Plasma Catalase Activity and Malondialdehyde Level in Patients

Home , Lipid peroxidation

1 2 3 3 3 Plasma catalase NA Ates¸ ,O¨ Yildirim , L Tamer ,AU¨ nlu¨ , B Ercan , STUDY LABORATORY N Mus¸lu3, A Kanik4, R Hatungil5 and U Atik3 activity and malondialdehyde level in patients with cataract

Abstract Introduction

Purpose Oxidative mechanisms play a major As age increases, cataract becomes a more role in the aetiology and pathogenesis of common disorder. Oxidant/antioxidant cataract, especially in age-related cataract. Our imbalance plays a key role in ageing. Oxidant/ study aims to investigate systemic oxidant and antioxidant systems imbalance has also proved antioxidant markers in cataract patients. to have potential importance for ocular tissues.1 Methods The activity of erythrocyte catalase It seems very likely that oxidative mechanisms and the level of malondialdehyde in plasma play a major role in the aetiology and 1Department of Medical were measured in 40 patients with cataract pathogenesis of cataract formation, especially in Biology and Genetics and 60 healthy control subjects. The age-related cataracts. Lens proteins in particular Faculty of Medicine malondialdehyde level, as an index of lipid are subject to extensive oxidative modifications. Mersin University peroxidation, was determined by Some of the protein modifications seen in Mersin, Turkey thiobarbitu¨ ric acid reaction according to Yagi. increased oxidative stress include the formation 2Department of The determination of catalase activity was of mixed disulphides with glutathione, Ophthalmology measured by a method that was defined by oxidation of cysteine, methionine, and Mersin University Beutler. Catalase enzyme activity and tryptophan,2 Oxidative stress has been shown to Mersin, Turkey malondialdehyde level were evaluated to cause cataracts in invitro models.3 find out whether there was a significant Exposure to light may lead to the formation of 3Department of difference in these variables. Analysis of reactive oxygen species by photo-sensitizing Biochemistry variance was used by forming a general mechanisms. Many tissues slowly regenerate Mersin University Mersin, Turkey linear model that takes age and gender as the oxidants, causing an increase in the risk for an covariate. accumulation of oxidant-inflicted damage in the 4Department of Biostatistics 1 Results CAT activity was found to be tissue components. This is based on the Mersin University 13 920.27847.9 U/l in cataract patients and previous findings that exposing lens proteins to Mersin, Turkey 16 061.371126.6 U/l in control subjects. CAT ultraviolet light or even sunlight in the presence activity in cataract patients was significantly or absence of aromatic amino acids generates 5Department of Phsiology lower than the control subjects (P ¼ 0.008). modified proteins. These proteins are insoluble, Mersin University Mersin, Turkey Plasma MDA level is significantly higher in coloured, and fluorescent.4,5 Crystallins and 7 patients with cataract 4.47 0.35 nmol/ml other proteins in lens fibre do not turn over and Correspondence: Nurcan compared to the control subjects must serve the lens for the lifetime of the person. Aras Ates¸, PhD 2.9470.26 nmol/ml (P ¼ 0.0001). There was no Thus, the lens is even more dependent than most Mersin U¨ niversitesi Tıp significant difference between different tissues on protection from oxidative damage.6 Faku¨ ltesi cataract subgroups when erythrocyte CAT The lens, like other tissues, contains a series of Tıbbi Biyoloji ve Genetik Anabilim Dalı Yenis¸ehir defence mechanisms that may protect it from activities and plasma MDA levels were Kampu¨su¨ compared (P ¼ 0.322, 0.062). the harmful effects of oxidations. These include Mersin 33161, Turkey Conclusion This study shows that oxidant/ superoxide dismutase (SOD), catalase (CAT), Tel: þ 90 324 3412815/1012 antioxidant balances alter in the presence of and glutathione peroxidase (GPX). Since the Fax: þ 90 324 3412400 cataract. lens has a limited capacity for protein turnover, GSM: þ 90 532 2269903 E-mail: naras@ especially in the nucleus, these protective Eye (2004) 18, 785–788. doi:10.1038/sj.eye.6700718 mersin.edu.tr mechanisms have a greater importance in Keywords: cataract; catalase; malondialdehyde; preventing the accumulation of modified Received: 16 April 2003 oxidative stress proteins.2 Accepted: 16 July 2003 Oxidative stress in patients with cataract NA Ates¸ et al 786

