PROTECTION BY VITAMIN-C OF LOSS OF VITAMIN-E IN CULTURED RAT HEPATOCYTES

Results from in, vivo studies of the capacity of vitamin C to spare an d/or recycle vitamin E are equivocal. While some in vitro and membrane models reveal an interaction between vitamins C and E, the characteri zation of this relationship in biologically relevant systems is lackin g. Thus, we investigated this relationship using hepatocytes isolated from 3- to B-month-old male Sprague-Dawley rats. Cells were incubated for 18-20 h in medium supplemented with 0.1-4 mM ascorbic acid. The lo ss of alpha-tocopherol and the formation of its primary oxidized metab olite, alpha-tocopherolquinone, was determined by HPLC. Levels of alph a-tocopherol in hepatocytes incubated without ascorbic acid declined f rom 390 to 35 pmol/mg protein; hepatocyte ascorbic acid levels decline d from 9 to 0.5 nmol/mg protein. alpha-Tocopherolquinone was undetecta ble in freshly isolated hepatocytes but following incubation in ascorb ate-free medium reached 10 pmol/mg protein. The formation of alpha-toc opherolquinone was not detected in hepatocytes incubated with ascorbic acid. Dehydroascorbic acid (DHA) levels represented 10-20% of the tot al ascorbate content in freshly isolated hepatocytes but after 3 h inc ubation the proportion of DHA increased to 50%; after 18-20 h incubati on DHA was undetectable. Hepatocytes incubated with 1.0, 2.0, 2.5, or 4.0 mM ascorbic acid lost significantly less alpha-tocopherol (62, 69, 67, and 56%, respectively) than unsupplemented controls (90%). Twelve percent of the alpha-tocopherol lost from hepatocytes during incubati on was detected in the medium of cells incubated with ascorbic acid, b ut vitamin E was undetectable in the medium of cells incubated without ascorbic acid. These results demonstrate an interaction between vitam ins C and E in cell culture and are not inconsistent with a potential recycling of oxidized alpha-tocopherol by ascorbic acid. (C) 1998 Acad emic Press.