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Research Article| Volume 138, ISSUE 2, P255-262, June 1998

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Antioxidant protection of LDL by physiologic concentrations of estrogens is specific for 17-beta-estradiol

      Abstract

      Risk for coronary artery disease is reduced by exposure to estrogens, although the mechanisms of protection are not fully defined. Recent observations have shown that physiologic concentrations of 17-beta-estradiol (E2) exhibit antioxidant activity in vitro, slowing the formation of atherogenic, oxidized low-density lipoprotein (LDL). Using concentrations physiologically relevant for premenopausal women, we compared the antioxidant potency of estrone (E1), E2, and estriol (E3) as measured by their ability to inhibit LDL oxidation. Plasma was incubated with 10 nmol/l estrogens for 4 h at 37°C, followed by LDL isolation and Cu2+-mediated oxidation in conjugated diene assays. Only E2 demonstrated antioxidant activity at these physiologic concentrations. Resistance to oxidation was not associated with sparing of endogenous α-tocopherol during plasma incubations. Incubation of plasma with radiolabeled estrogens yielded similar association of E1 and E2 with LDL which was 5–8-fold greater than the association of E3. Chromatographic analysis revealed the association of authenic E1 with LDL, while plasma-derived E2 esters were the major form of E2 associated with LDL which was resistant to oxidation. Thus, conjugation in plasma and association of E2 esters with LDL appear to be specific for E2 among these estrogens and render this LDL resistant to oxidation by Cu2+. This antioxidant activity may be another means whereby E2 protects against coronary artery disease in women.

      Keywords

      Abbreviations:

      CAD, coronary artery disease (), DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid) (), E1, estrone (), E2, 17-beta-estradiol (), E3, estriol (), HDL, high-density lipoprotein (), LCAT, lecithin–cholesterol acyltransferase (), LDL, low-density lipoprotein (), ox-LDL, oxidized LDL ()

