GSH: A MARKER FOR OXIDATIVE STRESS IN HUMAN CELL CULTURES

GSH: A MARKER FOR OXIDATIVE STRESS IN HUMAN CELL CULTURES Gueli Maria Concetta Dipartimento di Biomedicina Sperimentale e Neuroscienze Cliniche (BioNEC), Università degli Studi di Palermo. Reactive oxygen species (ROS) play an important role in physiological processes, but when being in excess ROS react readily with proteins, lipids, carbohydrates, and nucleic acids, often inducing irreversible functional alterations or even complete destruction. In cells, under physiological conditions, the production and detoxification of ROS are more or less balanced. GSH functions as antioxidant and the oxidative conversion of GSH to GSSG is widely recognized as a reliable index of oxidative stress. Therefore great interest exists in the determination of GSH and GSSG in various biological tissues, organs, and cells. We propose a rapid, user-friendly, HPLC with fluorescence detection (HPLC-FD) method to quantify GSH and GSSG using human epatoma HA22T/VGH cells. Dubecco’s Eagle’s modified medium, TNBP, SBDF, GSH, Bradford reagent (Sigma). HA22T/VGH cells were grown in appropriate medium. The number of cells per flask was determined by Reverse Microscope to assess cell growth rate. After 7 days of incubations HA22T/VGH cells at 80-90% of confluency were washed with cold PBS, scraped in NaCl 0.9% and centrifuged. The pellet cells were resuspended in 1 ml Lysis buffer, sonicate and centrifuged at 13000 rpm for 15 min. Afterwards the supernatants were collected and used for HPLC assay. Briefly:100mL of HA22T/VGH cell estracts and TNBP reagent was incubated for 30 min at 4°C after which TCA was added. The clear supernatant was added to an eppendorf containing 1.55mol/L NaOH; 0.125mol/L borate buffer, pH 9.5, SBDF solution, then incubated at 60°C. Waters-HPLC system consisted of a 600E Pump, 474 fluorescence detector (FD) and Empower TM2 Software. Separation of the SBDF derivatized thiols was performed on a Spherisorb ODS2, 0.1mol/L acetic acid-acetate buffer, pH 4.0 as mf. The R.T. for GSH was 11.57 ± 0.01 min (means ± SD). Calibration curve for GSH was linear up to 100 mmol/L. Levels of tGSH, GSH, and GSSG levels in HA22T/VGH cells were (means ± SEM) 26.87 ± 0.02; 23.18 ± 0.01 and 3.69 ± 0.01 μmol/L, respectively and 33.37 ± 0.01; 28.79 ± 0.01 and 4.58 ± 0.01 nmol/mg prot., respectively. This HPLC-FD method developed in our laboratory is very useful for research purposes and for routine clinical use. Gueli MC (2000) IBTS 15, 167. 4° SIBIOC Interr. SORRENTO 9-11 Ottobre 2013