Graefes Arch Clin Exp Ophthalmol (2015) 253:1161–1167 DOI 10.1007/s00417-015-3043-x
GLAUCOMA
Serum and aqueous xanthine oxidase levels, and mRNA expression in anterior lens epithelial cells in pseudoexfoliation Huseyin Simavli 1 & Mehmet Tosun 2 & Yasin Y. Bucak 3 & Mesut Erdurmus 4 & Zeynep Ocak 5 & Halil I. Onder 6 & Muradiye Acar 7
Received: 9 October 2014 / Revised: 18 April 2015 / Accepted: 29 April 2015 / Published online: 10 May 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Purpose The aim of this study was to determine serum and aqueous xanthine oxidase (XO) levels, and mRNA expression in anterior lens epithelial cells in pseudoexfoliation (PEX). Methods In this prospective study, serum, aqueous and anterior lens capsules were taken from 21 patients with PEX and 23 normal subjects who had undergone routine cataract surgery. Serum and aqueous XO levels were analyzed using the colorimetric method. mRNA expression of XO in anterior lens epithelial cells was evaluated using reverse transcription polymerase chain reaction analysis. Results Serum XO levels (means±standard deviations) were 207.0±86.1 IU/mL and 240.6±114.1 IU/mL in the normal and PEX groups, respectively (p=0.310). Aqueous XO levels (means±standard deviations) were 65.5±54.3 IU/mL in the normal group and 130.5±117.4 IU/mL in the PEX group (p = 0.028). There was a 2.9 fold decrease in mRNA
* Huseyin Simavli [email protected] 1
Department of Ophthalmology, Pamukkale University School of Medicine, Denizli 20700, Turkey
2
Department of Biochemistry, Faculty of Medicine, Abant Izzet Baysal University, Bolu 14080, Turkey
3
Eye Clinic, Bolu Izzet Baysal State Hospital, Bolu 14000, Turkey
4
Faculty of Medicine, Department of Ophthalmology, Hacettepe University, Ankara 06100, Turkey
5
Department of Genetics, Faculty of Medicine, Abant Izzet Baysal University, Bolu 14080, Turkey
6
Faculty of Medicine, Department of Ophthalmology, Duzce University, Duzce 80100, Turkey
7
School of Medicine, Department of Genetics, Turgut Özal University, Ankara 06030, Turkey
expression in anterior lens epithelial cells of PEX, which is significantly lower than the normal group (p=0.01). Conclusions Higher aqueous XO levels lacking associated different serum XO suggests higher oxidative stress in the aqueous. Higher aqueous XO levels in PEX with decreased mRNA expression in anterior lens epithelial cells indicate possible overexpression of XO in other structures related to the aqueous. Keywords Pseudoexfoliation . Xanthine oxidase . Oxidative stress . Anterior lens capsule . Aqueous humor . mRNA expression
Introduction Worldwide, pseudoexfoliation (PEX) is the most common cause of secondary open-angle glaucoma, which was first reported by the Finnish ophthalmologist, Lindberg [1], in 1917. PEX is an important risk factor for intraocular surgery. The most common clinical manifestation of PEX is the progressive accumulation of fibrillar extracellular material in the anterior chamber and especially on the anterior lens capsule. In addition to its occurrence within the eye, fibrillary material is found in different parts of the body, including the heart, lungs, liver and spleen [2], which suggests PEX is an ocular manifestation of a systemic disease. Although the pathogenesis of PEX, an age-related disorder, has not been fully clarified, there is increasing evidence that oxidative stress plays an important role in the pathogenesis of PEX [3–5]. There is an intricate balance between oxidative stress and antioxidants in the human body. The balance between them plays an important role in aging, cardiovascular diseases and eye diseases such as age-related macular degeneration, cataracts, glaucoma and uveitis [6–10].
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Xanthine oxidoreductase (XOR), an enzyme in purine metabolism, is transcripted as a single gene product in the xanthine dehidrogenase (XDH) form which is mostly found in the liver and intestine [11]. During inflammatory conditions and hypoxia [12, 13], XDH is converted to xanthine oxidase (XO), which catalyzes two consecutive reactions at the end of purine metabolism, from which the end product is uric acid with superoxide (O2−) and hydrogen peroxide (H2O2). XO, which can serve as an important source of reactive oxygen species (ROS), was shown to be decreased in the serum of patients with PEX [4]. Although anterior chamber hypoxia was reported in PEX [14], aqueous levels of XO have not yet been reported. One of the common points of XO and PEX is endothelial dysfunction. XO causes endothelial dysfunction via superoxide production [15] and there is evidence that suggests systemic endothelial dysfunction in PEX [16, 17]. Interleukin 6 (IL-6) is another common point. IL-6, which has been shown to upregulate transcription of XOR [18], was shown to be increased in PEX [19]. Although serum XO levels in PEX were shown to be increased [4], XO levels and mRNA expression in the aqueous and anterior lens capsule, where the clinical manifestations of PEX are mostly represented, have not yet been studied. In this study, we analyzed serum and aqueous levels of XO, and mRNA expression in the anterior lens capsule in order to identify the possible role of XO, an ROS-producing enzyme, in PEX, which can be a step in understanding the pathogenesis of the syndrome.
