Oxidative stress in the red blood cells of patients with primary open-angle glaucoma

Abstract

BACKGROUND: This study was designed to examine oxidative and antioxidative changes in the red blood cells (RBCs) of patients presenting with glaucomatous degeneration.

METHODS: The experimental design was a case-control study of strictly selected patients who required antiglaucomatous surgery during primary open-angle glaucoma despite relatively regulated intraocular pressure (IOP) (POAG group, n = 30) and patients who underwent an operation for nonpathological cataracts (cataract group, n = 25). The activities of total superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT), as well as the concentration of malondialdehyde (MDA), were measured. Glaucomatous damage was estimated from a transient pattern electroretinogram.

RESULTS: Significant increases in GPX (p = 0.026) and CAT (p = 0.000) activity were noted in the RBCs of POAG patients compared to those of cataract patients. Although SOD was elevated in patients with POAG, the differences compared to cataract controls were not significant (p = 0.079). MDA concentrations were significantly increased in the glaucoma group compared to the cataract controls.

CONCLUSION: An oxidative disorder primarily represented by catalase upregulation was observed during the course of glaucoma.

Keywords

Glaucoma oxidative stress SOD GPX CAT MDA erythrocyte

1 Introduction

Glaucoma represents a wide group of pathological disturbances that mainly cause progressive retinal ganglion cell (RGC) damage, visual field loss, and subsequent blindness. Despite well-developed diagnostic tools and relatively efficient treatment, glaucoma remains the leading cause of irreversible blindness worldwide. Glaucomatous neuropathy may progress with elevated or normal (arbitrarily estimated) intraocular pressure (IOP). Therefore, elevated IOP, the main known risk factor for glaucoma, is neither sufficient nor necessary to trigger glaucoma neuropathy. Several observations suggest a multifactorial etiology of glaucoma pathogenesis; nevertheless, the triggers initiating glaucomatous pathology remain unidentified. Primary glaucoma should not exclusively be considered as an ocular pathology [11]. Data on oxidative stress during glaucomatous disturbances do not conflict with other observations but rather complement mechanical, vascular, genetic, and immunological theories of glaucoma pathogenesis. Oxidative stress likely plays an important role in increasing IOP, producing trabecular meshwork alterations, and promoting neuronal cell death, thereby affecting retinal ganglion cells (RGCs) during glaucoma [2, 12]. In contrast, an increase in IOP is thought to generate oxidative stress in the retina [16]. The POAG in the context of oxidative stress presents two main front line of oxidation and defense against the oxidative stress. The first one is in anterior segment, when mostly UV light and visible light are main resources of exogenous ROS, produced mostly in an aqueous humour. The second front line is in posterior, well vascularized segment, when endogenous, systematic ROS are delivered with blood. Additionally, there is an age-dependent increase in a production of endogenous free radicals and ROS. Therefore, both POAG occurrence and systemic redox balance deterioration depending on age, probably have common pathways.

Red blood cells (RBCs) mainly carry out oxygen (O₂) transport. These cells are especially prone to oxidative [14] and nitrative [27] stress because they are the first cells in the body to be exposed to these stressful stimuli.

Antioxidative systems in RBCs should provide antioxidant protection not only to these cells but also to other tissues and organs in the body.

RBCs possess effective antioxidative enzyme systems that neutralize reactive oxidants into non- or less-reactive species. Superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT) are some of the main endogenous enzymatic defense systems in cells. These enzymes protect cells by directly scavenging superoxide radicals and hydrogen peroxide (H₂O₂) [21].

SOD catalyzes the dismutation of superoxide radical (^•O₂) to H₂O₂. Although H₂O₂ is not a radical, it is rapidly converted into the highly reactive and cytotoxic radical ^•OH via the Fenton reaction. GPX and CAT then neutralize H₂O₂. The excessive production of free radicals and unneutralized free radicals leads to lipid peroxidation, which affects biomembranes [26] and causes cell disintegration. Lipid peroxidation products are implicated as the mechanism underlying neurological disorders such as glaucoma [17] and the aging process [23]. One of the most frequently used biomarkers to investigate oxidative damage to lipids is malondialdehyde (MDA), the major lipid peroxidation product.

