as μmol/L.

STATISTICAL ANALYSIS:

Data analysis was conducted utilizing SPSS for Windows (IBM SPSS Statistics for Windows, Version 23.0. Armonk, NY). In the patient and control groups, descriptive statistics were used to calculate the mean and standard deviation values for age, sex, intraocular pressure (IOP), mean deviation (MD), PSD, and serum biomarkers. The Shapiro-Wilk test assessed data normality. To examine the sex differences between the groups, the chi-square test was used. Comparisons of age, IOP, MD, PSD, SOD, CAT, GSH, and MDA levels between the glaucoma and control groups were conducted using either the t test or the Mann-Whitney U test, depending on the normality of distribution. A P value of less than 0.05 was considered statistically significant.

Results

COMPARATIVE RESULTS OF SERUM BIOMARKERS:

We noted significant differences in serum levels of SOD, CAT, GSH, and MDA between the POAG group and the control group (all P<0.001). Table 3 presents the mean CAT, GSH, SOD, and MDA levels and their comparisons between groups. Compared to the control group, POAG group patients had lower serum SOD and GSH levels but higher CAT and MDA levels (Figures 1–4).

Discussion

We performed a comparative analysis of the serum levels of the antioxidant enzymes SOD and CAT, the antioxidant molecule GSH, and the oxidative stress marker MDA between patients with POAG and healthy controls. The POAG patients had higher oxidative stress, indicated by higher serum MDA levels (P<0.001) compared to healthy controls. Additionally, our findings revealed that levels of the antioxidant enzyme CAT were higher (P<0.001), while levels of the antioxidant enzyme SOD and the antioxidant molecule GSH were lower in POAG patients (P<0.001 for both).

Glaucoma is a neurodegenerative disorder characterized by progressive degeneration of retinal ganglion cells and optic neuropathy. POAG is widely recognized as the most prevalent form of glaucoma [32]. The etiology of the disease is complex, with significant risk factors including elevated intraocular pressure (IOP), advanced age, systemic vascular factors, ethnicity, and genetic predisposition. These elements are interrelated processes that result in damage to the optic nerve, disrupted blood flow, and alterations in the structure of glial and connective tissues [5]. Factors contributing to the issue include malfunctioning cellular pumps and glutamate transporters, oxidative stress leading to formation of free radicals, the presence of inflammatory cytokines, neuroinflammation, and abnormal immune responses [33].

ROS, produced through various extracellular and intracellular activities, are signaling mediator molecules that play roles in cell growth, differentiation, progression, and cell death [34]. ROS encompass a range of free radicals, such as singlet oxygen, O2•−, and hydroxyl radical. They also include non-radical species formed through the partial reduction of oxygen, notably H2O2. ROS are chemically much more reactive than molecular O2, enabling them to initiate fundamental biological processes [8]. O2•− forms during oxidative metabolism through the one-electron reduction of molecular O2. O2•− is swiftly transformed into H2O2 by SOD, which interacts with protein thiols, influencing cellular signaling. H2O2, being membrane-permeable, is an effective intracellular signaling agent. However, in the presence of iron or copper ions, H2O2 can produce hydroxyl radicals that oxidize lipids, proteins, and DNA, leading to cellular damage.

The superoxide radical is a type of ROS formed by the addition of a third electron to an oxygen molecule, and it occurs in all living organisms exposed to air [12]. SOD is a key enzyme that converts O2•− into H2O2 and plays a role in the body’s antioxidant defense mechanisms. Studies examining serum SOD levels in various diseases have reported both higher and lower levels compared to controls [27,35–38]. Similarly, studies evaluating serum SOD levels in POAG patients have yielded inconsistent results [14–18]. Among these studies, Majsterek et al [15], Awodele et al [16], and Engin et al [17] found that serum SOD activity was higher in patients with POAG compared to the control group (P<0.05, P<0.05, P<0.05, P<0.01, respectively). Erdurmuş et al [18] found that serum SOD activity was higher (P=0.001), whereas no significant difference was found in the study by Rokicki et al [14] in patients with POAG compared to the control group, and revealed that serum SOD levels were lower in patients with POAG. Reduced SOD activity can contribute to increased oxidative stress, potentially influencing the pathogenesis of the disease.

Catalase is essential for adaptive responses to H2O2 and is believed to support the function of glutathione peroxidase [39]. This enzyme forms part of the second line of defense against ROS. CAT is one of the antioxidant enzymes that significantly reduce oxidative stress by effectively breaking down H2O2 into harmless water and oxygen [40]. Other more complex enzymes, such as glutathione peroxidases, block the formation of hydroxyl radicals from H2O2 and protect the organism. The most significant increase in catalase enzyme activity is associated with mitochondria, where ROS production is highest. Catalase deficiency or dysfunction has been linked to various diseases (eg, cardiovascular disease, hypertension, diabetes, Alzheimer’s disease, Parkinson’s disease, vitiligo) [40]. However, studies have also shown elevated catalase levels in certain diseases [41]. In the study by Tekin et al, serum catalase activity was found to be higher in patients with keratoconus compared to the control group [42]. Kenney et al found that catalase enzyme activity in human corneas with keratoconus was 1.8 times higher than in healthy corneas (P<0.03) [43]. In contrast, studies conducted by Goyal et al and Ghanem et al, involving POAG patients, found no significant differences in CAT activity in aqueous humor samples compared to controls [19,20]. Meanwhile, Majsterek et al, examining blood samples, reported lower CAT activity levels (P<0.001) [15]. In our study, serum CAT activity was significantly higher in the POAG group than in the control group. The increased H2O2 associated with the disease may have induced catalase expression.