Materials and methods Study population This prospective study was conducted according to the Helsinki Declaration and approved by the local institutional ethics committee. Written informed consent was obtained from all participants. All of the subjects were recruited from among patients who had undergone cataract surgery. Pre-operative tests and measurements including blood tests, posterioranterior chest films, electrocardiography and arterial blood pressure were evaluated by an internist. Blood tests included glucose, blood urea nitrogen (BUN), creatinin, aspartate aminotransferase, alanin amino transferase, complete blood count including white blood cell count, hemoglobin, hemotocrit, and platelet count, and coagulation tests including protrombin time, international normalized ratio, and active parsiel tromboplastin time. Additionally, body temperature, arterial blood pressure, respiration rate, and heart rate were monitored three times a day during hospitalization. Patients with abnormal blood tests, chest films or electrocardiograph, or with a
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history of systemic disease or drug use, were not included in the study. All subjects underwent a detailed ophthalmologic examination that included ocular history, visual acuity testing, refraction, Goldmann applanation tonometry, slit-lamp biomicroscopy, and dilated ophthalmoscopy. Intraocular lens calculations were performed with B-Scan-Cine Scan (Quantel Medical, Cedex, France) in all patients. PEX was defined during dilated ophthalmoscopy as the presence of white-grey pseudoexfoliation material on the anterior lens capsule, iris, pupillary border, corneal endothelium and even the anterior chamber. Subjects with systemic or ocular disease other than cataract, or those with a history of ocular surgery or trauma were excluded from the study. Additionally, patients with any other signs implying exfoliation (e.g., signs of pigment liberation and deposition throughout the anterior segment, or transillumination defects in the pupil margin) were also excluded from the study. The control group consisted of healthy subjects without PEX who had undergone routine cataract surgery. Sample collection Venous blood samples were obtained from the antecubital vein just before the start of phacoemulsification in the operating room. They were centrifuged within 15 min of collection, at 2.750 g for 10 min, and the supernatant serum was then transferred into polypropylene tubes. Local anesthesia was performed in a subconjunctival fashion in all subjects beforehand using 0.5 cc of Jetokain® (Lidokain HCl 20 mg/ml, Epinefrin HCI 0.0125 mg/ml, Adeka, Samsun, Turkey). A side port was made with a 27gauge syringe and a small amount of aqueous humour (0.1– 0.2 ml) was aspirated. A balanced salt solution (BSS) was used to form the anterior chamber if needed. The port then was widened by a 20-gauge MVR knife. Another port was made from the opposite direction of the first one. A clear corneal incision was fashioned at the 11-o’clock meridian. Sterile air was injected into the anterior chamber through the side port incision. A volume of 0.1 ml of 0.1 % trypan blue (Vision Blue, Dorc International, Netherlands) was injected under the air bubble over the anterior capsule with a 27gauge cannula in order to improve visualization of the capsule. The anterior chamber was deepened with air to allow even contact of the dye with the lens capsule and then washed with balanced salt solution after waiting for 10 s. High viscosity viscoelastic, Healon GV® (Abbott Medical Optics Inc, Santa Ana, CA, USA), was injected into the anterior chamber. Capsulorhexis was initiated with a bent 26-gauge needle and a capsulorhexis forceps was used for completing a continuous curvilinear capsulorhexis. The anterior lens capsule and aqueous humor samples were transferred to different polypropylene tubes marked with the patients’ barcodes.
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All the materials, aqueous, serum and capsule, were stored at −80 °C until the assays were determined. XO enzyme activity in the aqueous and serum was determined using the colorimetric method BioVision (BioVision Inc., CA, USA) according to the manufacturer’s protocol. Genetic analysis Total RNA isolation Total RNA was extracted with TRIzol (Ambion/RNA by Life Technologies, Carlsbad, CA, USA) according to the previously reported technique [20]. One microgram of RNA was reverse transcribed with reverse transcriptase (Thermo Scientific, Waltham, MA, USA) with oligod (T) primers according to the manufacturer’s instructions (Table 1). Mouse β-actin was amplified as a control for the PCR. Samples lacking reverse transcriptase were amplified as a control for genomic DNA contamination. Real-time PCR (B-actin) Real-time PCR was performed on cDNA samples obtained (Thermo Scientific Revert Aid First Strand cDNA Synthesis Kit) as described in the previous report [20]. The PCR mixture consisted of SYBR Green PCR Master Mix, which included DNA polymerase, SYBR Green I Dye, dNTPs, PCR buffer, forward and reverse primers and cDNA of samples in a total volume of 50 μl/mL. The amplification of a housekeeping gene, β-Actin, was used for normalizing the efficiency of cDNA synthesis and the amount of RNA applied (Fig. 1). PCR was performed with initial denaturation at 95 °C for 5 min, followed by amplification for 38 cycles, each cycle consisting of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, polymerization at 72 °C for 1 min and, the last stage, polymerization at 72 °C for 5 min. Real-time PCR (xantine oxidase) Real-time PCR was performed on cDNA samples obtained (Thermo Scientific Revert Aid First Strand cDNA Synthesis Kit) as described in the previous report [20]. The PCR mixture consisted of SYBR Green PCR Master Mix, which included DNA polymerase, SYBR Green I Dye, dNTPs, PCR buffer, forward and reverse primers and cDNA of samples in a total Table 1 The forward and reverse primers used in the real-time PCR analyses of the XO and β-actin genes XO β-actin gene
Forward Reverse Forward Reverse
5′-GACCTCAACCCCGTGTTCAT-3′ 5′-AAAGCTGCCTCTGAGTGGTC-3′ 5′TTCCTGGGCATGGAGTCCT-3′ 5′-AGGAGGAGCAATGATCTTGATC-3′
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volume of 50 μl/mL. The amplification of a XO gene was used for normalizing the efficiency of cDNA synthesis and the amount of RNA applied (Fig. 1). PCR was performed with initial denaturation at 95 °C for 15 min, followed by amplification for 50 cycles, each cycle consisting of denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s, polymerization at 72 °C for 30 s and, the last stage, polymerization at 72 °C for 5 min. The data of mRNA expression of XO were evaluated by the relative standard curve method, which measures the expression level of the target gene normalized to a ß-actin reference gene; the same primer pairs used for the standard PCR were utilized for the real-time PCR. The arithmetic mean of the relative expression levels of XO gene in each group of patients and controls were calculated. Statistical analysis All analyses were performed using the SPSS for Windows V.11.0 software package (SPSS Inc., Chicago, Illinois). Continuous variables were presented as mean±standard deviation (SD). Nominal variables were presented as a percentage. Differences in measured parameters between the two groups were analyzed with an independent samples t-test. In order to determine the correlation between serum and aqueous XO levels, Pearson’s correlation coefficient was used. The differences were considered significant when the probability was less than 0.05.
