Protection by Vitamin C of Loss of Vitamin E in Cultured Rat Hepatocytes

Results fromin vivostudies of the capacity of vitamin C to spare and/or recycle vitamin E are equivocal. While somein vitroand membrane models reveal an interaction between vitamins C and E, the characterization of this relationship in biologically relevant systems is lacking. Thus, we investigated this relationship using hepatocytes isolated from 3- to 6-month-old male Sprague–Dawley rats. Cells were incubated for 18–20 h in medium supplemented with 0.1–4 mM ascorbic acid. The loss of α-tocopherol and the formation of its primary oxidized metabolite, α-tocopherolquinone, was determined by HPLC. Levels of α-tocopherol in hepatocytes incubated without ascorbic acid declined from 390 to 35 pmol/mg protein; hepatocyte ascorbic acid levels declined from 9 to 0.5 nmol/mg protein. α-Tocopherolquinone was undetectable in freshly isolated hepatocytes but following incubation in ascorbate-free medium reached 10 pmol/mg protein. The formation of α-tocopherolquinone was not detected in hepatocytes incubated with ascorbic acid. Dehydroascorbic acid (DHA) levels represented 10–20% of the total ascorbate content in freshly isolated hepatocytes but after 3 h incubation the proportion of DHA increased to 50%; after 18–20 h incubation DHA was undetectable. Hepatocytes incubated with 1.0, 2.0, 2.5, or 4.0 mM ascorbic acid lost significantly less α-tocopherol (62, 69, 67, and 56%, respectively) than unsupplemented controls (90%). Twelve percent of the α-tocopherol lost from hepatocytes during incubation was detected in the medium of cells incubated with ascorbic acid, but vitamin E was undetectable in the medium of cells incubated without ascorbic acid. These results demonstrate an interaction between vitamins C and E in cell culture and are not inconsistent with a potential recycling of oxidized α-tocopherol by ascorbic acid.