SELENIUM AND SELENOPROTEIN P1 LEVELS ARE RELATED TO PRIMARY OPEN-ANGLE GLAUCOMA / NIVOI SELENA I SELENOPROTEINA P1 POVEZANI SU SA PRIMARNIM GLAUKOMOM OTVORENOG UGLA

Summary Background Glaucoma is a highly prevalent eye disease related to optic nerve lesions and visual field defects. Primary Open-Angle Glaucoma (POAG) is a type of glaucoma that occurs frequently with unknown etiology. In this study, we investigated the serum levels of selenium, selenoprotein P1, glutathione, hemolysate glutathione peroxidase1 (GPx1) activity and aqueous humour selenium in POAG patients. Methods Ninety sex- and age-matched subjects (POAG patients; n=45 and, controls; n=45) with the controlled confounders (smoking, hypertension and alcohol beverages) were recruited on clinical histories and exams. The serum and aqueous humour selenium levels were measured using GFAAS technique. The serum selenoprotein P1 level was assayed with the ELISA method. The hemolysate GPx1 activity and serum reduced glutathione level were also measured using known colorimetric techniques. Results The serum selenium (P=0.01) and selenoprotein P1 (P<0.001) levels were significantly high in POAG patients. Furthermore, the aqueous humour selenium level was significantly high among patients as compared to controls (64.68±13.07 vs. 58.36±13.76 ng/mL, P=0.02). The results did not show a significant difference (P=0.36) in the hemolysate GPx1 activity between the groups. The cutoff points for intraocular pressure (IOP) and serum selenoprotein P1 parameters were estimated to be 39 mmHg (sensitivity 97.5%; 1-specificity 6.5%) and 188 mg/mL (sensitivity 93.5%; 1-specificity 14%), respectively. Kratak sadrzaj >Glaukom je veoma ~esto oboljenje oka koje je po - vezano sa lezijama na o~nom `ivcu i o{te}enjima vidnog polja. Primarni glaukom otvorenog ugla (PGOU) tip je glaukoma sa ~esto nepoznatom etiologijom. U ovoj studiji ispitivani su serumski nivoi selena, selenoproteina P1, glutationa, aktivnost hemolizata glutation-peroksidaze1 (GPx1) i selen u o~noj vodici kod pacijenata sa PGOU. Na osnovu klini~ke istorije i pregleda, obuhva}eno je 90 subjekata odgovaraju}eg pola i starosne dobi (pacijenti sa PGOU; n=45 i kontrolni subjekti; n=45) sa kontroli - sanim varijablama (pu{enje, hipertenzija i alkoholna pi}a). Ni voi selena u serumu i o~noj vodici izmereni su pomo}u tehnike GFAAS. Nivo selenoproteina P1 u serumu odre|en je me todom ELISA. Aktivnost hemolizata GPx1 i nivo redu - kovanog glutationa u serumu tako|e su mereni poznatim kolorimetrij skim tehnikama. Serumski nivoi selena (P=0,01) i selenoproteina P1 (P=0,001) bili su zna~ajno vi{i kod pacijenata sa PGOU. Uz to, nivo selena u o~noj vodici bio je zna~ajno vi{i kod pacijenata nego u kontrolnoj grupi (64,68±13,07 prema 58,36 ±13,76 ng/mL, P=0,02). Rezultati nisu otkrili zna~ajnu razliku (P=0,36) u aktivnosti hemolizata GPx1 izme|u grupa. Odre|ene su cutoff vrednosti za intraokularni pritisak (IOP) i parametre serumskog selenoproteina P1: 39 mmHg (ose - tljivost 97,5%; 1-specifi~nost 6,5%), odnosno 188 mg/mL (osetljivost 93,5%; 1-specifi~nost 14%).


Introduction
Glaucoma is an eye disease related to the optic nerve damage and visual field loss (1).It is the second leading cause of blindness after cataract.Based on the trabecular meshwork (TM) obstruction, glaucoma can clinically be observed in two forms, Open-Angle Glaucoma (POAG) and Primary Closed-angle glaucoma (PACG).Primary Open-Angle Glau coma (POAG) is a type that frequently occurs in the United States; however, a shift is considered in the demographic and geographic characteristics of the patients (2).The high prevalence of glaucoma is also reported in Asian countries (3).In PACG, the iris obstructs the trabecular meshwork in the angle of the eyes, whereas in POAG, the trabecular meshwork seems to be open and unobstructed by the iris (4).Although the POAG etiology is unknown, ganglion cell death is proposed to be due to high eye pressure, decreased blood flow to the optic nerve head, inflammatory events, and increased nitric oxide and glutamate levels (5,6).In addition to the above factors (7), other agents such as high selenium in bodily fluids, molecular genetics and some demographic parameters have also been suggested to be involved in glaucoma, but so far the evidence is inconclusive (8)(9)(10).
Selenium is present in body fluids (11) and is known as a functional trace element in the human lens (12).Some studies have reported the association between serum selenium level and glaucoma (13) and have shown that selenium overload within aqueous humour may cause a decrease in trabecular meshwork cell adhesion and activation of apoptotic signaling pathways (14,15).Based on these reports, we evaluated the levels of selenium, selenoprotein P1 and some antioxidant parameters to test the hypothesis which suggests POAG may be related to a high selenium level.

