Higher reactivity of apolipoprotein B-100 and alpha-tocopherol compared to sialic acid moiety of low-density lipoprotein (LDL) in radical reaction.
Radical reaction of low-density lipoprotein (LDL) is a key step in atherogenesis and causes both a decrease in the sialic acid moiety and modification of apolipoprotein B-100 (apoB). Although apoB modification (cross-link and fragmentation) increases in atherosclerosis, the change in apoB-bound sialic acid in atherosclerosis is controversial. To elucidate the physiological implications of desialylation of LDL by radical reaction, the reactivity of sialic acid of LDL was compared with that of apoB, which underwent facile fragmentation in radical reactions. ApoB was determined by immunoblot analysis with anti-apoB antiserum, and the sialic acid moiety was measured by blot analysis with a biotin-bound lectin [biotin-SSA from Japanese elderberry (Sambucus sieboldiana)] specific to sialic acid. When human LDL was oxidized with Cu(2+) at 37 degrees C, apoB and apoB-attached sialic acid decreased simultaneously. Comparison of the staining bands with anti-apoB and with biotin-SSA shows that sialic acid moieties still remain on fragmented apoB proteins, indicating that the decrease in sialic acid is much slower than that of apoB fragmentation. In addition, human plasma was oxidized with 400 microM of Cu(2+) at 37 degrees C. Similar analysis indicates that the decrease in sialic acid attached to apoB also results from the fragmentation of apoB. This study indicates that the fragmentation of apoB proceeds at a much faster rate than the decrease in sialic acid content when a free radical reaction is induced in isolated LDL as well as in plasma LDL exposed to Cu(2+)-induced oxidative stress. On the basis of these results, the modification of apoB is much more sensitive than the decrease in sialic acid as an indicator of oxidative stress.