Systemic Glutathione Level in Normal Tension Glaucoma

• Total Glutathione Levels [ Time Frame: Once in the morning. ] [ Designated as safety issue: No ]
  Subjects were instructed to fast from midnight to 8 AM on the morning of the test. All blood samples were obtained by a qualified registered nurse in the morning, between 8 and 10 AM.
  
  

Normal tension glaucoma is one of the most common cause of primary open angle glaucoma in Korea. Even though, we still do not know what the cause is. Only IOP-lowering drugs are the currently available therapeutic method. Glutathione is one of the mostly high concentrated intracellular antioxidants. We find apoptosis of neuronal cell in the mouse retina by systemic depletion of glutathione using buthionine sulfoximine. Glutathione is also known to be reduced in primary open angle glaucoma with high IOP. So we are planning to evaluate systemic glutathione level in normal tension glaucoma.

Blood samples will be obtained by venipuncture to the antecubital vein of normal tension glaucoma patients and age-matched normal controls. Five milliliters of blood will be collected in EDTA-treated tubes (to prevent oxidation). Thirty microliters of blood will be then transferred into centrifuge tubes, and will be added 33.3 microliter of 5-sulfosalicylic acid (SSA), 100mg/mL within 10 minutes from the blood collection.Each sample will be then diluted with 936.7 microliter sodium phosphate buffer (pH 7.5), and the content of each tube will be mixed rapidly in a centrifuge at 13,000 rpm for 5 minutes. A portion of supernatant (150 microliter) will be then collected into clean centrifuge tubes and immediately cooled at -70°C. To each well of a 96-well plate, 150 microliter of daily buffer (3 mg NADPH into 10 mL sodium phosphate buffer), 50microliter of DTNB solution, and 25 microliter of standards or samples will be added in quadruplicate, and the plate will be incubated at 37 °C for 3 minutes.Finally, 25 microliter GSSGR will be added to the previous mixture, and the plate will be read at 410 nm using a 96-well plate reader(UV spectrophotometer).