      1. Introduction

      Premenopausal women enjoy a decreased incidence of coronary artery disease (CAD) compared to age-matched men [
      • Barrett-Connor E.
      • Bush T.L.
      Estrogen and coronary heart disease in women.
      ]and postmenopausal women [
      • Kannel W.B.
      • Hjortland M.
      • McNamara P.M.
      • Gordon T.
      Menopause and the risk of cardiovascular disease: The Framingham Study.
      ]. Furthermore, exposure of postmenopausal women to estrogen treatment reduces the number of clinical events in this population, even in those with established CAD [
      • Stampfer J.M.
      • Colditz F.A.
      • Willett W.C.
      • Manson J.E.
      • Rosner B.
      • Speizer F.
      • Hennekens C.H.
      Postmenopausal estrogen therapy and cardiovascular disease.
      ,
      • Ettinger B.
      • Friedman G.D.
      • Bush T.
      • Quesenberry Jr., C.P.
      Reduced mortality associated with long-term postmenopausal estrogen therapy.
      ]. Although the mechanisms responsible for these observations are not fully understood, these studies suggest that estrogens play a significant role in reduction of established risk factors for CAD [
      • Barrett-Connor E.
      • Bush T.L.
      Estrogen and coronary heart disease in women.
      ], including favorable changes in high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol levels [
      • Walsh B.W.
      • Schiff I.
      • Rosner B.
      • Greenberg L.
      • Ravnikar V.
      • Sacks F.M.
      Effects of postmenopausal estrogen replacement on the concentrations and metabolism of plasma lipoproteins.
      ]. However, only 25–50% of the protective effects of estrogen are contributed by changes in lipoprotein levels [
      • Gruchow H.W.
      • Anderson A.J.
      • Barboriak J.J.
      • Sobocinski K.A.
      Postmenopausal use of estrogen and occlusion of coronary arteries.
      ,
      • Bush T.L.
      • Barrett-Connor E.
      • Cowan L.D.
      • Criqui M.H.
      • Wallace R.B.
      • Suchindran C.M.
      • Tyroler H.A.
      • Rifkind B.M.
      Cardiovascular mortality and noncontraceptive use of estrogen in women: results from the Lipid Research Clinics Program Follow-up Study.
      ]. Moreover, in animal models, estrogen has been shown to attenuate the development of atherosclerosis without changes in LDL concentrations [
      • Adams M.R.
      • Kaplan J.R.
      • Manuck S.B.
      • Koritnik D.R.
      • Parks J.S.
      • Wolfe M.S.
      • Clarkson T.B.
      Inhibition of coronary artery atherosclerosis by 17-beta estradiol in ovariectomized monkeys. Lack of an effect of added progesterone.
      ,
      • Kushwaha R.S.
      • Lewis D.S.
      • Carey K.D.
      • McGill Jr., H.C.
      Effects of estrogen and progesterone on plasma lipoproteins and experimental atherosclerosis in the baboon (Papio sp.).
      ].
      The oxidative modification of LDL has been implicated in the initiation and progression of atherosclerosis [
      • Steinberg D.
      • Parthasarathy S.
      • Carew T.E.
      • Khoo J.C.
      • Witztum J.L.
      Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity.
      ]. Early atherosclerotic lesions are characterized by an intimal accumulation of cholesteryl ester-laden `foam cells' formed presumably as a result of scavenger receptor-mediated internalization of LDL that has been oxidatively modified by cells of the vascular wall [
      • Steinbrecher U.P.
      • Parthasarathy S.
      • Leake D.S.
      • Witztum J.L.
      • Steinberg D.
      Modification of low density lipoprotein by endothelial cells involves lipid peroxidation and degradation of low density lipoprotein phospholipids.
      ,
      • Parthasarathy S.
      • Printz D.J.
      • Boyd D.
      • Joy L.
      • Steinberg D.
      Macrophage oxidation of low density lipoprotein generates a modified form recognized by the scavenger receptor.
      ,
      • Heinecke J.W.
      • Rosen H.
      • Chait A.
      Iron and copper promote modification of low density lipoprotein by arterial smooth muscle cells.
      ]. Epitopes from oxidized LDL (ox-LDL) generated in vitro have been identified in atherosclerotic lesions of rabbits and humans [
      • Ylä-Herttuala S.
      • Palinski W.
      • Rosenfeld M.E.
      • Parthasarathy S.
      • Carew T.E.
      • Butler S.
      • Witztum J.L.
      • Steinberg D.
      Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man.
      ]and antibodies against ox-LDL have been identified in sera of patients with atherosclerosis [
      • Salonen J.T.
      • Ylä-Herttuala S.
      • Yamamoto R.
      • Butler S.
      • Korpela H.
      • Salonen S.
      • Nyyssönen K.
      • Palinski W.
      • Witztum J.
      Autoantibody against oxidised LDL and progression of carotid atherosclerosis.
      ]. In animal models, inhibition of LDL oxidation by antioxidants is associated with retardation of atherosclerotic progression [
      • Carew T.E.
      • Schwenke D.C.
      • Steinberg D.
      Antiatherogenic effect of probucol unrelated to its hypocholesterolemic effect: evidence that antioxidants in vivo can selectively inhibit low density lipoprotein degradation in macrophage-rich fatty streaks and slow the progression of atherosclerosis in the Watanabe heritable hyperlipidemic rabbit.
      ], and recent reports associate high vitamin E consumption with reduced risk of CAD in both sexes [
      • Rimm E.B.
      • Stampfer M.J.
      • Ascherio A.
      • Giovannucci E.
      • Colditz G.A.
      • Willett W.C.
      Vitamin E consumption and the risk of coronary heart disease in men.
      ,
      • Stampfer M.J.
      • Hennekens C.H.
      • Manson J.E.
      • Colditz G.A.
      • Rosner B.
      • Willett W.
      Vitamin E consumption and the risk of coronary disease in women.
      ].
      Studies demonstrating the antioxidant properties of estrogens support the hypothesis that these hormones protect against CAD by means other than lipid-lowering. Estrogens with a phenolic structure protect LDL from both cellular and Cu2+-mediated oxidation in vitro, and of the three principal estrogens in premenopausal women, 17-beta-estradiol (E2) is more potent than either estrone (E1) or estriol (E3) [
      • Mazière C.
      • Auclair M.
      • Ronoveaux M.F.
      • Salmon S.
      • Santus R.
      • Mazière J.C.
      Estrogens inhibit copper and cell-mediated modification of low density lipoprotein.
      ,
      • Tang M.
      • Abplanalp W.
      • Ayres S.
      • Subbiah M.T.R.
      Superior and distinct antioxidant effects of selected estrogen metabolites on lipid peroxidation.
      ]. In addition, E2 has been shown to be more potent in protecting against LDL oxidation than α-tocopherol, the principal lipid-soluble antioxidant in LDL [
      • Ayres S.
      • Tang M.
      • Subbiah M.T.R.
      Estradiol-17β as an antioxidant: some distinct features when compared with common fat-soluble antioxidants.
      ]. While concentrations of estrogens required to demonstrate antioxidant activity in these in vitro studies (≥1 μmol/l) greatly exceed levels observed even during pregnancy, recent reports have shown that chronic or acute hormonal replacement with physiologic concentrations of E2 in ovariectomized swine [
      • Keaney Jr., J.F.
      • Shwaery G.T.
      • Xu A.
      • Nicolosi R.J.
      • Loscalzo J.
      • Foxall T.L.
      • Vita J.A.
      17β-Estradiol preserves endothelial vasodilator function and limits LDL oxidation in hypercholesterolemic swine.
      ]and postmenopausal women [
      • Sack M.N.
      • Rader D.J.
      • Cannon III R.O.
      Oestrogen and inhibition of oxidation of low-density lipoproteins in postmenopausal women.
      ]is associated with increased LDL resistance to oxidation ex vivo. We have recently found that antioxidant protection with physiologic E2 concentrations against Cu2+-mediated LDL oxidation requires hydrophobic modification of E2 by plasma components [
      • Shwaery G.T.
      • Vita J.A.
      • Keaney Jr., J.F.
      Antioxidant protection of LDL by physiological concentrations of 17beta-estradiol: requirement for estradiol modification.
      ]. In the present study, we compared plasma-mediated modification and association of E1, E2, and E3 with LDL and the subsequent Cu2+-mediated LDL oxidation in vitro and found that antioxidant protection of LDL by physiologic concentrations of estrogens is specific to E2 and is associated with the exclusive hydrophobic derivatization of E2 in plasma.