Results A total of 44 subjects were included to the study; 21 of them were in the PEX group, 23 of them were in the normal group. The mean visual acuity was 0.84±0.37 LogMar in the PEX group and 1.02±0.50 LogMar in the control group. The mean intraocular pressure (IOP) was 17.1±4.0 mmHg in the PEX group and 15.3±3.7 mmHg in the control group. The groups were statistically not different according to the preoperative visual acuity and IOP measurements (p=0.183, p=0.123, respectively). Eight of 21 subjects (38 %) had poor pupil dilatation in the PEX group. None of the patients in either group had phacodonesis. Demographics and preoperative blood tests of the study are shown in Table 2. There was no difference among PEX and normal groups in terms of age and sex (p=0.765, 0.763, respectively). All of the PEX group and normal group blood tests were in the reference range. Blood test results did not differ, with the exception of BUN. BUN levels of subjects with and without PEX were 34.1±7.7 mg/dL and 39.4±6.2 mg/dL, respectively, which is statistically significant (p=0.04). The mean serum levels of XO were 207.0±86.1 (IU/mL) in the normal group and 240.6±114.1 (IU/mL) in the PEX
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Fig. 1 Amplification plots of XO mRNA (a), housekeeping β-actin mRNA (c). Serial dilutions of XO (b) and β-actin cDNA (d) plasmids were amplified using real-time PCR. For each dilution, the fluorescence is
plotted against the cycle number. Relative input (log concentration) and cycle numbers of each dilution are given
group. XO activity in serum was not significantly different in the PEX group as compared to the control group (p=0.310). The mean aqueous levels of XO were 65.5±54.3 (IU/mL) in the normal group and 130.5±117.4 (IU/mL) in the PEX group. The activity of XO in the aqueous was significantly
higher in the PEX group than in the control group (p=0.028). There was a low negative correlation between serum and aqueous levels of XO which is not statistically significant (r=−0.269, p=0.103). mRNA expression levels of XO in anterior lens epithelial cells of PEX was found to be decreased 2.9 fold as compared to that of the normal group (p=0.01) (Table 3). An example of five subjects with PEX representing no XO mRNA expression in agarose is shown in Fig. 2.
Table 2 Demographics and blood test results of the normal and pseudoexfoliation population
Age (years) Sex (male) (42.9 %) Glucose (mg/ dL) BUN (mg/dL) Creatinine (mg/ dL) AST (U/L) ALT (U/L) WBC (K/uL) HGB (g/dL) HCT (%) PLT (K/uL) APTT (seconds) PT (seconds) INR
Reference range
Normals Pseudoexfoliation p Mean±SD Mean±SD
n/a n/a
74.3±7.8 11
75.1±7.0 (47.8 %)
0.765 9
0.763 60–109
97.1±11.6 93.4±9.6
0.329
19–44 0.1–1.2
39.4±6.2 34.1±7.7 0.87±0.13 0.79±0.18
0.04 0.143
1–40 1–40 4.5–11.0 11.5–17.5 37.0–53.0 140–400 20–36 10–14 0.8–1.2
17.5±3.6 12.6±4.7 6.8±1.2 13.5±1.1 40.0±2.9 249±67 21.9±11.5 12.3±0.4 1.03±0.08
0.187 0.262 0.148 0.875 0.424 0.405 0.131 0.277 0.897
19.9±5.7 14.7±5.3 6.2±1.2 13.4±1.1 40.9±3.4 295±121 27.2±7.5 12.9±0.9 1.04±0.09
SD standard deviation, n/a not applicable, BUN blood urea nitrogen, AST aspartate aminotransferase, ALT alanine aminotransferase, WBC white blood cell, HGB hemoglobin, HCT hemotocrit, PLT platelet, APTT active parsiel prothrombin time, PT prothrombin time, INR international normalized ratio
Discussion PEX is an elastotic process of the extracellular matrix characterised by excessive production and progressive accumulation of a grey-white fibrillary material deposited in the eye, especially on the anterior lens capsule. Besides its occurrence within the eye, exfoliative fibrillopathy has been reported in different parts of the body, such as the heart, lungs, liver and spleen, which suggests PEX is an ocular manifestation of a systemic disease [2]. There is growing evidence that suggests the oxidativeantioxidative balance is disturbed in PEX, not only in the anterior segment but also in the whole body [3–5, 21, 22], and that the resulting oxidative stress, which can be defined as elevated ROS levels over the range of physiologic values, constitutes a major mechanism involved in the pathophysiology of PEX. ROS are chemically reactive molecules containing oxygen that are generated intracellularly or via exogen sources through a variety of processes, including normal aerobic metabolism and various signal transduction pathways. XO, which catalyzes the rate-limiting steps of purine degradation, is a critical source of ROS [12]. Because PEX is
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Table 3 Serum and aqueous levels and mRNA expression of xanthine oxidase in pseudoexfoliation