Subjects
The sex and age-matched subjects were recruited upon clinical exams.The POAG patients (n=45) were excluded for secondary glaucoma, congenital glaucoma and PACG and were candidates for trabeculectomy or phacoemulsification.Controls (n=45) had clinically normal optic nerves and intraocular pressure which did not contribute to POAG risk.The study was approved by the university ethics commit-tee and informed consent was ob tained from all subjects.

Samples
Two blood specimens (each 5 mL) were collected in the blood tubes.The clotted and EDTA blood tubes were centrifuged at 3000 g (15 min) and the serum and plasma were separated and stored at -80 ˚C.The remaining red pellets were washed three times with normal saline (NaCl, 0.9%) and were diluted with distillated water for preparation of erythrocyte hemolysate (1: 1 V/V).

Serum and aqueous humour selenium levels
The serum and aqueous humour selenium values were measured using Graphite Furnace atomic absorption spectrometry (GFAAS).Five mL HNO 3 (16 mol/L) was added to 2.5 mL serum and then slowly heated up to 140 °C (20 min).The above stage was repeated with an acidic solution containing 2.5 mL H 2 SO 4 (18 mol/L) and 1 mL HClO 4 (11.6 mol/L).Then, 5 mL HCl (5 mol/L) was added after cooling and 10 mL from it was injected into a Parker Elmer ato mic spectrometer (Mercury Hydride system).The aqueous humour samples were directly injected into the system.

Selenoprotein P1
The serum selenoprotein P1 concentration was measured with the ELISA method (Antibodies Online, Cusabio Biotech Co).According to the protocol, 5 mL sera were diluted and incubated with reagents.Then, the serum selenoprotein P1 concentration was calculated on the standard curve (Boot strapping, 100).

Glutathione concentration (nmol/mL) = (A/molar extinction coefficient (GSH))×Dilution coefficient
Conclusions: The results also showed the selenium and selenoprotein P1 levels relate to intraocular pressure.We suggested serum selenoprotein P1 and intraocular pressure are more sensitive factors for the primary screening of POAG.

Statistics
Data was analyzed using a statistical software package (SPSS 18.0, Chicago).The parameters were evaluated using Student-t and Chi square tests between control and patient subjects.ROC curve was also used to identify the cutoff values.Linear regression analyses were performed and tested between some parameters.P value less than 0.05 was considered to be significant.

Results
The age (P=0.2) and sex (P=0.18)parameters and some confounders such as systolic blood pressure (P=0.76),diastolic blood pressure (P=0.59) and smoking (P=0.82)showed no significant differences between control and patient subjects.However, significant differences were observed in intraocular pressure (IOP) and central corneal thickness (P<0.0001)(Table I).
J Med Biochem 2014; 33 (2) 145 Table I Characteristics of the study population.Table II shows that the selenium level increases significantly in the aqueous humour of POAG patients (P=0.02).Furthermore, the serum selenium (P=0.01) and selenoprotein P1 (P<0.001) levels, similar to the aqueous humour selenium, increased significantly among patients.In contrast, the results did not show significant differences for the glutathione peroxidase1 (GPx1) activity (P=0.36) and the total antioxidant capacity (TAC) (P=0.84).The serum glutathione concentration was slightly in creased in patients (P=0.045)(Table II).

Discussion
In this study, the levels of selenium, selenoprotein P1, glutathione and GPx1 activity were evaluated on the basis of a long-term selenium supplementation study (Nutritional Prevention of Cancer; NPC) that showed a potential association between selenium and incidence of glaucoma.In agreement with the NPC results, our findings revealed that the body's selenium level may be one of the causal factors in the development of POAG.Some authors, however, did not find significant associations between selenium and glaucoma, but they suggested a higher risk for incidence of glaucoma as selenium level elevates in the aqueous humour (17).In spite of laboratorial and biological variations, the results showed an association between POAG and body's selenium.The study also revealed a significant association between POAG and the serum selenoprotein P1 level, so that the ROC curve showed that it may be a more sensitive factor than the serum and aqueous humour selenium levels.These results pointed out that the continuous diffusion of serum selenium into aqueous humour leads to selenium overload within the MT cells.The increase of cellular selenium higher than the normal range could change the oxidant balance within the MT cells and may trigger the apoptotic signals (14).Coneley et al. showed that the selenium overload within TM cells may affect outflow resistance, routine phagocytic function and extracellular matrix turnover (18).The oxidant balance is inversely related to the antioxidant enzyme activities because some oxidant and antioxidant substances are known as the enzyme substrates or products.Thus, cellular involvement of selenium without the consideration of selenoenzymes may describe the TAC results between control and patient subjects.
Despite the increase in the body's selenium concentration, the GPx1 activity was not high in POAG patients.In agreement with other studies (19,20), which showed maximum GPx1 activity occurrs with selenium values higher than 100 ng/mL, we found that the serum selenium concentrations were higher than 213 ng/mL.
The results also showed that the intraocular pres sure higher than 39 mmHg and serum selenoprotein P1 levels higher than 188 mg/mL can be considered as cutoff points in the primary screening of POAG; however, these results must be confirmed in other studies.

Conclusion
We found the aqueous humour selenium level is correlated to the serum selenium concentration.The intraocular hypertension also had a correlation with the selenium and selenoprotein P1 levels, so that the serum selenoprotein P1 level and intraocular hypertension are suggested as more sensitive factors for the primary screening of POAG.

146Figure 1
Figure 1 Linear correlation (A) between aqueous humour selenium and serum selenium, (B) between aqueous humour selenium and intraocular pressure.

Table II
Biochemical parameters.