The aim of this study was to evaluate redox balance changes in cells exposed to first-line oxidative stress during the course of primary glaucoma.

2 Materials and methods

The Ethics Committee of Medical University of Silesia, Katowice (permission number: KNW/0022/KB1/123/10) approved the study protocol. The study adhered to the tenets of the Declaration of Helsinki for experiments involving human tissue samples.

2.1 Participants

The primary open-angle glaucoma (POAG) group was exclusively composed of Caucasians who were preparing to undergo antiglaucomatous drainage surgery for visual field damage despite IOP control and intolerance to topical antiglaucomatous drugs. All patients exhibited bilateral visual field defects and noticeable, senile lens opacity.

The control group included Caucasians who were scheduled for cataract surgery.

The inclusion criteria for the POAG and control groups were as follows: (1) no previous intrabulbar surgery; (2) between 65 and 75 years old; (3) best corrected visual acuity of 0.5 or better (Snellen’s charts); (4) no myopia or hyperopia >3 diopters (D); (5) non-smokers; (6) no documented or diagnosed and treated ophthalmic or organic diseases (only treated arterial hypertension was permitted); (7) no additional medications except antihypertensive drugs; (8) no macular pathologies; (9) no abnormalities in routine preoperative laboratory tests, especially C-reactive protein (CRP), complete blood count (CBC) and differential; and (10) body mass index (BMI) <30.

The participants reported no addictions.

2.2 Ophthalmic examination

Clinical evaluations of POAG included gonioscopy, detailed ophthalmoscopy, central corneal thickness measurement, tonometry (Goldmann’s, Haag-Streit, Bern, Switzerland; 0.5% Alcaine) visual field examination (Octopus 301 HS, Interzeag) and policlinic history analysis. The average IOP for each patient was based on three measurements (the day before admission, the day of admission, and the day of surgery). All IOP measurements were obtained in the morning (between 8 AM and 11 AM). The IOP policlinic history (within the last 6 months), together with our measurements, aided in the exclusion of POAG patients in acute IOP phases.

To exclude patients with macular pathology, optical coherence tomography (OCT; Cirrus HD-OCT 5000, Carl Zeiss Meditec, Dublin, CA) was used.

The patients were examined on the day of blood sample collection.

2.3 Electrophysiological examination

The transient pattern electroretinogram (PERG) was examined using Reti-Port equipment (Roland Consult, Germany). The study conditions were performed as per the recommendations and standards of the ISCEV (International Society for Clinical Electrophysiology of Vision) [3]. Square checks with check size 30’, contrast 97%, reversal rate 4 reversals per second were used. Two trials for each stimulus condition were obtained to confirm reproducibility, 200 sweeps were collected and averaged. Fibre electrodes as a recording electrodes and gold-cup as a references and ground electrodes were used. The patients wore best optical correction for the distance of examination (1 m). Implicit time (the time to peak) and amplitude of the negative wave N95 (from the peak of P50 to the trough of N95) were measured.

2.4 Biochemistry

The total SOD (t-SOD), CAT, and GPX activities, as well as the MDA concentration, were determined from blood hemolysate samples.

On the day of blood draw, the hemolysate was prepared from centrifuged blood cells. Blood was centrifuged at 3,000 rpm for 10 min. The erythrocyte sediment was rinsed three times using 0.9% NaCl. Then, erythrocytes were hemolyzed with deionized water. The hemoglobin (Hb) concentration in 10% hemolysate was determined using the cyanmethemoglobin method of Drabkin [28].

Each sample was coded.

The t-SOD (EC1.15.1.1) activity was measured using the Oyanagui method [19] from 10% hemolysate and expressed in [NU/gHb].

