Inhibition by a coantioxidant of aortic lipoprotein lipid peroxidation and atherosclerosis in apolipoprotein E and low density lipoprotein receptor gene double knockout mice

Abstract

Antioxidants can inhibit atherosclerosis in animals, though it is not clear whether this is due to the inhibition of aortic lipoprotein lipid (per)oxidation. Coantioxidants inhibit radical-induced, tocopherol-mediated peroxidation of lipids in lipoproteins through elimination of tocopheroxyl radical. Here we tested the effect of the bisphenolic probucol metabolite and coantioxidant H 212/43 on atherogenesis in apolipoprotein E and low density lipoprotein (LDL) receptor gene double knockout (apoE-/-;LDLr-/-) mice, and how this related to aortic lipid (per)oxidation measured by specific HPLC analyses. Dietary supplementation with H 212/43 resulted in circulating drug levels of approximately 200 microM, increased plasma total cholesterol slightly and decreased plasma and aortic alpha-tocopherol significantly relative to age-matched control mice. Treatment with H 212/43 increased the antioxidant capacity of plasma, as indicated by prolonged inhibition of peroxyl radical-induced, ex vivo lipid peroxidation. Aortic tissue from control apoE-/-;LDLr-/- mice contained lipid hydro(pero)xides and substantial atherosclerotic lesions, both of which were decreased strongly by supplementation of the animals with H 212/43. The results show that a coantioxidant effectively inhibits in vivo lipid peroxidation and atherosclerosis in apoE-/-;LDLr-/- mice, consistent with though not proving a causal relationship between aortic lipoprotein lipid oxidation and atherosclerosis in this model of the disease.

Site-Specific Antiatherogenic Effect of Probucol in Apolipoprotein E–Deficient Mice

“…28 Lipid-soluble antioxidants and lipids were quantified by high-performance liquid chromatography (HPLC). 32 For ascorbate, samples were thawed, diluted with Dulbecco's PBS to adjust the pH to 7.4, and then immediately subjected to HPLC. 6 Plasma triglycerides were determined enzymatically (Boehringer).…”

“…For biochemistry, aortas (nϭ22 to 24 for both groups) were not fixed, because adventitious oxidation takes place when standard fixation procedures are used. 28,32 Once cleaned, the aortas were separated from the heart, taking care to include all aortic material while avoiding heart tissue. To obtain sufficient material for HPLC analysis (ie, 30 to 40 mg wet weight tissue), it was necessary to pool 7 to 8 aortas.…”

“…28,30,32 LOOH and CE-OH were measured because they are the primary and major lipid oxidation products formed when lipoproteins from apoEϪ/Ϫ mice undergo oxidation. 28 Our sample work-up procedure recovers 88Ϯ4% of a hydroperoxide standard of C18:2 added to mouse aorta and is recovered as LOOH or CE-OH.…”

“…28 To confirm the identity of aortic LOOH, HPLC with postcolumn chemiluminescence detection was used 30 before and after NaBH 4 treatment of the samples. 33 Bisphenol, probucol, and diphenoquinone were analyzed by gradient reverse-phase HPLC 32 with compounds eluting at Ϸ9, 17, and 27 minutes, respectively. All compounds were quantified by peak area comparison with authentic standards, and protein determined was performed as described.…”

Nano-Computed Tomography: Technique and Applications

“…Already 15 weeks after birth, atherosclerotic plaques can be seen in the descending aorta and in the aortic arch [12]. The first cup-shaped calcified plaques in the aortic arch and in the supraaortic vessels are seen after 20 weeks.…”