      2. Materials and methods

      2.1 Chemicals

      Unlabeled estrogens were purchased from Steraloids (Wilton, NH) and [3H]E1 (specific activity, 76 Ci/mmol), [3H]E2 (specific activity, 110 Ci/mmol), and [3H]E3 (specific activity, 90 Ci/mmol) were from Dupont New England Nuclear (Boston, MA). Sephadex G-25 was obtained from Pharmacia Biotech (Uppsala, Sweden) and Chelex-100 resin was from Bio-Rad Laboratories (Hercules, CA). High-performance liquid chromatography (HPLC) grade methanol and hexane were from Aldrich (Milwaukee, WI) and Ecolite+ scintillation cocktail was obtained from ICN Biomedicals (Costa Mesa, CA). All other chemicals were purchased from Sigma (St. Louis, MO).

      2.2 LDL isolation and incubation with estrogens

      LDL exposure to estrogens was carried out as previously described [
      • Shwaery G.T.
      • Vita J.A.
      • Keaney Jr., J.F.
      Antioxidant protection of LDL by physiological concentrations of 17beta-estradiol: requirement for estradiol modification.
      ]. Briefly, heparinized plasma was prepared from healthy, normolipidemic, fasted (14 h) males by centrifugation (1200×g) at 4°C for 15 min. Ethanolic solutions of unlabeled or radiolabeled E1, E2, or E3 or vehicle alone were dried under N2 and reconstituted with plasma to yield a final concentration of 10 nmol/l or 100 nmol/l in selected experiments. This plasma was incubated for 4 h at 37°C sealed under N2 gas. LDL was subsequently isolated by single vertical spin discontinuous density gradient ultracentrifugation [
      • Chung B.H.
      • Segrest J.P.
      • Ray M.J.
      • Brunzell J.D.
      • Hokanson J.E.
      • Krauss R.M.
      • Beaudrie K.
      • Cone J.T.
      Single vertical spin density gradient ultracentrifugation.
      ], and size exclusion gel filtration was performed using Sephadex G-25 columns. Contaminating metal ions were removed with Chelex-100 resin followed by filtration through a 0.45 μm filter and protein determination using a modified procedure of Lowry [
      • Peterson G.L.
      A simplification of the protein assay method of Lowry et al. which is more generally applicable.
      ]. LDL isolated in this manner contained no detectable lipid hydroperoxides (see below) and was used immediately for experiments. In selected experiments, plasma was was heated to 60°C for 35 min to inactivate acyltransferase activity prior to incubation with estrogens or coincubated with 1.4 mmol/l 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and estrogens prior to determination of LDL resistance to oxidation and estrogen incorporation.

      2.3 Oxidative modification of LDL

      Resistance to oxidation was measured spectrophotometrically as accumulation of conjugated dienes at 37°C using 100 μg LDL protein per ml in phosphate buffered saline (PBS; 154 mmol/l NaCl, 10 mmol/l NaH2PO4, pH 7.4) and a final concentration of 3.3 μmol/l CuCl2. Duration of the lag phase was calculated as previously described [
      • Esterbauer H.
      • Striegl H.
      • Puhl H.
      • Rotheneder M.
      Continuous monitoring of in vitro oxidation of human low density lipoprotein.
      ].

      2.4 LDL antioxidant and cholesteryl ester hydroperoxide content

      Isolated LDL (250 μg protein) lipids were extracted with methanol–hexane (1:10, v/v) and centrifuged at 500×g for 10 min at 4°C. An aliquot of the hexane extract was dried under N2 and resuspended in 0.1 ml ethanol. LDL α-tocopherol was determined by HPLC with electrochemical detection as described [

      Stocker R, Frei B. Endogenous antioxidant defences in human blood plasma. In: Sies H., editor. Oxidative Stress: Oxidants and Antioxidants. London: Academic Press, 1991:213–243.