Catalase activity (EC 1.11.1.6) was determined using the Aebi kinetic method [1]. Data are presented as international units per gram Hb [kIU/gHb].

Glutathione peroxidase (GPX) (EC 1.11.1.9) activity was measured using the kinetic method according to Paglia et al. [20]. Data are presented as [IU/gHb].

The concentration of MDA was determined in blood hemolysate according to Ohkawa et al. [18]. Data are reported as μmol/gHb.

2.5 Statistical analysis

Statistical analyses were performed with Statistica version 10 (StatSoft Inc., Poland). Comparisons were made between groups using a parametrical t-test or a non-parametrical Mann-Whitney test if the parametrical test assumptions were not met. Rates between the groups were compared using a χ2 test.

3 Results

Baseline patient characteristics are summarized in Table 1.

Only one eye per patient was included in the study. The sex (p = 0.41) and age (p = 0.21) distributions of the groups were comparable (Table 1). Glaucomatous patients presented significantly increased (p = 0.000) IOP; however, as mentioned above, only patients with a mean IOP of less than 21 mmHg (considering normal but not targeted) were included in the examined groups.

The N95 amplitude was significantly higher in the healthy controls (3.3 μV SD: 1.48, 95% CI: 2.70–3.89) compared to the glaucoma group (2.05 μV SD: 1.09, 95% CI: 1.64–2.45), p = 0.000. The N95 implicit time was not altered in patients with glaucoma (p = 0.66), as shown in Table 1.

The t-SOD activity in POAG patients (143.46 NU/gHb; SD: 17.54, 95% CI: 136.89–150.02) was slightly increased compared to healthy controls (138.35 NU/gHb; SD: 20.63, 95% CI: 130.02–146.09). Although these differences failed to reach significance (p = 0.079), they exhibited a trend (Fig. 1).

The CAT (Fig. 2) mean value in RBCs of glaucoma patients was 607.806 kIU/gHb (SD: 127.45, 95% CI: 560.242–655.426), whereas the mean value for the cataract patients was 461.361 kIU/gHb (SD: 64.12, 95% CI: 435.458–487.264). These differences were highly significant (p = 0.000).

The mean GPX (Fig. 3) activity in glaucomatous patients was increased to 60.29 IU/gHb (SD: 8.21, 95% CI: 57.22–63.35), whereas the value in non-glaucomatous patients was reduced to 54.53 IU/gHb (SD: 11.36, 95% CI: 49.94–59.12). These differences were significant (p = 0.026).

The concentration of MDA (Fig. 4) in RBCs was increased in the glaucoma group (0.271 μmol/gHb, SD: 0.03, 95% CI: 0.259–0.283) compared to controls (0.255 μmol/gHb, SD: 0.02, 95% CI: 0.244–0.265). The differences in MDA concentration between glaucomatous and non-glaucomatous participants were significant (p = 0.045).

4 Discussion

In the present study, increased oxidative stress was noted in the red blood cells of patients diagnosed with primary glaucoma. Redox state changes were observed and were associated with both enzymatic defense and oxidative stress product (MDA) accumulation.

This study design varied slightly compared to previous experimental protocols [8, 9]. The participants selected for this study included individuals who planned to undergo surgical procedures. A medical pre-operative evaluation was conducted in the hospital; this evaluation provided the medical history of involved subjects compared to the better known policlinic research model. Primary glaucoma is considered to be a general disorder rather than an exclusively ocular disorder. Therefore, we believe that the general clinical condition of patients should be carefully evaluated for precise primary glaucoma classification. We decided not to assess local oxidative changes in the aqueous humor and compare these changes with those observed in erythrocytes. Glaucomatous patients in the examined group were scheduled to undergo surgery due to progressive intolerance to topical antiglaucomatous agents. We were unable to exclude the topical immune system response, which may have affected the redox state of the aqueous humor in affected individuals.

