Amelioration of glucose induced hemolysis of human erythrocytes by vitamin E Thankamani Marar ⇑ Padmashree Dr. D.Y. Patil University, Department of Biotechnology and Bioinformatics, Sector 15, CBD Belapur, Navi Mumbai 400 614, Maharashtra, India article info Article history: Received 13 April 2011 Received in revised form 14 June 2011 Accepted 16 June 2011 Available online 26 June 2011 Keywords: Lipid peroxides Catalase Superoxide dismutase Glutathione RBC membrane abstract Cells under aerobic condition are always threatened with the insult of reactive oxygen species, which are efficiently taken care of by the highly powerful antioxidant systems of the cell. The erythrocytes (RBCs) are constantly exposed to oxygen and oxidative stress but their metabolic activity is capable of reversing the injury under normal conditions. In vitro hemolysis of RBCs induced by 5, 10 and 20 mM glucose was used as a model to study the free radical induced damage of biological membranes in hyperglycemic con- ditions and the protection rendered by vitamin E on the same. RBCs are susceptible to oxidative damage, peroxidation of the membrane lipids, release of hemoglobin (hemolysis) and alteration in activity of anti- oxidant enzymes catalase and superoxide dismutase. The glucose induced oxidative stress and the pro- tective effect of vitamin E on cellular membrane of human RBCs manifested as inhibition of membrane peroxidation and protein oxidation and restoration of activities of superoxide dismutase and catalase, was investigated. Thiobarbituric acid reactive substances are generated from decomposition of lipid peroxides and their determination gives a reliable estimate of the amount of lipid peroxides present in the membrane. Vita- min E at 18 lg/ml (normal serum level) strongly enhanced the RBC resistance to oxidative lysis leading to only 50–55% hemolysis in 24 h, whereas RBCs treated with 10 and 20 mM glucose without vitamin E leads to 70–80% hemolysis in 24 h. Levels of enzymic antioxidants catalase, superoxide dismutase and nonenzymic antioxidants glutathione showed restoration to normal levels in presence of vitamin E. The study shows that vitamin E can protect the erythrocyte membrane exposed to hyperglycemic condi- tions and so a superior antioxidant status of a diabetic patient may be helpful in retarding the progressive tissue damage seen in chronic diabetic patients. Ó 2011 Elsevier Ireland Ltd. All rights reserved. 1. Introduction The red blood cells are constantly exposed to oxidative injury, but their metabolic activity is capable of reversing the injury under normal conditions. Following an increased oxidative stress when content of active oxygen species exceeds the cellular antioxidant capacity, irreversible damage to the erythrocytes occurs, resulting in their ultimate loss by hemolysis and removal from circulation [1]. Elevated levels of glucose in the medium or blood are known to cause membrane damage and cell death of erythrocytes. How- ever the biochemical mechanism of this damage is not known. Similar to the non enzymatic glycosylation of haemoglobin leading to formation of glycosylated haemoglobin in diabetics, glycosyla- tion of cell membrane proteins leading to formation of advanced glycosylation end products may lead to irreversible crosslinking of membrane proteins which may contribute to loss of elasticity and increased membrane osmotic fragility [2]. Diabetes is associated with various microvascular and macro- vascular complications and oxidative stress is one of the main causes of diabetic complications. Free radicals play an important role in pathogenesis of tissue damage in many clinical disorders. Oxygen free radicals are capable of damaging biomolecules of all classes including nucleic acids, proteins, lipids, lipoproteins, carbo- hydrates and connective tissue macromolecules. Normally, there is a balance between tissue oxidant and antioxidant activity, the lat- ter achieved by the antioxidant scavenger system which includes enzymes like superoxide dismutase, catalase, glutathione peroxi- dase, antioxidant vitamins C, A, E and carotenoids and free other free radical scavengers like glutathione [3–5]. Vitamin E is a chain breaking antioxidant. It appears to be the first line of defense against peroxidation of polyunsaturated fatty acids contained in cellular and sub-cellular membrane phospholip- ids [6]. In RBCs vitamin E constitutes an important membrane antioxidant protecting the cell against oxidative damage. In the present study, in vitro oxidative hemolysis of human red blood cell (RBCs) was used as a model to study the free radical induced damage of biological membranes due to glucose and the protective effect of vitamin E. 0009-2797/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2011.06.004 ⇑ Tel.: +91 9892455625, +91 22 27562491; fax: +91 22 39286121. E-mail address: dr.marar@yahoo.com Chemico-Biological Interactions 193 (2011) 149–153 Contents lists available at ScienceDirect Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint