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

Abstract

We have investigated the effect of ␣ -tocopherolloading of mouse peritoneal macrophages and human monocytes on their ability to oxidize human low density lipoprotein (LDL). Mouse peritoneal macrophages incorporated ␣tocopherol ( ␣ -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 ␣ -TOH within the cell was similar to that of free cholesterol. Most ( Ϸ 88%) of ␣ -TOH partitioned into the membrane fractions (plasma membrane Ϸ 41%, mitochondria and lysosomes Ϸ 26%, and endosomes plus endoplasmic reticulum Ϸ 21%). Cellular ␣ -TOH was stable for at least 24 h in serum-or LDLfree 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, ␣ -TOHpreloaded cells transferred small amounts of ␣ -TOH (approximately 1 nmol/mg LDL protein after 9 h) to the lipoprotein. However, enrichment of the cells with ␣ -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 ␣ -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 ␣ -TOH did not change the kinetics of cell-mediated LDL oxidation. We conclude that elevated cellular levels of ␣ -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 ␣ -TOH is unable to affect their formation.-Baoutina, A., R. T. Dean, and W. Jessup. ␣ -Tocopherol supplementation of macrophages does not influence their ability to oxidize LDL.