known as a systemic disease, we decided to determine serum XO levels of PEX. In this study, we found that serum levels of XO in subjects with PEX were not different than a normal group (Table 3), although higher serum XO in PEX was reported by Yagci et al. [4]. We used a colorimetric method for quantification of serum and aqueous XO, while Yagci used a spectrophotometric method [23]. Sex was shown to affect XO, although it is still controversial [24–27]. One of the possible reasons for the contradictory result can be the gender distribution among test groups which was not stated by Yagci et al. [4]. In our study, distribution of sex in the PEX and normal groups was the same (Table 2). Another reason can be the lack of clinical classification of PEX (i.e., mild, severe) [28] in both studies [4]. Intraocular secretion of PEX material is closely related to aqueous circulation and, therefore, is influenced by substances in the aqueous [29]. Henceforth, examination of aqueous humor composition in patients with PEX may identify important pathogenetic factors related to this disorder [30]. For this reason, we examined aqueous XO levels of PEX which, as far as we know, was not studied before, and compared them with a normal group. We found significantly higher aqueous XO levels in PEX (Table 3). Because PEX was shown to be associated with anterior chamber hypoxia [14], which accelerates the conversion of XDH to XO [31], higher XO levels in PEX resulted. The eye is more susceptible to oxidative stress through light exposure, radiation, a high level of O2 consumption, continuous exposure to environmental chemicals, and
atmospheric oxygen [23]. Moreover, total antioxidant capacity in serum is higher than in aqueous [32]. Additionally, manifestations of PEX are predominantly in the anterior segment of the eye. These additional factors can be the cause of higher XO levels of PEX in the aqueous despite similar XO levels in serum. Increased XO levels in PEX point out two extra possibilities. The first one is that PEX results in an increased XO production in the anterior chamber, which was reported for some of the antioxidant defence systems in the anterior lens capsule in PEX [21]. Like ascorbate [33], active selective secretion of XO from non-pigmented epithelium of the ciliary body to the anterior chamber is the second possibility. In active secretion, aqueous levels must be higher than serum levels. In our study, serum XO levels were 3–4 fold higher than aqueous levels which eliminates this possibility. In addition to these results, the lack of significant correlation of serum and aqueous levels (p=0.103) supports a selective production of XO in the anterior chamber in PEX. mRNA expression of the lens epithelial cells in PEX was found to be less than the normal group, although there were higher XO levels in the aqueous (Table 3). If the source of XO is not the anterior lens epithelial cells then the source of increased XO levels in PEX should be the other ocular structures related to the anterior/posterior chamber. Supporting this, gene expression of the lens was reported to be very distinct from all other compartments of the eye (i.e., cornea, iris, retina) [34]. Moreover, PEX represented gene expressions different from the normal group in iris, lens epithelium and ciliary process in different patients [35]. The other possible reason of decreased mRNA expression of anterior lens epithelial cells in this study is the inhibitor effect of high XO levels in the aqueous. Blood urea nitrogen levels represent the functions of the liver and kidneys. Urea is produced in the liver and excreted to urine by the kidneys. In our study, BUN levels were significantly lower in subjects with PEX, although all patients were within normal limits (Table 2). Lower levels of BUN mean lower production in the liver or higher excretion from the
Fig. 2 Agarose gel presentation for XO mRNA expression. Lane 1: 100 bp ladder. Lane 2 (st6): Standard 6 for XO gene indicates the positive control. Lane 3–7 (H3, H6, H8, H9, H10): Subjects without
XO mRNA expression in PEX group. Lane 8–12 (C1, C3, C4, C5, C6): Represents the control group with XO mRNA expression. Lane 13 (−k): Negative control (no added cDNA)
Aqueous (IU/mL) Serum (IU/mL) mRNA expression
Normals Mean±SD
Pseudoexfoliation Mean±SD
p
65.5±54.3 207.0±86.1 1±0.19
130.5±117.4 240.6±114.1 0.34±0.40
0.028a 0.310 0.01a
SD standard deviation a
Statistically significant
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kidneys. Gonen et al. [36] reported that renal functions were not affected in PEX. Additionally, they did not find any differences in the urea of patients with PEX [36]. One of the limitations of this study is the limited study population. Another limitation is the lack of analysis of other ocular structures due to ethical considerations. In conclusion, XO can play a role in ocular manifestations of PEX. Higher aqueous levels of XO in PEX point out increased oxidative stress in the anterior chamber. Decreased mRNA expression in anterior lens epithelial cells indicates that anterior lens epithelial cells do not have a remarkable contribution to aqueous XO production. Higher aqueous XO levels lacking a correlation with serum XO levels of both PEX and normal groups suggests higher oxidative stress in the aqueous of PEX patients. Further studies are needed to understand the precise role of XO in the pathogenesis of PEX.
Disclosures None. Conflict of interest All authors certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.