In this study, we aim to investigate the oxidant/ Principle antioxidant balance marker in systemic circulation. For Catalase catalyses the breakdown of H O to H O and O . this reason, we determined the plasma malondialdehyde 2 2 2 2 The rate of decomposition of H O by catalase is (MDA) level, as the index of lipid peroxidation and 2 2 measured spectrometrically at 230 nm, since H O erythrocyte catalase (CAT) activity in patients with 2 2 absorbs light at this wavelength. Ethanol is added to cataract. stabilize the haemolysate by breaking down ‘complex II’

of catalase and H2O2. After the addition of 50 ml tris Materials and methods buffer, 900 mlofH2O2 and 30 mlofH2O to the cuvettes, the system is incubated at 371C for 10 min, the haemolysate Subject is added, and, in the following 10 min, the decrease of The study was carried out on 40 subjects with cataract optical density is measured against blank at 412 nm. (24 men, 16 women; ages range 43–86 years), who attended the clinic of the ophthalmology department, and 60 MDA determination healthy control subjects (30 men, 30 women; age range The MDA levels, as an index of lipid peroxidation, were 45–73 years), who visited our hospital for an annual determined by thiobarbituric acid (TBA) reaction checkup. The patients with thyroid function disorders, according to Yagi.9.The principle of the method depends hypertension, diabetes mellitus, liver and renal on the measurement of the pink colour produced by the dysfunction, anaemia, osteoporosis, and inflammatory interaction of TBA with MDA. The sample (for blank arthritis were carefully identified and excluded from this H O is used), SDS, acetic acid, thiobarbituric acid, and study. Patients with cataract formation as a secondary to 2 H O were added to the test tubes, respectively. Then it identifiable causes, such as diabetes, trauma, and steroid 2 was incubated at 951C for 30 min in a water bath. After administration, were also excluded. incubation, butanol-piridine (15 : 1) solution is added to After enrolment, all subjects were given a detailed lens the tubes. Than the tubes are centrifugated at 4000 r.p.m. examination to determine the updated cataract status. At for 10 min. The butanol top layer is measured against the examination, Lens Opacities Classification System II, blank spectrophotometry at 532 nm wavelength. 1,1,3,3 which uses photographic standards for grading cataract tetraetoxypropane was used as the primary standard. type and severity and was described by Chylack et al,7 was used to grade lens status with slitlamp examination. The system uses four nuclear standards for grading Statistical method nuclear opalescence and colour, five cortical standards, CAT enzyme activity and MDA level were recorded as and four subcapsular standards. Subjects were qualified averages7standard deviation, and it was evaluated as cases if both pupils could be dilated to at least 6 mm whether there was a significant difference. Analysis of and both eyes had (1) lens opacities of grade 3 and more, variance (ANOVA) was used by forming a general linear and (2) no conditions other than cataract that might model that takes age and gender as the covariate. account for any vision loss that was present. Statistical tests were performed by SPSS 9.0. A P-value Cases were classified into pure (nuclear, cortical, and o0.05 was considered as statistically significant. posterior subcapsular) and mixed types, depending on the lens status of both eyes. Pure cases had a single type of opacity. Patients with more than one type of opacity Results were classified as mixed. In all 60% of the patient group was male and the mean A subject qualified as a control if (1) both pupils could age was 66. 571.47, 50% of the control group was male be dilated to at least 6 mm, (2) both lenses were graded as and their mean age was 57.471.0. All cases in the patient having no nuclear, posterior subcapsular, cortical group had bilateral cataract. In total, 18 cataract cases opacities or having grade I or II opacities. (45.0%) had cortical cataract, 12 cataract cases (30.0%) Patients were informed about the study and they had mixed type cataract, six cataract cases (15.0%) had consented to giving blood samples. Venous peripheral nuclear cataract and four cataract cases (10.0%) had blood samples (10 ml) were collected in ethylene- posterior subcapsular cataract. diamine-tetraacetic acid (EDTA)-coated tubes. Erythrocyte CAT activity and plasma MDA level are shown in Table 1. CAT activity was found to be 13920.27847.9 U/l in cataract patients and The determination of catalase activity 16061.371126.6 U/l in control subjects. CAT activity in The determination of CAT activity was measured cataract patients was significantly lower than the control according to the Beutler method.8. subjects (P ¼ 0.008).