      ]. Samples were also analyzed for cholesteryl ester hydroperoxides by HPLC with post-column chemiluminescence detection as described [
      • Frei B.
      • Yamamoto Y.
      • Niclas D.
      • Ames B.N.
      Evaluation of an isoluminol chemiluminescence assay for the detection of hydroperoxides in human blood plasma.
      ].

      2.5 Association and HPLC analysis of [3H]estrogens associated with LDL

      Plasma was incubated with 10 or 100 nmol/l [3H]estrogens for 4 h as described above. The association of radiolabeled estrogens with isolated LDL was measured using scintillation spectroscopy (LKB 1214 Betarack) after removal of unbound 3H in LDL preparations by gel filtration with Sephadex G-25. An aliquot of LDL was precipitated with heparin–MnCl2 [
      • Warnick G.R.
      • Nguyen T.
      • Albers A.A.
      Comparison of improved precipitation methods for quantitation of high-density lipoprotein cholesterol.
      ]to determine if [3H]estrogens were associated with albumin, which has been reported to be a contaminant of LDL prepared in this manner [
      • Chung B.H.
      • Segrest J.P.
      • Ray M.J.
      • Brunzell J.D.
      • Hokanson J.E.
      • Krauss R.M.
      • Beaudrie K.
      • Cone J.T.
      Single vertical spin density gradient ultracentrifugation.
      ]. Electrophoresis of the apolipoprotein B-containing precipitate showed no detectable albumin and, <7% of the radiolabel in the LDL isolate was present in the albumin-containing supernatant. LDL (200 μg) was extracted with methanol and hexane as described above. Extracts were chromatographed using a 25 cm LC-18 column (Supelco) and a Hewlett-Packard Series 1050 HPLC system using absolute methanol as mobile phase at a flow rate of 1.0 ml/min with UV detection at 210 nm. Radiochromatograms of LDL-associated [3H]estrogens were generated by scintillation counting 0.3 ml fractions of eluate.

      2.6 Data analysis

      All data are reported as mean±S.E.M. The measures of LDL oxidative resistance and LDL estrogen content were compared among treatment groups by ANOVA and a post hoc Bonferroni t-test. Statistical significance was accepted if the null hypothesis was rejected with P<0.05.

