Pseudoexfoliation (PEX) syndrome is a common age-related disorder of the extracellular matrix that is frequently associated with severe chronic secondary open-angle glaucoma and cataract. Pseudoexfoliation syndrome may affect up to 30% of people older than 60 years of age in a worldwide distribution and is biomicroscopically diagnosed by abnormal fibrillar deposits on ocular structures that line the aqueous-bathed surfaces of the anterior segment. Apart from the long-known intraocular manifestations and conjunctival involvement, PEX syndrome has been shown to be a systemic process. Nevertheless, the cause and pathogenesis of PEX syndrome are still not known precisely, as well as its ocular and systemic manifestations.

Both because the aqueous humor can be easily obtained at intraocular surgery and all ocular tissues involved in this syndrome are bathed by the aqueous humor and should therefore be influenced by the factors contained therein, many experimental studies have been done by analyzing the aqueous humor composition of PEX patients. Many other studies analyzing the serum of PEX patients have been performed depending on the evidences about systemic involvement of the syndrome. Analyzing either the serum or the aqueous humor of cases with PEX syndrome, the studies have showed an increase in oxidative stress markers such as malondialdehyde and 8-isoprostaglandin-F2α, and a concomitant decrease in antioxidative protective factors such as ascorbic acid (the most effective free radical scavenger in the eye), catalase, and glutathione peroxidase (GSHPx), suggesting a faulty antioxidative defense system and increased oxidative stress in PEX. So, there is increasing evidence that oxidative stress constitutes a major mechanism involved in the pathobiology of PEX syndrome.

Being an essential trace element, selenium (Se) functions as a part of proteins known as selenoproteins. Selenium as selenocysteine is essential for the biologic functions of these selenoproteins. Through these selenoproteins, Se functions in the defensive mechanism for oxidative stress, in the regulation of thyroid hormone activity, and in the regulation of the redox status of vitamin C and other molecules. In several of its roles, Se functions as a dietary antioxidant and thus has been studied for its possible role in chronic diseases and glaucoma.

Specifically, the 4 known selenium-dependent GSHPx designated as GSHPx 1 to 4 defend against oxidative stress, as do the selenoproteins P and W. Thyroid hormone metabolism is regulated by 3 selenium-dependent iodothyronine deiodinases, while 3 identified thioredoxin reductases (TR) function in the regeneration of ascorbic acid from its oxidized metabolites and for the reduction of intramolecular disulfide bonds. Previously, lowered content of Se in serum and lens of cataract patients with PEX compared to ones without PEX has been reported in a study. The authors have suggested a role of increased oxidative stress in the development of PEX syndrome.

In this study, analyzing the levels of Se in aqueous humor, conjunctival specimens, and serum of PEX cases and control subjects, we have planned to investigate the role of Se in the development and pathogenesis of PEX syndrome.

Materials and Methods

In this prospective, institutional study, we enrolled consecutive patients with or without PEX syndrome and senile cataract in whom cataract surgery was indicated for visual rehabilitation. We excluded patients with other ophthalmic conditions and systemic pathologies that might have influenced the levels of Se, such as the ones having chronic illnesses, particular dietary habits, and treatment regimens including trace-element-containing preparation intake. All study groups were composed of normotensive (intraocular pressures lower than 21 mm Hg) cases. Patients included in the study were not using any topical medications. Patients with ophthalmic diseases other than cataract and PEX syndrome such as glaucoma, uveitis, age-related macular disease, and previous laser and intraocular surgery were not involved in the study. Having systemic diseases such as hypertension, cardiovascular diseases, cancer, and diabetes mellitus, which may affect the levels of Se, were exclusion criteria, as well.

The study group comprised 27 patients diagnosed as having cataracts and PEX syndrome during their routine ophthalmologic examination at our ophthalmology department, and the control group comprised 20 cataract patients without PEX. The 2 groups were age-matched. All patients underwent a comprehensive ophthalmic examination prior to surgery. Pseudoexfoliation syndrome and cataracts were diagnosed by slit-lamp examination after the pupils were dilated by pharmacologic agents. The diagnosis of PEX syndrome was made when fibrillogranular PEX material was seen on the anterior lens capsule.

In the operating room, serum of all cases was collected by a peripheral venous route before surgery. At the beginning of the routine phacoemulsification surgery, after entering into the anterior chamber through a limbal paracentesis, a small amount of aqueous humor (10–40 μL) was aspirated by using a 27-gauge needle on a tuberculin syringe, before making a clear corneal tunnel incision. Cataract surgery was performed under local anesthesia. When the surgery was completed, a piece of conjunctiva was excised from superior temporal conjunctiva under the upper lid. Serum, aqueous humor, and conjunctiva samples were stored at −70°C until biochemical analysis for measuring Se content.

Determination of Selenium Levels of Conjunctiva, Serum, and Aqueous Humor Samples

Conjunctival specimens were stored in acid-washed tubes. Following storage at −70°C, samples were dried at 104°C for 24 hours, weighed, ashed in a muffle furnace at 200°C for 12 hours, and dissolved in 5 mL nitric acid (65%) and 1 mL perchloric acid (70%). They were diluted with 0.1% nitric acid and the samples were again diluted with 1% Triton X-100 solution (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) at a ratio of 1:4 before reading.

Serum and aqueous humor samples were diluted to a ratio of 1:4 with 1% Triton X-100 solution before reading.

Selenium levels of all samples were measured by using atomic absorption spectrophotometer (SpectrAA 240FS; Varian, Melbourne, Australia) with a deuterium continuum background corrector and a graphite tube atomizer (GTA-96 Model) with autosample dispenser (both from Varian). Measurements were performed using a spectral bandwidth of 1.0 nm at 196.0 nm and a hollow cathode lamp. Hollow cathode lamps were operated at 12.5 mA. The method of standard addition was employed, using the sampler “automixing” mode. The temperature and sampler program are shown in Tables 1 and 2 .

TABLE 1

Suitable Temperature Programs for Selenium Determination in Conjunctiva, Serum, and Aqueous Humor by Graphite Furnace Atomic Absorption Spectrometry

Step No	Temperature (°C)	Time (sec)	Gas Flow (L/min)	Read Command
1	70	5.0	3	No
2	120	30.0	3	No
3	120	5.0	3	No
4	600	10.0	3	No
5	600	5.0	3	No
6	1200	15.0	3	No
7	1200	5.0	3	No
8	1200	2.0	0	No
9	2700	0.9	0	Yes
10	2700	2.0	0	Yes
11	2700	2.0	3	No

 

TABLE 2

Autosampler Program for the Method of Selenium Standard Addition for Calibration Curve

	Standard	Volume (uL) Sample	Blank	Modifier
Blank	—	—	15	20
Addition 1	2	5	8	20
Addition 2	4	5	6	20
Sample	—	5	10	20

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