The antioxidative defense system of RBCs was used to evaluate the redox response in glaucomatous and non-glaucomatous participants. Human RBCs exhibit a well-regulated average life span of 120±20 days and are uniquely exposed to a wide variety of oxidative stresses. By virtue of their specialization, mature RBCs are simultaneously restricted in their ability to respond to oxidative stress because they can neither synthesize new protein nor replace irreversibly damaged cellular components [25]. ROS-induced lipid peroxidation primarily impacts the RBC membrane, leading to changes on the surface of erythrocytes. Even these minimal changes produce morphological and functional abnormalities in RBCs [21] therefore, these changes potentially participate in an ischemic mechanism of glaucoma. It should also be mentioned that erythrocytes are one of the easiest types of human cells to acquire.

Although the groups were small in this pilot study, we believe that the participants were precisely classified based on ophthalmological parameters. In addition, the general conditions of the patients were considered.

In the POAG group, we included patients with documented visual field damage progression despite relatively well-controlled IOP. Although visual field analysis was performed, these results were intentionally not presented in this paper. As an objective estimation of RGC function, PERG is more precise. Optic nerve diseases preferentially affect N95, and the amplitude of this signal is decreased or absent in optic atrophy [4]. In current study, no significant differences in the N95 implicit time were noted between the study groups. The N95 amplitude was significantly reduced in the POAG group compared to the control group, indicating advanced glaucomatous atrophic changes in the optic nerve (Fig. 5.)

Optic nerve changes during the course of its atrophy were demonstrated in an experimental study performed by Mittag et al. In a rat glaucoma model, the ERG responses begin to decline after 3 to 4 months marked by an approximately 100% increase in the IOP [15].

Glaucomatous pathological processes are thought to fluctuate between stable and progressive states [24]. In our glaucoma group, all patients were planning to undergo surgery due to glaucomatous neuropathy exacerbation despite relative IOP normalization. Therefore, our examined patients presented an acute phase of neuropathy.

Patients in the examined and control groups presented cataracts. Oxidative stress is involved in the pathogenesis of cataracts [5]. As suggested by Nucci et al. [17], comparisons of age-matched and cataract-matched groups offer more precise results in the context of oxidative stress.

SOD, CAT and GPX activities were evaluated in the present study. These enzymes represent the main endogenous defense system found in all aerobic cells [22].

In our paper, the increase in t-SOD was not significant, but a trend was observed that corresponded with data obtained using similar research models and the aqueous humor [8 –10]. Interestingly, data on SOD activity in the serum of patients with glaucoma exhibited contrary results [7, 13]. This discrepancy potentially results from the diversified proportion of SOD isoenzymes (CuZn-complexed) found in RBC, serum and aqueous humor samples. The extracellular form (SOD3) is more relevant to serum, whereas the cytoplasmic (SOD1) form is more relevant to RBCs. In contrast, the absence of a significant increase in t-SOD activity in RBCs may reflect the start of antioxidative defense exhaustion in these cells, especially in glaucomatous patients.

Similar to the SOD results, our GPX results in RBCs were similar to the effects reported by Ferreiraet al. [8], Ghanem et al. [9] and Goyal et al. [10] in aqueous humor. The serum [7, 13] results were not similar. This difference may be explained by the use of different experimental protocols and/or tissues. These discrepancies may be attributed to variations in the transistor oxidative status and antioxidative capacity among RBCs, serum, and aqueous humor. Based on these few reports, the SOD and GPX responses to oxidative stress under glaucomatous conditions appear to be similar in RBCs and aqueous humor but different in serum.

CAT appears to play a leading role compared to GPX due to its ability to degrade H₂O₂ without consuming cellular reducing equivalents (NADPH); this characteristic represents an energy-efficient mechanism for removing H₂O₂. In the present paper, CAT activity was strongly (p = 0.000) increased by approximately 25%. Although no corresponding results were reported in previous publications, CAT activity was not altered in the aqueous humor [8 –10] but was decreased [13] or unchanged [29] in the serum. CAT activity is highest in the liver, kidney, and RBCs [6]. Therefore, changes in the CAT activity in erythrocytes may be more remarkable than those observed in the serum or aqueous humor. To the best of our knowledge, this is the first paper to demonstrate significant alterations in CAT activity in patients with age-related primary glaucoma. We recorded significantly increased activity in GPX and especially CAT in RBCs, which indicate a response to H₂O₂ overproduction and may reflect an intensification of oxidative processes during the course of glaucoma.