      3. Results

      We have recently reported that plasma components are required for antioxidant protection of LDL by physiologic concentrations of E2 [
      • Shwaery G.T.
      • Vita J.A.
      • Keaney Jr., J.F.
      Antioxidant protection of LDL by physiological concentrations of 17beta-estradiol: requirement for estradiol modification.
      ]. In order to compare the antioxidant potency of the structurally related estrogens E1 and E3 to that reported for E2 at physiologically relevant concentrations, we incubated plasma with 10 nmol/l E1, E2, and E3 prior to LDL isolation and measured LDL resistance to oxidation in conjugated diene assays. Consistent with our previous observations, plasma incubated with 10 nmol/l E2 for 4 h at 37°C yielded LDL that was significantly more resistant to Cu2+-mediated oxidation as lag time to oxidation of E2-exposed LDL increased by 20% above vehicle-treated LDL (Fig. 1; P<0.05 vs. control, n=4). However, LDL resistance to oxidation was not significantly altered by exposure to either E1 or E3 in plasma (lag time 115±3 and 117±4 min, respectively) and was comparable to that of control LDL (lag time 115±4 min). Moreover, heat inactivation of plasma (60°C for 35 min) or coincubation with 1.4 mmol/l DTNB inhibited the antioxidant protection afforded by E2 described above, actually decreasing the lag time by 22% and 20%, respectively, compared to control incubations (data not shown).
      Figure thumbnail gr1
      Fig. 1Plasma was incubated with 10 nmol/l E1, E2, E3, or vehicle for 4 h at 37°C. LDL was isolated by ultracentrifugation, Chelex treated, and low molecular weight contaminants were removed by gel filtration with Sephadex G-25 columns. Resistance to oxidation was measured in conjugated diene assays using 100 μg/ml LDL protein and 3.3 μmol/l copper. Data are representative of 4–6 experiments. For clarity, only every fourth data point is shown. Lag time to oxidation is significantly increased in LDL isolated from plasma incubations with E2. P<0.05 by ANOVA.
      The increased LDL resistance to oxidation afforded by E2 suggests some stable alteration in the nature of LDL rendering increased antioxidant protection. Two important determinants of LDL susceptibility to oxidation in vitro are the content of lipid-soluble antioxidants, principally α-tocopherol [
      • Esterbauer H.
      • Dieber-Rotheneder M.
      • Striegl G.
      • Waeg G.
      Role of vitamin E in preventing the oxidation of low-density lipoprotein.
      ], and the presence of `preformed' lipid hydroperoxides [
      • Frei B.
      • Gaziano J.M.
      Content of antioxidants, preformed lipid hydroperoxides, and cholesterol as predictors of the susceptibility of human LDL to metal ion-dependent and -independent oxidation.
      ]. Incubation of plasma with exogenous α-tocopherol increases its content in LDL and enhances LDL resistance to oxidation [
      • Esterbauer H.
      • Dieber-Rotheneder M.
      • Striegl G.
      • Waeg G.
      Role of vitamin E in preventing the oxidation of low-density lipoprotein.
      ,
      • Hatta A.
      • Frei B.
      Oxidative modification and antioxidant protection of human low density lipoprotein at high and low oxygen partial pressures.
      ]. E2 has been shown to regenerate α-tocopherol from the tocopheroxyl radical [
      • Mukai K.
      • Daifuku K.
      • Yokoyama S.
      • Nakano M.
      Stopped-flow investigation of antioxidant activity of estrogens in solution.
      ]in a manner similar to the interaction of tocopherol and ascorbate [
      • Scarpa M.A.
      • Rigo M.
      • Maiorino M.
      • Ursini F.
      • Gregolin G.
      Formation of alpha-tocopherol radical and recycling of alpha-tocopherol by ascorbate during peroxidation of phosphatidylcholine liposomes.
      ]. Thus, we investigated the LDL content of α-tocopherol and lipid hydroperoxides after exposure of plasma to 10 nmol/l estrogens to determine if differences in LDL resistance to oxidation resulted from selective changes in endogenous antioxidants or lipid hydroperoxides. LDL isolated from plasma incubated with vehicle for 4 h had an α-tocopherol content of 11.9±0.9 nmol/mg protein, consistent with previous reports [
      • Shwaery G.T.
      • Vita J.A.
      • Keaney Jr., J.F.
      Antioxidant protection of LDL by physiological concentrations of 17beta-estradiol: requirement for estradiol modification.
      ,
      • Esterbauer H.
      • Dieber-Rotheneder M.
      • Striegl G.
      • Waeg G.
      Role of vitamin E in preventing the oxidation of low-density lipoprotein.
      ]. There was no difference in the LDL α-tocopherol content with E1, E2, or E3 treatment (11.9±0.9, 11.5±0.7 and 12.1±0.9 nmol/mg protein, respectively, n=4). Similarly, LDL samples were free of detectable cholesteryl ester hydroperoxides, thus precluding prevention of LDL lipid hydroperoxide formation as a mechanism of antioxidant activity specific for E2.