Reduced glutathione is the molecule’s active form, readily donating electrons to neutralize free radicals. GSH stands out as the most abundant endogenous antioxidant in the body [44]. It is a potent reduced peptide made up of 3 amino acids in the structure of γ-l-glutamyl-l-cysteinyl glycine. Its remarkable electron-donating ability makes glutathione indispensable in combating oxidative stress. Glutathione peroxidase effectively utilizes GSH as a crucial cofactor to reduce H2O2, resulting in oxidized glutathione (GSSG). This process is vital for maintaining cellular health and preventing oxidative stress [45]. Decreased serum GSH levels have been observed in various systemic diseases [46–49], as well as in glaucoma and other ocular diseases [24,25,50–52]. Sato et al found significantly lower GSH levels in the aqueous humor of patients with POAG and NTG compared to the control group (P<0.01) [25]. In the study by Ghergel et al, serum GSH levels of patients with POAG and NTG were found to be significantly lower compared to the control group (P<0.001) [24]. Consistent with the literature, our results demonstrated low serum GSH levels in POAG patients, which may indicate increased oxidative stress in POAG.

ROS produced as a result of oxidative stress lead to lipid peroxidation. MDA, a byproduct of lipid peroxidation, is a reliable marker of systemic oxidative stress and is associated with various pathological conditions [53–55]. Nucci et al investigated MDA and total antioxidant levels in aqueous humor and blood in glaucomatous patients and non-glaucomatous controls [56]. Blood and aqueous MDA levels were significantly higher in glaucoma patients (P<0.001 for both), whereas total antioxidant levels were significantly higher in the control group (P<0.001 for both). Erdurmuş et al [18] and Yildirim et al [57] found serum MDA levels were significantly higher in patients with POAG compared to the control group (P=0.001, for both). In 2 other studies, by Ghanem et al [20] and Zanon-Moreno et al [26], MDA levels in aqueous humor samples were significantly higher in patients with POAG compared to the control group (P<0.001 for both). Our results align with previous studies reporting elevated MDA levels in POAG.

While H2O2 is effectively converted into harmless water by glutathione peroxidase, CAT can transform it into water and oxygen through an alternative pathway. This essential process protects the body from harm. However, our study revealed low GSH levels, a molecule crucial for catalyzing the glutathione peroxidase enzyme. The decrease in GSH levels coincided with reduced SOD activity, leading to diminished glutathione peroxidase activity and, consequently, a reduced ability to convert H2O2 into water. This chain reaction likely resulted in a relative increase in H2O2 concentrations. Therefore, the catalase pathway became the sole viable route for H2O2 elimination, which may explain the increased CAT activity observed in our results. Maintaining adequate GSH levels is clearly essential for optimal enzymatic function and the body’s ability to manage oxidative stress.

Superoxide dismutase (SOD) serves as a primary antioxidant defense. Reduced levels of SOD in serum may indicate compromised antioxidant capacity, increasing the susceptibility of ocular tissues to oxidative damage [13]. This could be clinically useful for assessing disease progression. CAT works with SOD to neutralize oxidative species. Changes in catalase activity can indicate oxidative imbalance [13], which could indicate a heightened susceptibility to oxidative damage in ocular tissues, and may be helpful in diagnosing individuals who are suspected of having glaucoma. Reduced serum GSH levels are associated with decreased antioxidant capacity and may be linked to susceptibility of the optic nerve head [22,23]. Its levels may also affect vascular endothelial function, which is crucial for optic nerve perfusion. Evaluating GSH levels may aid in diagnosing and tracking the progression of the disease. Elevated serum MDA levels indicate increased oxidative stress. High MDA levels may be linked to retinal ganglion cell damage, optic nerve degeneration, and trabecular meshwork dysfunction, which are all hallmarks of glaucoma [13]. MDA may serve as a diagnostic and prognostic marker; higher levels may indicate disease severity or progression. All these biomarkers may be used as therapeutic targets. Monitoring them can help evaluate the effectiveness of antioxidant therapy or lifestyle interventions. However, further studies are required to utilize these biomarkers in diagnosing POAG, assessing disease progression, and evaluating treatment response.

A limitation of the study is that biomarkers from aqueous humor samples were not analyzed to evaluate the local effect. Another limitation of the study is that the relationship between disease severity and biomarker levels was not assessed.

Conclusions

This study demonstrates that patients with POAG had higher oxidative stress and impaired antioxidant defense mechanisms, as evidenced by altered serum levels of MDA, SOD, CAT, and GSH. A significant systemic imbalance in the oxidant-antioxidant equilibrium appears to exist in POAG patients. Evaluating serum levels of SOD, CAT, GSH, and MDA can be a valuable means for diagnosing and treating POAG, especially if future studies with larger participant numbers confirm these results. Furthermore, the development of innovative systemic and topical antioxidant therapies holds great potential as a novel approach for effectively treating POAG. Further studies are necessary to assess the cost-effectiveness of utilizing these biomarkers in clinical practice, their connection to glaucoma progression, and their precise role in evaluating treatment response.