Comparison of the antioxidant effects of equine estrogens, red wine components, vitamin E, and probucol on low-density lipoprotein oxidation in postmenopausal women.

OBJECTIVE: Oxidized low-density lipoprotein (LDL) seems to play an important role in the etiology of atherosclerosis. To further study this, we performed two studies: (1) we determined the ability of 10 estrogen components of the drug, conjugated equine estrogen (CEE), trans-resveratrol (t-resveratrol) and quercetin (red wine components), trolox (vitamin E analog), and probucol (a serum cholesterol-lowering drug) to delay or prevent the oxidation of plasma LDL isolated from untreated postmenopausal women, and (2) we assessed the effect of long-term (>1 year) estrogen replacement therapy and hormone replacement therapy on LDL oxidation by ex vivo methods. DESIGN: For the in vivo study, three groups of postmenopausal women were selected based on whether they were on long-term CEE therapy (group A: 0.625 mg CEE; n = 21), on combination CEE plus progestogen therapy (group B: 0.625 mg CEE + 5.0 mg medroxyprogesterone acetate, 10 days; n = 20), or not on any hormone therapy (group C; n = 37). For the in vitro study, only LDL samples obtained from group C were used. The kinetics of LDL oxidation were measured by continuously monitoring the formation of conjugated dienes followed by determination of the lag time. RESULTS: All compounds tested protected the LDL from oxidative damage. The relative antioxidant potency of estrogen components was generally greater than that of the other compounds. The minimum dose (nmoles) required to double the lag time from the control lag time of 57 +/- 2 min was 0.47 for 17beta-dihydroequilenin, 17alpha-dihydroequilenin, Delta 8 -estrone; 0.6 to 0.7 for Delta 8 -17beta-estradiol, equilenin, and quercetin; 0.9 for 17beta-dihydroequilin and 17alpha-dihydroequilin; 1.3 for equilin, estrone, 17beta-estradiol, 17alpha-estradiol; 1.4 for trolox; 1.9 for probucol; and 3.0 for t-resveratrol. The data from the in vivo study indicate that after long-term estrogen replacement therapy (group A) and hormone replacement therapy (group B), the LDL was significantly ( p < 0.01) protected (higher lag time) against oxidation compared with the control (group C). There was no difference between groups A and B. CONCLUSIONS: The oxidation of LDL isolated from postmenopausal women is inhibited differentially by various estrogens and other antioxidants. The unique ring B unsaturated estrogen components of CEE were the most potent, and t-resveratrol, the red wine component, was the least potent. Long-term CEE or CEE + medroxyprogesterone acetate administration to postmenopausal women protects the LDL against oxidation to the same extent. These combined data support the hypothesis that some of the cardioprotective benefits associated with CEE therapy and perhaps red wine consumption may be due to the ability of their components to protect LDL against oxidative modifications.

Differential neuroprotective effects of equine estrogens against oxidized low density lipoprotein-induced neuronal cell death.

OBJECTIVE: In the present study, the neurotoxic effect of oxidized low density lipoprotein on PC-12 neuronal cells maintained in culture was used to test the neuroprotective effect of several equine estrogens, such as estrone (E(1)), 17beta-estradiol (17beta-E(2)), 17alpha-estradiol (17alpha-E(2)), equilin (Eq), 17beta-dihydroequilin (17beta-Eq), 17alpha-dihydroequilin (17alpha-Eq), equilenin (Eqn), 17beta-dihydroequilenin (17beta-Eqn), 17alpha-dihydroequilenin (17alpha-Eqn), Delta(8)-estrone (Delta(8)-E(1)), and Delta(8),17beta-estradiol (Delta(8),17beta-E(2)). METHODS: The PC-12 cells (10,000 cells/well) were grown on collagen-coated 96-well plates in Dulbecco's Modified Eagle Medium supplemented with 10% horse serum, 5% fetal bovine serum, and 10 mM HEPES. In culture, the cells displayed normal PC-12 morphology and behavior, exhibiting increased dendritic growth and cessation of cell division upon exposure to nerve growth factor. Twenty-four hours after plating, various concentrations (0.1-50 microM) of estrogens were added followed by addition of oxidized low density lipoprotein (5-12.5 microg/well) in a total volume of 100 microL. After 24 hours, cell viability was determined using the MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2H-tetrazolium, inner salt) cell proliferation assay. RESULTS: The results indicate that the extent of low density lipoprotein oxidation and concentration of oxidized low density lipoprotein is directly proportional to cell toxicity. The mean +/- standard deviation cell death obtained using 10.0 microg/well of oxidized low density lipoprotein was 53.6% +/- 8.7%. With the exception of 17alpha-estradiol, all estrogens tested were found to be neuroprotective against oxidized low density lipoprotein toxicity in a typical dose-dependent manner. The order of neuroprotective potency was Delta(8)-E(1) (1.2 microM), Delta(8),17beta-E(2) (1.3 microM), Eqn (5.3 microM), 17beta-Eqn (5.3 microM), Eq (6 microM), 17beta-Eq (8.5 microM), E(1) (11 microM), 17beta-E(2) (11 microM), 17alpha-Eq (12 microM), and 17alpha-Eqn (16 microM) followed by 17alpha-E(2) which provided less than 50% protection. CONCLUSION: Our data indicate that the neurotoxic effects of oxidized low density lipoprotein can be inhibited differentially by various estrogens, with the Delta(8) estrogens being the most potent neuroprotectors. These novel findings further suggest that some of the neuroprotective benefits associated with estrogen therapy might occur by the suppression of oxidized low density lipoprotein neurotoxicity. Because estrogens such as Delta(8)-E(1) are relatively less uterotropic and potentially less feminizing than the classic estrogen 17beta-E(2), they may be useful in the prevention of Alzheimer disease and other neurodegenerative diseases in both women and men.