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1167 form binding protein in pseudoexfoliation syndrome. Exp Eye Res 73(6):765–780. doi:10.1006/exer.2001.1084 31. Doehner W, Anker SD (2005) Uric acid in chronic heart failure. Semin Nephrol 25(1):61–66 32. Mancino R, Di Pierro D, Varesi C, Cerulli A, Feraco A, Cedrone C, Pinazo-Duran MD, Coletta M, Nucci C (2011) Lipid peroxidation and total antioxidant capacity in vitreous, aqueous humor, and blood samples from patients with diabetic retinopathy. Mol Vis 17:1298–1304 33. Kinsey VE (1947) Transfer of ascorbic acid and related compounds across the blood-aqueous barrier. Am J Ophthalmol 30(10):1262– 1266 34. Diehn JJ, Diehn M, Marmor MF, Brown PO (2005) Differential gene expression in anatomical compartments of the human eye. Genome Biol 6(9):R74. doi:10.1186/gb-2005-6-9-r74 35. Zenkel M, Poschl E, von der Mark K, Hofmann-Rummelt C, Naumann GO, Kruse FE, Schlotzer-Schrehardt U (2005) Differential gene expression in pseudoexfoliation syndrome. Invest Ophthalmol Vis Sci 46(10):3742–3752. doi:10.1167/iovs. 05-0249 36. Gonen T, Gonen KA, Guzel S (2014) What is the effect of pseudoexfoliation syndrome on renal function in patients without glaucoma? Curr Eye Res 39(2):188–193. doi:10.3109/02713683. 2013.834940
GLAUCOMA
Serum and aqueous xanthine oxidase levels, and mRNA expression in anterior lens epithelial cells in pseudoexfoliation Huseyin Simavli 1 & Mehmet Tosun 2 & Yasin Y. Bucak 3 & Mesut Erdurmus 4 & Zeynep Ocak 5 & Halil I. Onder 6 & Muradiye Acar 7
Received: 9 October 2014 / Revised: 18 April 2015 / Accepted: 29 April 2015 / Published online: 10 May 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Purpose The aim of this study was to determine serum and aqueous xanthine oxidase (XO) levels, and mRNA expression in anterior lens epithelial cells in pseudoexfoliation (PEX). Methods In this prospective study, serum, aqueous and anterior lens capsules were taken from 21 patients with PEX and 23 normal subjects who had undergone routine cataract surgery. Serum and aqueous XO levels were analyzed using the colorimetric method. mRNA expression of XO in anterior lens epithelial cells was evaluated using reverse transcription polymerase chain reaction analysis. Results Serum XO levels (means±standard deviations) were 207.0±86.1 IU/mL and 240.6±114.1 IU/mL in the normal and PEX groups, respectively (p=0.310). Aqueous XO levels (means±standard deviations) were 65.5±54.3 IU/mL in the normal group and 130.5±117.4 IU/mL in the PEX group (p = 0.028). There was a 2.9 fold decrease in mRNA
* Huseyin Simavli [email protected] 1
Department of Ophthalmology, Pamukkale University School of Medicine, Denizli 20700, Turkey
2
Department of Biochemistry, Faculty of Medicine, Abant Izzet Baysal University, Bolu 14080, Turkey
3
Eye Clinic, Bolu Izzet Baysal State Hospital, Bolu 14000, Turkey
4
Faculty of Medicine, Department of Ophthalmology, Hacettepe University, Ankara 06100, Turkey
5
Department of Genetics, Faculty of Medicine, Abant Izzet Baysal University, Bolu 14080, Turkey
6
Faculty of Medicine, Department of Ophthalmology, Duzce University, Duzce 80100, Turkey
7
School of Medicine, Department of Genetics, Turgut Özal University, Ankara 06030, Turkey
expression in anterior lens epithelial cells of PEX, which is significantly lower than the normal group (p=0.01). Conclusions Higher aqueous XO levels lacking associated different serum XO suggests higher oxidative stress in the aqueous. Higher aqueous XO levels in PEX with decreased mRNA expression in anterior lens epithelial cells indicate possible overexpression of XO in other structures related to the aqueous. Keywords Pseudoexfoliation . Xanthine oxidase . Oxidative stress . Anterior lens capsule . Aqueous humor . mRNA expression
Introduction Worldwide, pseudoexfoliation (PEX) is the most common cause of secondary open-angle glaucoma, which was first reported by the Finnish ophthalmologist, Lindberg [1], in 1917. PEX is an important risk factor for intraocular surgery. The most common clinical manifestation of PEX is the progressive accumulation of fibrillar extracellular material in the anterior chamber and especially on the anterior lens capsule. In addition to its occurrence within the eye, fibrillary material is found in different parts of the body, including the heart, lungs, liver and spleen [2], which suggests PEX is an ocular manifestation of a systemic disease. Although the pathogenesis of PEX, an age-related disorder, has not been fully clarified, there is increasing evidence that oxidative stress plays an important role in the pathogenesis of PEX [3–5]. There is an intricate balance between oxidative stress and antioxidants in the human body. The balance between them plays an important role in aging, cardiovascular diseases and eye diseases such as age-related macular degeneration, cataracts, glaucoma and uveitis [6–10].
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Xanthine oxidoreductase (XOR), an enzyme in purine metabolism, is transcripted as a single gene product in the xanthine dehidrogenase (XDH) form which is mostly found in the liver and intestine [11]. During inflammatory conditions and hypoxia [12, 13], XDH is converted to xanthine oxidase (XO), which catalyzes two consecutive reactions at the end of purine metabolism, from which the end product is uric acid with superoxide (O2−) and hydrogen peroxide (H2O2). XO, which can serve as an important source of reactive oxygen species (ROS), was shown to be decreased in the serum of patients with PEX [4]. Although anterior chamber hypoxia was reported in PEX [14], aqueous levels of XO have not yet been reported. One of the common points of XO and PEX is endothelial dysfunction. XO causes endothelial dysfunction via superoxide production [15] and there is evidence that suggests systemic endothelial dysfunction in PEX [16, 17]. Interleukin 6 (IL-6) is another common point. IL-6, which has been shown to upregulate transcription of XOR [18], was shown to be increased in PEX [19]. Although serum XO levels in PEX were shown to be increased [4], XO levels and mRNA expression in the aqueous and anterior lens capsule, where the clinical manifestations of PEX are mostly represented, have not yet been studied. In this study, we analyzed serum and aqueous levels of XO, and mRNA expression in the anterior lens capsule in order to identify the possible role of XO, an ROS-producing enzyme, in PEX, which can be a step in understanding the pathogenesis of the syndrome.
