1. Introduction Materials and methods References For further information Name Mohammed T. Al-Othmani Email abofadi_9@hotmail.com IBRAHIM H. IBRAHIM et al. Journal of Biophysics and Biomedical Sciences. March 2010; 3(1): 254-257 The need for daily supplements of vitamins was the objective of many studies to underline their side effects on humans upon their use in an uncontrolled manner (1-3). Oxidative damage of erythrocytes was the basis of a number of physiological and pathological phenomena (1), since it affects the overall cell structure, membrane characteristics and hemoglobin content. There exist different methods to measure the oxidative stress and antioxidant activity of many compounds, such as the hemolysis assays for measuring the damage caused by free radicals. Moreover, several hypotheses have been proposed to explain the mechanism of erythrocytes hemolysis following oxidative stress both in vivo and in vitro (2). Ibrahim (3) had previously shown a newly developed method for assessing the hemolytic effects caused by ascorbic acid (Vitamin C) by measuring the variation in erythrocytes conductivity. He attributed his findings to the oxidative damage of erythrocyte hemoglobin and membranes. The objective of this work was to investigate the effects of large doses of vitamins on hemoglobin and membrane characteristics of erythrocytes. A series of samples of vitamin C, Thiamine (vitamin B1), Riboflavin (vitamin B2), Pantothenic acid (vitamin B5), Pyridoxine (vitamin B6) and Cobalamin (vitamin B12) of various concentrations at 0.25, 0.5, 1, 2, 4 and 8 mM were prepared for this study. A. Preparation of Erythrocyte Suspensions Human blood samples from 6 healthy individuals were collected in EDTA tubes and stored at 4 °C for use in the same day. Erythrocytes were isolated by centrifugation (10 min, 1500 RPM), plasma and buffy coat removed and the cells washed three times with isotonic saline (0.15 M NaCl) at room temperature. Erythrocytes were suspended in saline to reach a concentration of about 3 × 105 cells/ml. B. Conductivity Measurements A conductivity meter (Model FE287/kit, EDT Direct Ion Ltd., UK) was used for erythrocytes measurements. Variations in conductivity (mS/cm) with time were recorded for each concentration of a certain vitamin, as detailed elsewhere (3), where a 0.7 ml of vitamin of a certain concentration was added to 19.3 ml of erythrocytes suspension and the devise was used to record the conductivity of that mixture every 5 min. C. Osmotic Fragility Test A 0.1 ml of whole blood samples were added to varying concentrations of NaCl solution (from 0, 0.1 and 0.2 up to 1%) of volume 9.9 ml for each concentration, which was incubated at room temperature for 30 min. The level of hemolysis of erythrocytes was determined by measuring the hemoglobin released from the cells, relative to the total cellular hemoglobin content. The amount of hemoglobin was estimated on basis of the absorbance of the supernatant of each saline concentration at 540 nm using a spectrophotometer (UV/Visible Spectrophotometer, model Ultrospec 2000, Pharmacia Biotech, UK) after 30 min. Absorbance of the supernatant after complete hemolysis with distilled water (0%) was taken as 100% hemolysis, as described earlier (4). The last procedure was repeated but with adding 0.35 ml of the used vitamin concentration (1 mM) to 0.1 ml of blood to each saline concentration (9.55 ml). Conclusions Results [1] B. Halliwell, J. M. Gutteridg, Arch. Biochem. Biophys. 246, 501 (1986). [2] M. D’Aguino, S. Gaetani, M. A. Spadoni, Biochim. Biophys. Acta. 731, 161 (1983). [3] I. H. brahim, Variation in conductivity of human erythrocytes caused by ascorbic acid: A new method for following hemolysis. The 3rd International Conference on Engineering Mathematics and Physics. Faculty of Engineering, Cairo University, Egypt, (1997). [4] A. Haut, G. R. Tudhope, G. E. Cartwright et al., J. Clin. Invest. 41, 1766 (1962). [5] H. Einsele, M. R. Clemens, H. Remmer, Free Radic. Res.Commun. 1, 63 (1985). [6] M. C. Vissers, A. Stern, F. Kuypers et al., Free Radic. Biol. Med. 16, 703 (1994). [7] E. Niki, E. Komur, M. Takahashi et al., J. Biol. Chem. 263, 19814 (1988). [8] B. Goldberg, A. Stern, Arch. Biochem. Biophys. 178, 218 (1977). [9] S. M. Waugh, P. S. Low, Biochem. 24, 34 (1985). [10] I. H. Ibrahim, Eg. J. Biophys. 12, 153 (2006). [11] F. M. Ali, W. S. Mohamed, M. R. Mohamed, Bioelectromag. 24, 535 (2003). The study of the total electric conductivity for erythrocytes suspension is very important in a way that it may reflect the health status of humans. Our results showed a direct oxidizing effect of studied vitamins on hemoglobin and membranes, consequently their hemolytic effect on erythrocytes. The results showed also that the use of large amounts of vitamins for long periods of time may cause hemoglobin oxidation and destruction of erythrocytes membrane, which may lead to hemolytic anemia. Effects of Some Water-Soluble Vitamins on Human Erythrocytes