European Review for Medical and Pharmacological Sciences 2010; 14: 19-23 Effects of reduced glutathione on nitric oxide level, total antioxidant and oxidant capacity and adenosine deaminase activity O. ATAKISI1*, H.M. ERDOGAN2, E. ATAKISI1, M. CITIL2, A. KANICI3, O. MERHAN1, M. UZUN4 1Department of Biochemistry; 2Department of Internal Medicine, College of Veterinary Medicine, University of Kafkas, Kars (Turkey); 3Department of Pharmacology and Toxicology, College of Veterinary Medicine, University of Ankara, Ankara (Turkey); 4Canakkale Onsekiz Mart University, School of Health Science, Canakkale (Turkey) Abstract. – The aim of this study was to the formation of γ-glutamylcysteine from l-gluta- determine the effect of exogenous GSH, an im- mate and l-cysteine. In the second step, catalyzed portant antioxidant containing thiol group, on to- by GSH synthetase (EC 6.3.2.3, GS), glycine is tal antioxidant capacity (TAC) and total oxidant γ capacity (TOC), adenosine deaminase (ADA), a added to the C-terminal of -glutamylcysteine to significant indicator of cellular immunity, and ni- form GSH1,2. The key function of GSH molecule is tric oxide (NO) concentrations in rabbits. the maintenance of protein structure and function, Sixteen healthy New Zealand rabbits were di- the regulation of protein synthesis and degradation, vided into 2 groups as control and GSH-treated the maintenance of immune function, protection group. Animals in control group received single against oxidative damage, and detoxification of re- intraperitoneal (ip) administration of 0.9% saline. 3,4 Rabbits in GSH-treated group received reduced active chemicals . GSH molecule also plays role 3 L-glutathione (10 mg/kg) (ip). Blood samples were in immune function . taken from the marginal ear vein 0, 3, 6 and 12 Adenosine deaminase (ADA) is an essential hours after injection. Plasma TAC and NO levels enzyme of purine metabolism. ADA catalyzes were not statistically different between control and the irreversible hydrolytic deamination of adeno- GSH group at 0 and 12 hours. Plasma TAC level sine and 2-deoxyadenosine to inosine and 2-de- was found to be significantly higher (p<0.05) while 5,6 NO level was found to be significantly lower oxyinosine, respectively . ADA is necessary for (p<0.05) in GSH treated group when compared to lymphocyte proliferation and differentiation. control group at 3 and 6 hours. Plasma TOC and Therefore, it is considered as an important immu- ADA activity were not statistically different be- noenzyme marker of cell mediated immunity7,8. tween control and GSH group during experiment. Nitric oxide (NO) is an endogenous reactive In conclusion, exogenous GSH resulted in an free radical, produced from the amino acid L-argi- alteration of TAC and NO but not TOC and ADA, nine by the enzymatic action of nitric oxide syn- so exogenous GSH may be a valuable enhancer 9 of the antioxidant system. thase (NOS) . Although various NOS isoforms have been described, only three forms, inducible Key Words: NOS (iNOS), endothelial NOS (eNOS) and neu- Total antioxidant capacity, Total oxidant capacity, ronal NOS (nNOS), have been described in de- 10,11 Nitric oxide, Adenosine deaminase, Reduced glu- tail . NO is stabilized by carrier molecule like tathione, Rabbit. reduced thiol species that preserves its biological activity. Low molecular weight thiols such as cys- teine, GSH, and penicillamine are prime candi- dates for such carrier molecules, and they can form S-nitrosothiols on reaction with oxides of ni- Introduction trogen12. This molecule readily reacts in the pres- ence of NO to yield biologically active S-ni- Reduced glutathione (GSH) is synthesized by trosothiols that is more stable and potent than NO two sequential adenosine triphosphate (ATP)-de- itself. S-nitrosothiols are unstable in aqueous solu- pendent reactions. In the first reaction, γ-glutamyl- tion. For example, S-nitrosoglutathione (GS-NO) cysteine synthetase (EC 6.3.2.2, γ-GCS) catalyzes undergoes decomposition over hours, whereas S- Corresponding Author: Onur Atakisi, PhD; e-mail: [email protected] 19 O. Atakisi, H.M. Erdogan, E. Atakisi, M. Citil, A. Kanici, O. Merhan, M. Uzun nitrosocysteine has a half-life of less than 2 ments, Winooski, VT, USA) using commercial min13,14. GS-NO is a bioactive intermediary that kit (Rel Asssay, Gaziantep, Turkey) in plasma may regulate cellular functions and inhibits the samples. Antioxidants in the sample reduce dark sarcoplasmic reticulum bound creatine kinase3. blue-green colored 2,2′-azinobis (3-ethylben- Generally S-nitrosoglutathione (GS-NO) has been zothiazoline-6-sulfonic acid) diammonium salt found endogenously in neutrophils and human air- (ABTS) radical to colorless reduced ABTS form. ways at µM concentrations15. S-nitrosothiols may The change of absorbance at 660 nm is related play the same role in the mechanism of action of with total antioxidant level of the sample 19. Endothelium-Derived Relaxing Factor (EDRF) as NO3. Some Authors have assumed that the