Materials and methods Study population This prospective study was conducted according to the Helsinki Declaration and approved by the local institutional ethics committee. Written informed consent was obtained from all participants. All of the subjects were recruited from among patients who had undergone cataract surgery. Pre-operative tests and measurements including blood tests, posterioranterior chest films, electrocardiography and arterial blood pressure were evaluated by an internist. Blood tests included glucose, blood urea nitrogen (BUN), creatinin, aspartate aminotransferase, alanin amino transferase, complete blood count including white blood cell count, hemoglobin, hemotocrit, and platelet count, and coagulation tests including protrombin time, international normalized ratio, and active parsiel tromboplastin time. Additionally, body temperature, arterial blood pressure, respiration rate, and heart rate were monitored three times a day during hospitalization. Patients with abnormal blood tests, chest films or electrocardiograph, or with a
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history of systemic disease or drug use, were not included in the study. All subjects underwent a detailed ophthalmologic examination that included ocular history, visual acuity testing, refraction, Goldmann applanation tonometry, slit-lamp biomicroscopy, and dilated ophthalmoscopy. Intraocular lens calculations were performed with B-Scan-Cine Scan (Quantel Medical, Cedex, France) in all patients. PEX was defined during dilated ophthalmoscopy as the presence of white-grey pseudoexfoliation material on the anterior lens capsule, iris, pupillary border, corneal endothelium and even the anterior chamber. Subjects with systemic or ocular disease other than cataract, or those with a history of ocular surgery or trauma were excluded from the study. Additionally, patients with any other signs implying exfoliation (e.g., signs of pigment liberation and deposition throughout the anterior segment, or transillumination defects in the pupil margin) were also excluded from the study. The control group consisted of healthy subjects without PEX who had undergone routine cataract surgery. Sample collection Venous blood samples were obtained from the antecubital vein just before the start of phacoemulsification in the operating room. They were centrifuged within 15 min of collection, at 2.750 g for 10 min, and the supernatant serum was then transferred into polypropylene tubes. Local anesthesia was performed in a subconjunctival fashion in all subjects beforehand using 0.5 cc of Jetokain® (Lidokain HCl 20 mg/ml, Epinefrin HCI 0.0125 mg/ml, Adeka, Samsun, Turkey). A side port was made with a 27gauge syringe and a small amount of aqueous humour (0.1– 0.2 ml) was aspirated. A balanced salt solution (BSS) was used to form the anterior chamber if needed. The port then was widened by a 20-gauge MVR knife. Another port was made from the opposite direction of the first one. A clear corneal incision was fashioned at the 11-o’clock meridian. Sterile air was injected into the anterior chamber through the side port incision. A volume of 0.1 ml of 0.1 % trypan blue (Vision Blue, Dorc International, Netherlands) was injected under the air bubble over the anterior capsule with a 27gauge cannula in order to improve visualization of the capsule. The anterior chamber was deepened with air to allow even contact of the dye with the lens capsule and then washed with balanced salt solution after waiting for 10 s. High viscosity viscoelastic, Healon GV® (Abbott Medical Optics Inc, Santa Ana, CA, USA), was injected into the anterior chamber. Capsulorhexis was initiated with a bent 26-gauge needle and a capsulorhexis forceps was used for completing a continuous curvilinear capsulorhexis. The anterior lens capsule and aqueous humor samples were transferred to different polypropylene tubes marked with the patients’ barcodes.
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All the materials, aqueous, serum and capsule, were stored at −80 °C until the assays were determined. XO enzyme activity in the aqueous and serum was determined using the colorimetric method BioVision (BioVision Inc., CA, USA) according to the manufacturer’s protocol. Genetic analysis Total RNA isolation Total RNA was extracted with TRIzol (Ambion/RNA by Life Technologies, Carlsbad, CA, USA) according to the previously reported technique [20]. One microgram of RNA was reverse transcribed with reverse transcriptase (Thermo Scientific, Waltham, MA, USA) with oligod (T) primers according to the manufacturer’s instructions (Table 1). Mouse β-actin was amplified as a control for the PCR. Samples lacking reverse transcriptase were amplified as a control for genomic DNA contamination. Real-time PCR (B-actin) Real-time PCR was performed on cDNA samples obtained (Thermo Scientific Revert Aid First Strand cDNA Synthesis Kit) as described in the previous report [20]. The PCR mixture consisted of SYBR Green PCR Master Mix, which included DNA polymerase, SYBR Green I Dye, dNTPs, PCR buffer, forward and reverse primers and cDNA of samples in a total volume of 50 μl/mL. The amplification of a housekeeping gene, β-Actin, was used for normalizing the efficiency of cDNA synthesis and the amount of RNA applied (Fig. 1). PCR was performed with initial denaturation at 95 °C for 5 min, followed by amplification for 38 cycles, each cycle consisting of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, polymerization at 72 °C for 1 min and, the last stage, polymerization at 72 °C for 5 min. Real-time PCR (xantine oxidase) Real-time PCR was performed on cDNA samples obtained (Thermo Scientific Revert Aid First Strand cDNA Synthesis Kit) as described in the previous report [20]. The PCR mixture consisted of SYBR Green PCR Master Mix, which included DNA polymerase, SYBR Green I Dye, dNTPs, PCR buffer, forward and reverse primers and cDNA of samples in a total Table 1 The forward and reverse primers used in the real-time PCR analyses of the XO and β-actin genes XO β-actin gene
Forward Reverse Forward Reverse
5′-GACCTCAACCCCGTGTTCAT-3′ 5′-AAAGCTGCCTCTGAGTGGTC-3′ 5′TTCCTGGGCATGGAGTCCT-3′ 5′-AGGAGGAGCAATGATCTTGATC-3′
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volume of 50 μl/mL. The amplification of a XO gene was used for normalizing the efficiency of cDNA synthesis and the amount of RNA applied (Fig. 1). PCR was performed with initial denaturation at 95 °C for 15 min, followed by amplification for 50 cycles, each cycle consisting of denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s, polymerization at 72 °C for 30 s and, the last stage, polymerization at 72 °C for 5 min. The data of mRNA expression of XO were evaluated by the relative standard curve method, which measures the expression level of the target gene normalized to a ß-actin reference gene; the same primer pairs used for the standard PCR were utilized for the real-time PCR. The arithmetic mean of the relative expression levels of XO gene in each group of patients and controls were calculated. Statistical analysis All analyses were performed using the SPSS for Windows V.11.0 software package (SPSS Inc., Chicago, Illinois). Continuous variables were presented as mean±standard deviation (SD). Nominal variables were presented as a percentage. Differences in measured parameters between the two groups were analyzed with an independent samples t-test. In order to determine the correlation between serum and aqueous XO levels, Pearson’s correlation coefficient was used. The differences were considered significant when the probability was less than 0.05.
Results A total of 44 subjects were included to the study; 21 of them were in the PEX group, 23 of them were in the normal group. The mean visual acuity was 0.84±0.37 LogMar in the PEX group and 1.02±0.50 LogMar in the control group. The mean intraocular pressure (IOP) was 17.1±4.0 mmHg in the PEX group and 15.3±3.7 mmHg in the control group. The groups were statistically not different according to the preoperative visual acuity and IOP measurements (p=0.183, p=0.123, respectively). Eight of 21 subjects (38 %) had poor pupil dilatation in the PEX group. None of the patients in either group had phacodonesis. Demographics and preoperative blood tests of the study are shown in Table 2. There was no difference among PEX and normal groups in terms of age and sex (p=0.765, 0.763, respectively). All of the PEX group and normal group blood tests were in the reference range. Blood test results did not differ, with the exception of BUN. BUN levels of subjects with and without PEX were 34.1±7.7 mg/dL and 39.4±6.2 mg/dL, respectively, which is statistically significant (p=0.04). The mean serum levels of XO were 207.0±86.1 (IU/mL) in the normal group and 240.6±114.1 (IU/mL) in the PEX
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Fig. 1 Amplification plots of XO mRNA (a), housekeeping β-actin mRNA (c). Serial dilutions of XO (b) and β-actin cDNA (d) plasmids were amplified using real-time PCR. For each dilution, the fluorescence is
plotted against the cycle number. Relative input (log concentration) and cycle numbers of each dilution are given
group. XO activity in serum was not significantly different in the PEX group as compared to the control group (p=0.310). The mean aqueous levels of XO were 65.5±54.3 (IU/mL) in the normal group and 130.5±117.4 (IU/mL) in the PEX group. The activity of XO in the aqueous was significantly
higher in the PEX group than in the control group (p=0.028). There was a low negative correlation between serum and aqueous levels of XO which is not statistically significant (r=−0.269, p=0.103). mRNA expression levels of XO in anterior lens epithelial cells of PEX was found to be decreased 2.9 fold as compared to that of the normal group (p=0.01) (Table 3). An example of five subjects with PEX representing no XO mRNA expression in agarose is shown in Fig. 2.
Table 2 Demographics and blood test results of the normal and pseudoexfoliation population
Age (years) Sex (male) (42.9 %) Glucose (mg/ dL) BUN (mg/dL) Creatinine (mg/ dL) AST (U/L) ALT (U/L) WBC (K/uL) HGB (g/dL) HCT (%) PLT (K/uL) APTT (seconds) PT (seconds) INR
Reference range
Normals Pseudoexfoliation p Mean±SD Mean±SD
n/a n/a
74.3±7.8 11
75.1±7.0 (47.8 %)
0.765 9
0.763 60–109
97.1±11.6 93.4±9.6
0.329
19–44 0.1–1.2
39.4±6.2 34.1±7.7 0.87±0.13 0.79±0.18
0.04 0.143
1–40 1–40 4.5–11.0 11.5–17.5 37.0–53.0 140–400 20–36 10–14 0.8–1.2
17.5±3.6 12.6±4.7 6.8±1.2 13.5±1.1 40.0±2.9 249±67 21.9±11.5 12.3±0.4 1.03±0.08
0.187 0.262 0.148 0.875 0.424 0.405 0.131 0.277 0.897
19.9±5.7 14.7±5.3 6.2±1.2 13.4±1.1 40.9±3.4 295±121 27.2±7.5 12.9±0.9 1.04±0.09
SD standard deviation, n/a not applicable, BUN blood urea nitrogen, AST aspartate aminotransferase, ALT alanine aminotransferase, WBC white blood cell, HGB hemoglobin, HCT hemotocrit, PLT platelet, APTT active parsiel prothrombin time, PT prothrombin time, INR international normalized ratio
Discussion PEX is an elastotic process of the extracellular matrix characterised by excessive production and progressive accumulation of a grey-white fibrillary material deposited in the eye, especially on the anterior lens capsule. Besides its occurrence within the eye, exfoliative fibrillopathy has been reported in different parts of the body, such as the heart, lungs, liver and spleen, which suggests PEX is an ocular manifestation of a systemic disease [2]. There is growing evidence that suggests the oxidativeantioxidative balance is disturbed in PEX, not only in the anterior segment but also in the whole body [3–5, 21, 22], and that the resulting oxidative stress, which can be defined as elevated ROS levels over the range of physiologic values, constitutes a major mechanism involved in the pathophysiology of PEX. ROS are chemically reactive molecules containing oxygen that are generated intracellularly or via exogen sources through a variety of processes, including normal aerobic metabolism and various signal transduction pathways. XO, which catalyzes the rate-limiting steps of purine degradation, is a critical source of ROS [12]. Because PEX is
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Table 3 Serum and aqueous levels and mRNA expression of xanthine oxidase in pseudoexfoliation