Eye Oxidative stress in patients with cataract NA Ates¸ et al 787

Table 1 Erythrocyte CAT activitiy and MDA level in patients with cataract and control group

Variables Cataract group Control group P (n ¼ 40) Mean7SE (n ¼ 60) Mean7SE

CAT (U/l) 13 9207847 1606171126 0.0001 MDA (nmol/ml) 4.4770.35 2.9470.26 0.008

Table 2 Erythrocyte CAT activities and MDA levels in cataract subgroups

Variables Cortical cataract Mixed cataract Nuclear cataract Posterior subcapsular P (n ¼ 18) Mean7SE (n ¼ 12) Mean7SE (n ¼ 6) Mean7SE cataract (n ¼ 4) Mean7SE

CAT (U/l) 14 60275684 15 18876223 10 38574112 12 87771265 0.322 MDA (nmol/ml) 4.0971.32 5.1572.74 6.5973.86 2.9071.57 0.062

The plasma MDA level is significantly higher in There is evidence to suggest that GPX and SOD decreases patients with cataract 4.4770.35 nmol/ml compared to as cataract develops.16,20–22 the control subjects 2.9470.26 nmol/ml There is also evidence to suggest that low glucose 6- (P ¼ 0.0001).Erythrocyte CAT activities and plasma MDA phosphate dehydrogenase (G6PD) activity is associated levels of four different cataract types are summarized in with the increased risk for cataract. Low G6PD activity Table 2. There was no significant difference between the may lower the supply of reduced nicotinamide–adenine different cataract subgroups when erythrocyte CAT dinucleotide phosphate needed for the protection of activities and plasma MDA levels were compared reduced glutathione, with reduced nicotinamide– (P ¼ 0.322, 0.062). adenine dinucleotide phosphate being a cofactor for the enzyme glutathione reductase.16 It is shown that the MDA level was increased about two-fold in the plasma and by 1.5 in the lens, in response Discussion to selenite treatment in experimental cataract. This shows Oxygen-free radicals and antioxidant systems are increased peroxidation of unsaturated fatty acids in the thought to be involved in pathologic processes in the eye, lipid bilayers of lenticular plasma membrane. The including cataract.10 uveitis,11 retinopathy of peroxidative damage to the lens is one of the major prematurity,12 corneal inflammation,13 and keratitis.14 events in the pathogenesis of cataract. The current study During oxidative stress, a series of highly reactive proposes that this might be mediated by toxic À intermediates are produced, including the superoxide metabolites of oxygen such as O2 ,H2O2 and OH Á ,asa À radical (O2 ), hydrogen peroxide (H2O2), and hydroxy consequence of the impaired enzymatic defences against radical (OH Á ). These radicals are capable of reacting with their toxicity. The same study shows that blood GPX proteins, nucleic acids, and lipids leading to lipid, activities were increased and SOD activity was decreased peroxidation of biological membranes.15 in the selenite-treated group.23 It is commonly believed that oxidative mechanisms In the present study, the plasma MDA level was found play an important role in the aetiology of cataract, to be significantly higher (P ¼ 0.0001) and erythrocyte especially maturity-onset cataract.16 Oxidative stress has CAT activity was significantly lower in cataract patients been shown to cause cataract in invitro models.3 Many compared to the control subjects (P ¼ 0.008). But there types of cataracts, including maturity-onset cataract, was no significant difference between cataract subgroups induce extensive oxidative damage to proteins,17 lipids18 in terms of plasma MDA levels and erythrocyte CAT and DNA.19 activities. This may have been due to the low case Cataracts have been reported to be associated with numbers in cataract subgruops. elevated level of hydrogen peroxide (H2O2) in the In conclusion, this study shows that oxidant/ aqueous humor.3 Crystallins and other proteins in lens antioxidant balance changes in cataract. Evaluated in the fibre do not turn over and must serve the lens for the light of the previous studies on this subject, this study lifetime of the person. Thus, the lens is even more supports the hypothesis of cataract as an oxidative dependent than most tissues on protection from disorder. But further studies are needed to conclude the oxidative damage.6 The lens defence against oxidative exact role of the oxidant stress in the development of damage includes the enzymes GPX, SOD, and CAT. cataract formation.

Eye Oxidative stress in patients with cataract NA Ates¸ et al 788

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