Figure 2

Effect of cellular NO synthesis on media and cellular levels of α- and γ-tocopherols. At confluence, a mixture of α-plus γ-tocopherol in 50 μl ethanol (10 μM final concentration/100 ng/dish for each tocopherol) was added to each 100 mm culture dish (10 mL of culture medium per dish). After seven days cell culture medium was changed and cells were re-treated with tocopherols. At this time six of the 12 dishes were also treated with IFN (10 ng/ml) & LPS (1 μg/ml) to stimulate NO production. Three culture plates that were treated with IFN/LPS and three that contained only the mixture of tocopherols, were also treated with 50 μl of 2 mM PBIT in PBS, yielding a final media concentration of 10 μM PBIT. The other 6 dishes were treated with either 50 μl PBS vehicle or 50 μl of 2 mM PBIT. Seven days later, media nitrite and 8-epi-prostaglandin F2α levels were measured as described in the methods section in duplicate. Tocopherol levels were measured in the cell culture medium (A) and in cells (B). Values represent the mean of six determinations ± SEM. * (p < 0.01) **(p < 0.05) Indicates significantly different from corresponding control value by ANOVA analysis. In addition all cellular γ-tocopherol levels were significantly higher than the corresponding α-tocopherol level for each treatment by student t-test (p < 0.01). PBIT treatment significantly elevated both α- and γ-tocopherols in IFN/LPS-treated cells (p < 0.01 by ANOVA).