Alterations in MDA concentrations between glaucomatous and healthy controls exhibited weak statistical significance. This biomarker of aging correlates with decreased antioxidant capacity. In our experimental group, elevated activity of enzymatic defense systems revealed that the antioxidant capacity was not decreased and that lipid peroxidation protection was sufficient.

The above paper provides additional evidence supporting the role of oxidative stress in age-related glaucoma; however, the underlying mechanisms remain unclear. It may be possible to regulate IOP, decrease RBC vulnerability, and prevent retinal ischemia by regulating redox balance [2 , 21].

5 Conclusion

During the course of glaucoma, an oxidative disorder primarily characterized by catalase upregulation was noted in erythrocytes.

Disclosure

The authors have no competing interests to disclose.

Financial support

Costs of patient’s recruitment were covered by local government. Local authority provided all materials necessary for the study like copies of questionnaire etc. and prepared space for conduction of the study. The authors have no proprietary or commercial interest in any materials discussed in this article.

References

Aebi

, Catalase in vitro, Methods Enzymol 105 (1984), 121–126.

Awai-Kasaoka

, Inoue

, Kameda

, Fujimoto

, Inoue-Mochita

and Tanihara

, Oxidative stress responsesignaling pathways in trabecular meshwork cells and their effects on cell viability, Mol Vis 19 (2013), 1332–1340.

Bach

, Brigell

M.G.

, Hawlina

, Holder

G.H.

, Johnson

M.A.

, McCulloch

D.L.

, Meigen

and Viswanathan

, ISCEV standard for clinical pattern electroretinography (PERG): 2013; 2012 update, Doc Ophthalmol 126 (2012), 1–7.

Bach

and Hoffmann

, The origin of the pattern electroretinogram. In: Heckenlively

, Arden

, editors. Principles and practice of clinical electrophysiology of vision. 2nd ed. The Mit Press. Cambridge (Massachusetts), London (England),2006, pp. 185–196.

Borchman

and Yappert

M.C.

, Age-related lipid oxidation in human lenses. Invest. Ophthalmol, Vis Sci 39 (1998), 1053–1058.

Deisseroth

and Dounce

A.L.

, Catalase: Physical and chemical properties, mechanism of catalysis, and physiological role, Physiol Rev 50 (1970), 319–375.

Engin

K.N.

, Yemişci

, Yiğit

, Ağaçhan

and Coşkun

, Variability of serum oxidative stress biomarkers relative to biochemical data and clinical parameters of glaucoma patients, Mol Vis 16 (2010), 1260–1271.

Ferreira

S.M.

, Lerner

S.F.

, Brunzini

, Evelson

P.A.

and Llesuy

S.F.

, Oxidative stress markers in aqueous humor of glaucoma patients, Am J Ophthalmol 137 (2004), 62–69.

Ghanem

A.A.

, Arafa

L.F.

and El-Baz

, Oxidative stress markers in patients with primary open-angle glaucoma, Curr Eye Res 35 (2010), 295–301.

10.

Goyal

, Srivastava

, Sihota

and Kaur

, Evaluation of oxidative stress markers in aqueous humor of primary open angle glaucoma and primary angle closure glaucoma patients, Curr Eye Res 39 (2014), 823–829.

11.

Gupta

, Ang

L.C.

, Noël de Tilly

, Bidaisee

and Yücel

Y.H.

, Human glaucoma and neural degeneration in intracranial optic nerve, lateral geniculate nucleus, and visual cortex, Br. J Ophthalmol 90 (2006), 674–678.

12.

Izzotti

, Bagnis

and Saccà

S.C.

