Relative alpha-tocopherol deficiency in cultured cells: free radical-mediated lipid peroxidation, lipid oxidizability, and cellular polyunsaturated fatty acid content

We propose that most cultured cells are deficient in vitamin E. Using our optimized assay for tocopherol, we find that L1210 lymphoblastic leukemia cells, cultured in standard growth media, contain only 2.3 +/- 0.03 microgram of tocopherol/10(8) cells, whereas when they are transplanted and grown for the same time in the ascites fluid of mice fed standard diets, this increases to 5.8 +/- 0.6 microgram of tocopherol/10(8) cells. This apparent tocopherol deficiency in cultured cells is likely due to the low concentrations of tocopherol contained in most tissue culture media, even with the addition of serum. To further study this apparent deficiency and the relationship of cellular tocopherol to membrane lipid bis-allylic hydrogen positions, we supplemented the growth media of L1210 lymphoblastic leukemia cells with alpha-tocopherol and compared the resultant cellular tocopherol content to the degree of unsaturation of cellular lipids. alpha-Tocopherol was incorporated by cells in a time- and concentration-dependent manner with plateaus at 24 h and 100 microgram, respectively. A maximum 400% increase in cellular tocopherol was easily achieved. By experimentally modifying the fatty acid content of cellular lipids, we were able to determine that cellular tocopherol uptake and content is not a function of cellular lipid composition; cells enriched with polyunsaturated lipids incorporated tocopherol to the same extent as those enriched with more saturated lipids. Thus, as the cellular polyunsaturated fatty acid content increases, the tocopherol:bis-allylic position ratio in the cells decreases, resulting in less antioxidant protection for each lipid double bond. Consequently, when polyunsaturated fatty acid-enriched cells are exposed to an oxidative stress, such as Fe2+, their tocopherol levels decline much faster than cells enriched with saturated fatty acids. This decline is consistent with their respective tocopherol:bis-allylic position ratio. These results provide a basis, at the cellular level, for investigators to consider vitamin E when studying cell response to oxidative stress.