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 l eukemia cells, cultured in standard growth media, contain only 2.3 +/- 0.03 mu g of tocopherol/10(8) cells, whereas when they are transplant ed and grown for the same time in the ascites fluid of mice fed standa rd diets, this increases to 5.8 +/- 0.6 mu g of tocopherol/l0(8) cells . This apparent tocopherol deficiency in cultured cells is likely due to the low concentrations of tocopherol contained in most tissue cultu re media, even with the addition of serum. To further study this appar ent deficiency and the relationship of cellular tocopherol to membrane Lipid bis-allylic hydrogen positions, we supplemented the growth medi a of L1210 lymphoblastic leukemia cells with cy-tocopherol and compare d the resultant cellular tocopherol content to the degree of unsaturat ion of cellular lipids. cu-Tocopherol was incorporated by cells in a t ime- and concentration-dependent manner with plateaus at 24 h and 100 mu M, respectively. A maximum 400% increase in cellular tocopherol was easily achieved. By experimentally modifying the fatty acid content o f cellular lipids, we were able to determine that cellular tocopherol uptake and content is not a function of cellular lipid composition; ce lls enriched with polyunsaturated Lipids incorporated tocopherol to th e same extent as those enriched with more saturated lipids, Thus, as t he cellular polyunsaturated fatty acid content increases, the tocopher ol:bis-allylic position ratio in the cells decreases, resulting in les s antioxidant protection for each lipid double bond, Consequently, whe n polyunsaturated fatty acid-enriched cells are exposed to an oxidativ e stress, such as Fe2+, their tocopherol levels decline much faster th an cells enriched with saturated fatty acids. This decline is consiste nt with their respective tocopherol:bis-allylic position ratio, These results provide a basis, at the cellular level, for investigators to c onsider vitamin E when studying cell response to oxidative stress. (C) 1995 Academic Press, Inc.