known as a systemic disease, we decided to determine serum XO levels of PEX. In this study, we found that serum levels of XO in subjects with PEX were not different than a normal group (Table 3), although higher serum XO in PEX was reported by Yagci et al. [4]. We used a colorimetric method for quantification of serum and aqueous XO, while Yagci used a spectrophotometric method [23]. Sex was shown to affect XO, although it is still controversial [24–27]. One of the possible reasons for the contradictory result can be the gender distribution among test groups which was not stated by Yagci et al. [4]. In our study, distribution of sex in the PEX and normal groups was the same (Table 2). Another reason can be the lack of clinical classification of PEX (i.e., mild, severe) [28] in both studies [4]. Intraocular secretion of PEX material is closely related to aqueous circulation and, therefore, is influenced by substances in the aqueous [29]. Henceforth, examination of aqueous humor composition in patients with PEX may identify important pathogenetic factors related to this disorder [30]. For this reason, we examined aqueous XO levels of PEX which, as far as we know, was not studied before, and compared them with a normal group. We found significantly higher aqueous XO levels in PEX (Table 3). Because PEX was shown to be associated with anterior chamber hypoxia [14], which accelerates the conversion of XDH to XO [31], higher XO levels in PEX resulted. The eye is more susceptible to oxidative stress through light exposure, radiation, a high level of O2 consumption, continuous exposure to environmental chemicals, and
atmospheric oxygen [23]. Moreover, total antioxidant capacity in serum is higher than in aqueous [32]. Additionally, manifestations of PEX are predominantly in the anterior segment of the eye. These additional factors can be the cause of higher XO levels of PEX in the aqueous despite similar XO levels in serum. Increased XO levels in PEX point out two extra possibilities. The first one is that PEX results in an increased XO production in the anterior chamber, which was reported for some of the antioxidant defence systems in the anterior lens capsule in PEX [21]. Like ascorbate [33], active selective secretion of XO from non-pigmented epithelium of the ciliary body to the anterior chamber is the second possibility. In active secretion, aqueous levels must be higher than serum levels. In our study, serum XO levels were 3–4 fold higher than aqueous levels which eliminates this possibility. In addition to these results, the lack of significant correlation of serum and aqueous levels (p=0.103) supports a selective production of XO in the anterior chamber in PEX. mRNA expression of the lens epithelial cells in PEX was found to be less than the normal group, although there were higher XO levels in the aqueous (Table 3). If the source of XO is not the anterior lens epithelial cells then the source of increased XO levels in PEX should be the other ocular structures related to the anterior/posterior chamber. Supporting this, gene expression of the lens was reported to be very distinct from all other compartments of the eye (i.e., cornea, iris, retina) [34]. Moreover, PEX represented gene expressions different from the normal group in iris, lens epithelium and ciliary process in different patients [35]. The other possible reason of decreased mRNA expression of anterior lens epithelial cells in this study is the inhibitor effect of high XO levels in the aqueous. Blood urea nitrogen levels represent the functions of the liver and kidneys. Urea is produced in the liver and excreted to urine by the kidneys. In our study, BUN levels were significantly lower in subjects with PEX, although all patients were within normal limits (Table 2). Lower levels of BUN mean lower production in the liver or higher excretion from the
Fig. 2 Agarose gel presentation for XO mRNA expression. Lane 1: 100 bp ladder. Lane 2 (st6): Standard 6 for XO gene indicates the positive control. Lane 3–7 (H3, H6, H8, H9, H10): Subjects without
XO mRNA expression in PEX group. Lane 8–12 (C1, C3, C4, C5, C6): Represents the control group with XO mRNA expression. Lane 13 (−k): Negative control (no added cDNA)
Aqueous (IU/mL) Serum (IU/mL) mRNA expression
Normals Mean±SD
Pseudoexfoliation Mean±SD
p
65.5±54.3 207.0±86.1 1±0.19
130.5±117.4 240.6±114.1 0.34±0.40
0.028a 0.310 0.01a
SD standard deviation a
Statistically significant
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kidneys. Gonen et al. [36] reported that renal functions were not affected in PEX. Additionally, they did not find any differences in the urea of patients with PEX [36]. One of the limitations of this study is the limited study population. Another limitation is the lack of analysis of other ocular structures due to ethical considerations. In conclusion, XO can play a role in ocular manifestations of PEX. Higher aqueous levels of XO in PEX point out increased oxidative stress in the anterior chamber. Decreased mRNA expression in anterior lens epithelial cells indicates that anterior lens epithelial cells do not have a remarkable contribution to aqueous XO production. Higher aqueous XO levels lacking a correlation with serum XO levels of both PEX and normal groups suggests higher oxidative stress in the aqueous of PEX patients. Further studies are needed to understand the precise role of XO in the pathogenesis of PEX.
Disclosures None. Conflict of interest All authors certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.
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