      To determine if the selective antioxidant protection of LDL by E2 was related to the degree of association with LDL compared to that of other estrogens, we incubated plasma for 4 h at 37°C with [3H]estrogens and measured the radiolabel associated with LDL after isolation and gel filtration. At both 10 and 100 nmol/l estrogen concentrations, E1 and E2 demonstrated comparable levels of association with LDL that were equivalent to approximately 1–2% of the total estrogen added, while E3, the most polar of these estrogens, showed 5–8-fold less association with LDL than either of the more non-polar estrogens (Table 1). For all three estrogens the association with LDL in plasma was dose-related (Table 1).
      Table 1Plasma-mediated association of estrogens with low density lipoprotein
      Plasma treatmentLDL estrogen content (pmol/mg protein)
      10 nmol/l100 nmol/l
      VehicleNDND
      Estrone0.081±0.0040.872±0.062
      Estradiol0.067±0.0020.861±0.136
      Estriol0.018±0.008*0.105±0.004*
      Plasma was incubated with the indicated concentrations of radiolabelled estrogens and LDL isolated and gel filtered as described in Section 2. Estrogen incorporation was determined by scintillation counting.
      *P<0.05 versus estrone and estradiol incubation by ANOVA (n=4).
      Using scintillation spectroscopy, we next investigated the chemical nature of plasma-mediated association of estrogens with LDL. Of the three estrogens employed in this study, E1 is the most non-polar. When LDL isolated from plasma treated with 10 nmol/l [3H]E1 was extracted with methanol–hexane, 0.065±0.007 pmol E1 per mg LDL was recovered in the methanol phase, while 0.020±0.003 pmol E1 per mg LDL was found in the hexane phase (76% and 24%, respectively), closely approximating the partitioning of authenic E1 when incubated in PBS alone and extracted as above (Fig. 2). However, incubation of plasma with E2 resulted in the conversion of E2 into a form that was more hydrophobic as evidenced by its increased solubility in hexane compared to authenic E2 in PBS (Fig. 2). This derivatized E2 was the principal form associated with LDL. Approximately 76% of the radiolabel that associated with LDL in E2–plasma incubations was found in the hexane phase, nearly 12-fold the amount of authentic E2 that partitions into hexane (Fig. 2). Although some hydrophobic modification of E3 in plasma was evident by its enhanced solubility in hexane compared to authentic E3 (Fig. 2), E3 did not appreciably associate with LDL in plasma compared to the more non-polar estrogens.
      Figure thumbnail gr2
      Fig. 2Purified [3H]estrogens in (A) PBS and (B) LDL isolated from plasma incubated with [3H]estrogens at 10 nmol/l were extracted with methanol–hexane as described in . Partitioning of radiolabel was measured by scintillation spectroscopy. The total association in the combined methanol and hexane extracts reflects the observations of . Values in (A) are the mean of two separate determinations which differed by <3%; in (B), n=4.
      To characterize further the nature of the estrogen species which associated with LDL in plasma, aliquots of the extracted LDL lipids were analyzed by HPLC. The radiochromatograms in Fig. 3 demonstrate that E2 is specific in its modification to hydrophobic forms that associate with LDL. E1 associates with LDL with equal or greater avidity compared to E2 (Table 1) and appears only as the authentic, parent compound in both the methanol and hexane phases with a single elution time of 3.6 min (Fig. 3). However, E2 is primarily found in LDL as hydrophobic derivatives, with only 25–30% of the radiolabel eluting as authenic E2. This association and derivatization of E2 is qualitatively reproducible with three or four major hydrophobic peaks separated by HPLC in each of four samples examined, while amounts of each peak vary among individuals.
      Figure thumbnail gr3
      Fig. 3Methanol and hexane fractions from were analyzed by HPLC using absolute methanol as mobile phase at a flow rate of 1 ml/min. Fractions were collected every 20 s and radiochromatograms generated by scintillation counting. One chromatogram representative of three is shown. The distribution of radiolabelled E1 associated with LDL reflects normal partitioning of E1 in a methanol–hexane extraction, whereas the majority of radiolabeled E2 is shifted into the hexane phase where only 6% of E2 is normally partitioned. Only small quantities of E3 associate with LDL in plasma and appears equally distributed as authenic E3 and a derivatized hydrophobic form.
      To confirm that association of E2 with LDL was an enzymatic process, we subjected samples to heat inactivation (60°C for 35 min) or co-incubation with 1.4 mmol/l DTNB to inhibit acyltransferase activity in plasma. Heat inactivation or DTNB treatment inhibited the association of E2 with LDL by 65 and 72%, respectively (mean of two observations, varying by <7%). This reduction in E2 association with LDL was primarily limited to hydrophobic forms of E2. Hexane extracts of LDL from these experiments accounted for approximately 82% of the reduction in E2 association. Thus, hydrophobic derivitization of estrogens and association with LDL is specific to E2, and this process is prevented by either inhibition or inactivation of acyltransferase activity in plasma.

