α-tocopherol supplementation of macrophages does not influence their ability to oxidize LDL

We have investigated the effect of α-tocopherol-loading of mouse peritoneal macrophages and human monocytes on their ability to oxidize human low density lipoprotein (LDL). Mouse peritoneal macrophages incorporated α- tocopherol (α-TOH) from culture medium supplemented with the vitamin in a time- and concentration-dependent manner. Subcellular fractionation by density gradient ultracentrifugation showed that the distribution of incorporated α-TOH within the cell was similar to that of free cholesterol. Most (≃88%) of α-TOH partitioned into the membrane fractions (plasma membrane ≃41%, mitochondria and lysosomes ≃26%, and endosomes plus endoplasmic reticulum ≃21%). Cellular α-TOH was stable for at least 24 h in serum- or LDL-free media whether permissive (Ham's F-10) or non-permissive (Dulbecco's minimum essential medium, DMEM) for LDL oxidation. When incubated with LDL in DMEM, α-TOH-preloaded cells transferred small amounts of α-TOH (approximately 1 nmol/mg LDL protein after 9 h) to the lipoprotein. However, enrichment of the cells with α-TOH did not change the kinetics of oxidation of either normal or TOH-depleted LDL in Ham's F-10 medium compared with non- loaded cells, as assessed by α-TOH consumption, cholesteryl ester degradation, and cholesteryl ester hydroperoxide and 7-ketocholesterol accumulation. Nor did it alter superoxide release by the cells or their ability to reduce extracellular copper(II). Similar to mouse macrophages, enrichment of human monocytes with α-TOH did not change the kinetics of cell-mediated LDL oxidation. B We conclude that elevated cellular levels of α-TOH in mouse peritoneal macrophages and in human monocytes do not affect their ability to oxidize LDL lipids in vitro. This suggests that either cell- mediated oxidation of LDL under the conditions of this study is not dependent on cell-derived radical species or that cellular α-TOH is unable to affect their formation.