biolog- Determination of Total Oxidant Capacity ical effects of these compounds are due to the Plasma total oxidant capacities were deter- spontaneous release of NO. However, this hypoth- mined via spectrophotometer (PowerWave XS, esis is not fully supported yet16-18. BioTek, Instruments, Winooski, VT, USA) with This study therefore aimed at determining the commercial kit (Rel Asssay, Gaziantep, Turkey). effect of exogenous GSH, an important antioxi- Oxidants present in the sample oxidize the fer- dant containing thiol group, on total antioxidant rous ion-chelator complex to ferric ion. The oxi- capacity, total oxidant capacity, and adenosine dation reaction is prolonged by enhancer mole- deaminase a significant indicator of cellular im- cules, which are present in reaction medium. The munity and NO concentrations in rabbits. ferric ion makes a colored complex with choro- mogen in an acidic medium. The color intensity is related to the total oxidant molecules present in the sample at 530 nm. Trolox and hydrogen Materials and Methods peroxide standards were used for total antioxi- dant and total oxidant capacities19. Sixteen New Zealand rabbits (Laboratory Ani- mal Unit of the University of Kafkas, Kars, Turkey) Determination of Nitric Oxide Levels of both sexes, aged between 7 and 9 months were in Plasma used. The mean body weight was 2.39 ± 0.42 kg. Nitric oxide concentrations were determined They were kept in cages at room temperature (22- using a spectrophotometer (PowerWave XS, 25ºC) with a 12:12h light:dark cycle and fed a spe- BioTek, Instruments, Winooski, VT, USA) in cial pelleted rabbit diet (Bayramoglu Yem AS, plasma samples. Plasma samples were depro- Erzurum, Turkey) ad libitum. Animals were divided teinized with 10% zinc sulphate. Total NO (ni- into 2 groups as control and GSH-treated group. trate and nitrite) concentrations were determined Each group consisted of 8 animals and treated as colorimetrically by the acidic Griess reaction20. follows: animals in control group received a single intraperitoneal (ip) administration of 0.9% saline Determination of ADA. (Baxter, Mediflex, Eczacibasi, Istanbul, Turkey). Adenosine deaminase activity (ADA) in plasma Rabbits in GSH group were treated with a single ip was determined at 37°C according to the method administration of L-glutathione reduced (Sigma- of Giusti and Galanti21 based on the Bertholet re- Aldrich Chemie GmbH, Taufkirchen, Germany) action, formation of coloured indophenol complex dissolved in 0.9% saline at a dose of 10 mg/kg body from ammonia liberated from adenosine, and weight. Blood samples were taken from the margin- quantified colorimetrically with spectrophotome- al ear vein at 0, 3, 6 and 12 hours after injection into ter (UV-1201, Shimadzu, Japan). One unit of heparin treated tubes. Plasma was collected by cen- ADA is defined as the amount of enzyme required trifugation at 3000 rpm for 10 min and stored at - to release 1 mmol of ammonia/min from adeno- 50°C until analyses. sine at standard assay condition. Results were ex- pressed as international unit of enzyme activity. Biochemical Analysis Statistical Analysis The data for biochemical parameters were ana- Determination of Total lyzed by ANOVA followed by post hoc Tukey test Antioxidant Capacity using SPSS Windows 10.0. All data were present- Total antioxidant capacities were determined ed as mean ± SE. Values were considered statisti- colorimetrically (PowerWave XS, BioTek, Instru- cally significant if P value was less than 0.05. 20 Effects of reduced glutathione on antioxidative status Results GSH, a thiol antioxidant, is known to act as pro- tector in various cytotoxic conditions22-24. In ad- Plasma TAC, TOC, NO levels and ADA activ- dition, GSH is found within the cell at millimolar ity at 0, 3, 6 and 12 hours following the treat- concentrations that serves several essential func- ments are presented in Table I. Plasma TOC and tions including protein and DNA synthesis and ADA activities were not statistically different be- amino acid transport25-27. In condition of oxida- tween control and GSH group during the experi- tive stress, the GSH/GSSG ratio from 100 to 10 ment. or even 1 have been described and found to cor- Plasma TAC and NO levels were also not sta- relate with the amount of protein mixed disulfide tistically different between control and GSH formation. The ratio of the reduced pool to glu- group at 0 and 12 hours. Plasma TAC level was tathione disulfide (GSSG) is critical to cellular found to be significantly higher at 3 (1.16±0.08 redox balance28. vs 0.64±0.10; p <0.05) and 6 hours (0.93±0.07 vs Studies have reported reduced GSH pool during 0.71±0.08 μmol Trolox Equiv./L; p <0.05) while cytotoxicity and oxidative stress situations22-27. In NO level was found to be significantly lower in a research involving isolated rat liver exogenous GSH treated group when compared to control GSH was shown to protect liver damage induced group at 3 (13.72±1.26 vs 18.61±0.85; p <0.05) cyanide or hypoxia and reoxygenation.