, The role of oxidative stress in glaucoma, Mutat Res 612 (2006), 105–114.

13.

Majsterek

, Malinowska

, Stanczyk

, Kowalski

, Blaszczyk

, Kurowska

A.K.

, Kaminska

, Szaflik

and Szaflik

J.P.

, Evaluation of oxidative stress markers in pathogenesis of primary open-angle glaucoma, Exp Mol Pathol 90 (2011), 231–237.

14.

Mazzulla

, Schella

, Gabriele

, Baldino

, Sesti

, Perrotta

, Costabile

and de Cindio

, Oxidation of human red blood cells by a free radical initiator: Effects on rheological properties, Clin Hemorheol Microcirc 60 (2015), 375–388.

15.

Mittag

T.W.

, Danias

, Pohorenec

, Yuan

H.M.

, Burakgazi

, Chalmers-Redman

, Podos

S.M.

and Tatton

W.G.

, Retinal damage after 3 to 4 months of elevated intraocular pressure in a rat glaucoma model, Invest Ophthalmol Vis Sci 41 (2000), 3451–3459.

16.

Moreno

M.C.

, Campanelli

, Sande

, Sánez

D.A.

and Keller Sarmiento

M.I.

, Rosenstein RE.Retinal oxidative stress induced by high intraocular pressure, Free Radic Biol Med 37 (2004), 803–812.

17.

Nucci

, Di Pierro

, Varesi

, Ciuffoletti

, Russo

, Gentile

, Cedrone

, Pinazo

Duran M.D.

, Coletta

and Mancino

, Increased malondialdehyde concentration and reduced total antioxidant capacity in aqueous humor and blood samples from patients with glaucoma, Mol Vis 19 (2013), 1841–1846.

18.

Ohkawa

, Ohishi

and Yagi

, Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction, Anal Biochem 95 (1979), 351–358.

19.

Oyanagui

, Reevaluation of assay methods and establishment of kit for superoxide dismutase activity, Anal Biochem 142 (1984), 290–296.

20.

Paglia

and Valentine

, Studies on the quantities and quantitative characterisation of erythrocyte glutathione peroxidase, J Lab Clin 70 (1967), 158–169.

21.

Pandey

K.B.

and Rizvi

S.I.

, Biomarkers of oxidative stress in red blood cells, Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 155 (2011), 131–136.

22.

Pandey

K.B.

and Rizvi

S.I.

, Markers of oxidative stress in erythrocytes and plasma during aging in humans, Oxid Med Cell Longev 3 (2010), 2–12.

23.

Rizvi

S.I.

and Maurya

P.K.

, Markers of oxidative stress in erythrocytes during aging in humans, Ann N Y Acad Sci 1100 (2007), 373–382.

24.

Singh

, Is the patient getting worse? Open Ophthalmol J 3 (2009), 65–66.

25.

Sivilotti

M.L.

, Oxidant stress and haemolysis of the human erythrocyte, Toxicol Rev 23 (2004), 169–188.

26.

Spengler

M.I.

, Svetaz

M.J.

, Leroux

M.B.

, Bertoluzzo

S.M.

, Parente

F.M.

and Bosch

, Lipid peroxidation affects red blood cells membrane properties in patients with systemic lupus erythematosus, Clin Hemorheol Microcirc 58 (2014), 489–495.

27.

Teixeira

, Napoleão

and Saldanha

, S-nitrosoglutathione efflux in the erythrocyte, Clin Hemorheol Microcirc 60 (2015), 397–404.

28.

Winterhalter

, Hemoglobins, porphyrins and related compounds in Clinical Biochemistry: Principles and Methods, Curtius

and Roth

eds., Berlin, 1974, pp. 1305–1322.

29.

Yildirim

, Ateş

N.A.

, Ercan

, Muşlu

, Unlü

, Tamer

, Atik

and Kanik

, Role of oxidative stress enzymes in open-angle glaucoma, Eye 19 (2005), 580–583.