      4. Discussion

      The data presented here demonstrate that, of the three principal estrogens in premenopausal women, antioxidant protection of LDL against Cu2+-mediated oxidation is specific for E2 at physiologically relevant concentrations. This antioxidant activity is associated with the accumulation of hydrophobic derivatives of E2 in LDL. Although E1 and E2 showed comparable association with LDL, E1 demonstrated neither protection of LDL against Cu2+-mediated oxidation nor evidence of hydrophobic conjugation, which we have recently shown to be required for antioxidant protection by physiologic concentrations of E2 [
      • Shwaery G.T.
      • Vita J.A.
      • Keaney Jr., J.F.
      Antioxidant protection of LDL by physiological concentrations of 17beta-estradiol: requirement for estradiol modification.
      ]. It is likely that E3, the most hydrophilic of these estrogens, did not afford antioxidant activity due to its poor association with lipoproteins. These observations suggest that the specific combination of esterification and association of E2 with LDL in plasma is associated with alterations of the lipoprotein particle which render it more resistant to oxidation by Cu2+.
      We have recently shown that plasma incubations with E2 resulted in dose-dependent formation and association with LDL of hydrophobic conjugates with characteristics of fatty acid esters of E2 with the hydroxyl group of C-17 as the preferential site for fatty acid conjugation [
      • Shwaery G.T.
      • Vita J.A.
      • Keaney Jr., J.F.
      Antioxidant protection of LDL by physiological concentrations of 17beta-estradiol: requirement for estradiol modification.
      ]. Furthermore, modification of E2 was required for antioxidant activity as heat inactivation of plasma, or incubation with DTNB which blocks thiols in the active site of lecithin–cholesterol acyltransferase (LCAT) inhibited the formation and association of E2 esters with LDL by 65–90% and completely blocked subsequent antioxidant protection of LDL. The observations in the present report are in concert with the structural differences among the three estrogens used here and support our hypothesis that esterification and association of estrogens with LDL is required for antioxidant protection at physiologic concentrations. In particular, E2 incubated in plasma was the only estrogen converted to a hydrophobic form which substantially associated with LDL and enhanced its resistance to oxidation. Although E1 and E2 associated with LDL to a similar degree, C-17 of E1 is a ketone and is resistant to esterification (Fig. 4). Thus, all of the radiolabel recovered from incubation of plasma with [3H]E1 eluted as authenic E1, while the majority of [3H]E2 was modified to the hydrophobic ester (Fig. 3). Finally, although E3 is similar to E2 in that C-17 bears a hydroxyl group and demonstrates some degree of modification during exposure to plasma (Fig. 2, Fig. 4), it poorly associates with lipoproteins, including HDL [
      • Leszczynski D.E.
      • Schafer R.M.
      Nonspecific and metabolic interactions between steroid hormones and human plasma lipoproteins.
      ], which we propose mediates the hydrophobic modification of estrogens through the activity of LCAT.
      Figure thumbnail gr4
      Fig. 4While all three estrogens have at C-3 a phenolic hydroxyl group important in the free radical scavenging behavior of supraphysiologic concentrations of estrogens, our data indicate that esterification at C-17 is required for antioxidant protection of LDL at physiologic concentrations of these hormones (see and ). While E2 and E3 are esterified in plasma, E1 is a ketone at carbon 17 which is resistant to esterification and shows no antioxidant protection of LDL.
      Our observations in plasma support those of Leszczynski and Schafer, describing the association of estrogens with isolated lipoproteins in vitro [
      • Leszczynski D.E.
      • Schafer R.M.
      Nonspecific and metabolic interactions between steroid hormones and human plasma lipoproteins.
      ,
      • Leszczynski D.E.
      • Schafer R.M.
      Characterization of steroid hormone association with human plasma lipoproteins.
      ]. Incubation of isolated lipoproteins with physiologic concentrations of estrogens in the absence of plasma components led to E1 and E2 concentrations in LDL that were approximately 5–10 times those of E3. The authors showed that E2 association with LDL was linear and reached equilibrium by 10 h. In contrast, when incubated with HDL, E2 appeared to become more lipophilic over time and remained in non-equilibrium even after 96 h [
      • Leszczynski D.E.
      • Schafer R.M.
      Nonspecific and metabolic interactions between steroid hormones and human plasma lipoproteins.
      ]. As previously shown for dehydroepiandrosterone [
      • Leszczynski D.E.
      • Schafer R.M.
      • Perkins E.G.
      • Jerrell J.P.
      • Kummerow F.A.
      Esterification of dehydroepiandrosterone by human plasma HDL.
      ,
      • Lavallee B.
      • Provost P.R.
      • Belanger A.
      Formation of pregnenolone- and dehydroepiandrosterone-fatty acid esters by lecithin–cholesterol acyltransferase in human plasma high density lipoproteins.
      ], the authors suggest that the enhanced association of E2 with HDL involved esterification of E2 by HDL-associated LCAT, as conjugation was inhibited by DTNB and the steroid esters were similar to those found in HDL3 [
      • Leszczynski D.E.
      • Schafer R.M.
      • Perkins E.G.
      • Jerrell J.P.
      • Kummerow F.A.
      Esterification of dehydroepiandrosterone by human plasma HDL.
      ]. Under these conditions using isolated LDL in saline buffer, there was no such modification or accumulation of E2 esters. This is in agreement with our previous report showing that modification and association of E2 with LDL requires plasma where there is significant interaction among lipoproteins and/or transport components (e.g. cholesteryl ester transfer protein) that may shuttle modified steroid hormones between lipoproteins [
