Selected papers from “L’innovazione scientifica per la nutrizione”, 15th October, 2015, Lodi (MI), Italy 10 HAF © 2013 Vol. II, No. 2s ISSN: 2283-3927 l Introduction In nature, Vitamin E is present under eight different forms, four tocopherols (α, β, γ, and δ) and four tocotrienols (α, β, γ, and δ). The α–tocopherol has the highest biological activity (Dersjant & Peisker, 2010). The natural form of α–tocopherol is composed of 100% RRR-α-tocopherol whereas the synthetic form (all-rac-α- tocopherol) consists of a mixture of eight stereoisomers at equal amount. Previous studies suggest that the bioefficacy of the different α-tocopherol forms should be reconsidered (Vagni et al., 2011). At the cell level, α-tocopherol functions as an antioxidant (Baldi, 2005). The main aim of this study was to investigate the in vitro relative bioefficacy of RRR-α-tocopherol (RRR- α-T) versus all-rac-α-tocopherol (all-rac-α-T) to counteract the cytotoxic effect induced by hydrogen peroxide (H2O2). To this aim, Bovine Mammary Epithelial cell line (BME-UV1) and Madine Darby Canine Kidney cell line (MDCK) were used since they represent suitable and well characterized in vitro models in relation to their particular susceptibilities to oxidative damage. Material and Methods BME-UV1 and MDCK cells were cultivated into 75 cm3 tissue culture flasks in complete medium. Preliminary experiments were performed to determine H2O2 cytotoxicity and the LC50 were calculated in both cell lines. Further a putative difference in the protective effect of the two forms of tocopherol against H2O2-induced stress was evaluated. Cells were trypsinized and seeded into wells of 96 wells -cell culture plates (seeding density: BME-UV1 2 x 105 cells/ml; MDCK 1 x105 cells/ml). Cells were pre-incubated for 3 h with selected RRR- α-T and all-rac- α- T concentrations and then exposed to increasing H2O2 concentrations ranging from 250 to 750 µM in BME-UV1 cell line and from 125 to 175 µM in MDCK cell line for the following 24h. The effects of RRR- α-T and all-rac- α-T on both BME-UV1 and MDCK cell lines in counteracting H2O2 toxicity were determined by the MTT test and the LDH test. At least three replicates at each incubation time were performed and all experiments were performed at least twice. Results are expressed as mean and SD. The effect of various treatments were evaluated by one-way analysis of variance using the GLM procedure of SAS (SAS institute Inc, NC, USA). Values significantly different from controls are indicated as P<0.05. Results and Discussion In BME-UV1, the proliferative effect induced by RRR-α-T form was higher at the lowest concentrations (1nM and 0.01uM) than all-rac α-T (about 30%, which is consistent with the current bioactivity conversion factors). In MDCK, no relevant effect on mitochondrial activity was observed (Figure 1). LC50 values determined in BME-UV1 and MDCK cells were 376µM and 140µM, respectively. KEYWORDS Vitamin E, bioefficacy, in vitro models; CORRESPONDING AUTHOR Carlotta Giromini, carlotta.giromini@unimi.it JOURNAL HOME PAGE riviste.unimi.it/index.php/haf Relative bioefficacy of RRR-α-tocopherol versus all-rac-α-tocopherol in in vitro models A. Baldi 1 , C. Giromini 1 , D. Gottardo 1 , R. Rebucci 1 , L. Pinotti 1 , E. Fusi 1 1 Department of Health, Animal Science and Food Safety, Università degli Studi di Milano, Italy Article Selected papers from “L’innovazione scientifica per la nutrizione”, 15th October, 2015, Lodi (MI), Italy 11 HAF © 2013 Vol. II, No. 2s ISSN: 2283-3927 Pre-treatments with 100μM of RRR-α-T and 100μM all-rac-α-T were able to significantly (P<0.05) counteract the effect induced by 750μM of H2O2 in BME-UV1. In MDCK the pre-treatment with 1nM of all-rac-α-T was able to significantly (P<0.05) reduce the effect of 125 and 150mM H2O2 (Figure 2). In MDCK cells, the pre-incubation with all-rac-α-T (1nM) determined a significant reduction of the membrane damage (LDH test), induced by 175 μM of H2O2. Conclusions The dose-response curve experiments shown that RRR-α-T and all-rac-α-T tocopherols were able to maintain (MDCK cells) and increase (BME-UV1 cells) the cell viability. It has been observed that adequate RRR-α-T and all-rac- α-T concentrations could reduce the oxidative damages induced by H2O2 in both BME-UV1 and MDCK cells. Differences detected in the two α-tocopherol forms, when present, were consistent with the conversion factors. However, in some cases all-rac was exhibited a higher antioxidant effect, compared with RRR-α-T. In conclusion, RRR-α-T and all-rac-α-T have shown the ability to counteract the oxidative effects of H2O2 in the cell lines considered. However, further investigation will help in describing their specific mechanism of action in vitro. References A. Baldi, “Vitamin E in Dairy Cows,” Livestock Production Science, Vol. 98, No. 1-2, 2005, pp. 117-122. doi:10.1016/j.livprodsci.2005.10.004 Dersjant‐Li, Y., & Peisker, M. (2010). A critical review of methodologies used in determination of relative bio‐availability ratio of RRR‐α‐tocopheryl acetate and all‐rac‐α‐tocopheryl acetate. Journal of the Science of Food and Agriculture, 90(10), 1571-1577 S. Vagni et al., (2011). “Vitamin E Bioavailability: Past and Present Insights”, Food and Nutrition Science, 2, pp. 1088 -1096 Acknowledgments This work was supported by DSM Nutritional Products AG.