      • Shwaery G.T.
      • Vita J.A.
      • Keaney Jr., J.F.
      Antioxidant protection of LDL by physiological concentrations of 17beta-estradiol: requirement for estradiol modification.
      ]. Although the affinity of E1 for LDL is slightly greater than that of E2 in both saline [
      • Leszczynski D.E.
      • Schafer R.M.
      Nonspecific and metabolic interactions between steroid hormones and human plasma lipoproteins.
      ]and plasma (Table 1), esterification and antioxidant activity appear specific for E2 as analysis of LDL extracts showed that E1 associated with LDL in an unaltered form and provided no antioxidant activity (Fig. 1, Fig. 3).
      The data presented here are in agreement with those of Larner et al. [
      • Larner J.M.
      • Rosner W.
      • Hochberg R.B.
      Binding of estradiol-17-fatty acid esters to plasma proteins.
      ], who showed that authenic estrogens added to plasma poorly associate with LDL (<1% of total) whereas E2 esters preferentially associate with LDL due to their extreme hydrophobicity (43% of radiolabelled E2-17-stearate preferentially associated with LDL in human serum). Our preliminary data indicate that E2 associates to a greater degree with HDL than LDL in plasma (0.95±0.04 vs. 0.64±0.03 pmol E2 per mg protein, n=3) and is hydrophobically modified in subsequently isolated HDL. This esterification of E2 may maintain a concentration gradient for authenic E2 as the fatty acid esters are most likely to associate with the non-polar cholesterol-ester rich core of the particle sequestered from the pool of unassociated E2. However, we are skeptical that E2 or its hydrophobic derivatives that associate with lipoproteins directly provide antioxidant protection by free radical scavenging. There are three lines of evidence that lead to this conclusion. First, E1 associates with LDL to a slightly greater extent than E2 yet provides no significant antioxidant activity against Cu2+-mediated oxidation of LDL despite previous in vitro studies that have demonstrated similar degrees of free radical scavenging activity [
      • Tang M.
      • Abplanalp W.
      • Ayres S.
      • Subbiah M.T.R.
      Superior and distinct antioxidant effects of selected estrogen metabolites on lipid peroxidation.
      ,
      • Mooradian A.D.
      Antioxidant properties of steroids.
      ]. Secondly, our observations with LDL derived from plasma incubations with E2 demonstrated that while oxidation of LDL by Cu2+ was inhibited, no protection was afforded from oxidation by 2,2′-azobis(2-amidinopropane) dihydrochloride, a source of aqueous peroxyl radicals [
      • Shwaery G.T.
      • Vita J.A.
      • Keaney Jr., J.F.
      Antioxidant protection of LDL by physiological concentrations of 17beta-estradiol: requirement for estradiol modification.
      ]. Finally, the amount of estrogen associated with LDL is quite small compared with what one would expect to provide free radical scavenging activity in the lipoprotein particle. Incubations of plasma with 100 nmol/l E2 indicate that, on average, <1% of LDL particles containes a hydrophobic form of E2 (Table 1). This is in agreement with both the experimental measurements of the association of estrogens and lipoproteins and the calculated values of hormone transport in plasma of premenopausal women [
      • Leszczynski D.E.
      • Schafer R.M.
      Nonspecific and metabolic interactions between steroid hormones and human plasma lipoproteins.
      ]. The nature of this antioxidant mechanism remains unknown and requires further investigation.
      To our knowledge, there have been no studies demonstrating a protective effect of E1 or E3 against atherosclerosis, while E2 is protective in both primates and rabbits [
      • Adams M.R.
      • Kaplan J.R.
      • Manuck S.B.
      • Koritnik D.R.
      • Parks J.S.
      • Wolfe M.S.
      • Clarkson T.B.
      Inhibition of coronary artery atherosclerosis by 17-beta estradiol in ovariectomized monkeys. Lack of an effect of added progesterone.
      ,
      • Haarbo J.
      • Leth-Espensen P.
      • Stender S.
      • Christiansen C.
      Estrogen monotherapy and combined estrogen–progestogen replacement therapy attenuate aortic accumulation of cholesterol in ovariectomized cholesterol-fed rabbits.
      ]. In premenopausal women, E2 is the predominant estrogen with follicular concentrations exceeding 1 nmol/l while E1 and E3 concentrations remain <0.5 nmol/l in the non-pregnant female [
      • Dunn J.F.
      • Nisula B.C.
      • Rodbard D.
      Transport of steriod hormones: binding of 21 endogenous steroids to both testosterone-binding globulin and corticosteroid-binding globulin in human plasma.
      ]. However, after menopause the ovaries cease to secrete bioactive estrogens and the majority of circulating estrogens are secreted as E1 from peripheral aromatization of adrenal androstenedione. We have shown that E1 is not esterified in plasma and is unable to provide antioxidant protection of LDL in spite of similar degrees of association with LDL in a plasma milieu. E3 is most likely ineffective as an antioxidant in LDL due its low plasma levels and poor affinity for the lipoproteins.
      Thus, antioxidant protection is specific for E2 among the three principle estrogens in premenopausal women. This protection is associated with the exclusive formation and association of E2-fatty acid esters with LDL, a plasma-mediated phenomenon which is associated with increased resistance of LDL to Cu2+-mediated oxidation. This specific antioxidant behavior may be another means by which E2 protects premenopausal women from CAD.

      Acknowledgements

      This work was supported by a grants from the Council for Tobacco Research-USA, Inc. (4073), the American Heart Association, and the National Institutes of Health (HL52936). J.A.V. is an Established Investigator of the American Heart Association and J.F.K. is the recipient of a Clinical Investigator Development Award (HL03195) from the NIH.

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