Skip Navigation

Cardiovascular Research 2002 53(3):589-596; doi:10.1016/S0008-6363(01)00403-5
© 2002 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Dimitrova, K. R.
Right arrow Articles by Kim, Y. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dimitrova, K. R.
Right arrow Articles by Kim, Y. D.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2002, European Society of Cardiology

Estradiol prevents homocysteine-induced endothelial injury in male rats

Kamellia R. Dimitrova, Kerry W. DeGroot, Alfonso M. Pacquing, Johan P. Suyderhoud, Eugen A. Pirovic, Thomas J. Munro, Jacqueline A. Wieneke, Adam K. Myers and Young D. Kim*

Departments of Anesthesia and Physiology and Biophysics, Georgetown University, School of Medicine, Washington DC 20007, USA

* Corresponding author. Present address: Department of Anesthesia, 3800 Reservoir Road, NW, CCC Building, Washington, DC 20007, USA. Tel.: +1-202-687-8854; fax: +1-202-784-2769 kimyd{at}gusun.georgetown.edu

Received 15 March 2001; accepted 6 June 2001


   Abstract
Top
 Abstract
1. Methods
 2. Results
 3. Discussion
 Acknowledgments
 References
 
Objective: We investigated whether estradiol may prevent accelerated atherosclerosis due to hyperhomocysteinemia by enhancing the antioxidant system. Methods: Male Wistar rats were treated with placebo (P) or 1 mg (1E2) and 2 mg (2E2) 17β estradiol. Half of the animals (n=6) from each group received homocysteine (Hcy, 100 mg/kg/day) administered in the drinking water for 60 days (P/Hcy, 1E2/Hcy and 2E2/Hcy). Glutathione (GSH) content and glucose-6-phosphate dehydrogenase (G6PDH) activity were determined in myocardial tissues, as well as the serum Hcy concentrations and blood levels of hydrogen peroxide (H2O2). The relaxation response of aortic ring segments to acetylcholine (ACh) was used for the assessment of endothelial function, and hematoxylin-eosin stained thin sections of rat aorta were used for detection of the histological changes (namely endothelial damage and wall thickening). Results: Depression of relaxation to ACh occurred in P/Hcy compared to P (15.7±4% vs. 96.3±7%, P<0.0001), but estrogen significantly restored endothelium dependent relaxation in hyperhomocysteinemic rats (86.8±9.3%, P<0.001). Histological examination revealed aortic endothelial denudation in P/Hcy while the endothelial structures of the aorta from the 1E2/Hcy and 2E2/Hcy appeared normal. Significant reductions in GSH and G6PDH levels were detected in P/Hcy (1.5±0.01 µmol/g and 3.21±1.2 U/mg, respectively) compared to 1E2/Hcy (2.5±0.3 µmol/g and 12.81±1.5 U/mg, P<0.001) and 2E2/Hcy (3.11±1.1 µmol/g and 15.66±4 U/mg, P<0.001). In addition, blood H2O2 level in 1E2/Hcy and 2E2/Hcy remained low while it was raised significantly in P/Hcy compared to P (P<0.001). Conclusions: These data suggest that the observed reduction of GSH levels and suppression of G6PDH activity induced by Hcy coupled, with endothelial ultrastructural changes and impaired function, all reversed by estradiol, may have relevance to the mechanisms of atherogenesis and the beneficial effects of estrogen replacement therapy.

KEYWORDS Hormones; Atherosclerosis; Free radicals; Endothelial function; Gender; Vasoconstriction dilation

Hyperhomocysteinemia causes endothelial dysfunction and is recognized as an independent risk factor for atherosclerosis [1]. The initiating step of the atherogenic process is induced by generation of reactive oxygen species (ROS). ROS disrupts endothelial cell integrity, which in turn, can cause endothelial cell damage and predispose affected vessels to the subsequent development of atherosclerosis [2]. Atherosclerotic plaques similar to those found in the early stages of human arteriosclerosis have been observed in Hcy-treated experimental animals [3]. Increased levels of Hcy is a contributing factor to the development of premature atherosclerosis and an independent risk factor for stroke and myocardial infarction [4]. The mechanism whereby Hcy leads to endothelial cell damage is believed to be through its auto-oxidation to homocystine and H2O2 [5]. The beneficial estrogen-mediated effects on lipid metabolism, lipid-peroxidation, smooth-muscle-cell proliferation, hemostasis and vasomotion account for only 25–55% of the observed risk reduction, suggesting that other factors are involved [6]. The antioxidant property of estrogen is believed to be one of the mechanisms for its atheroprotective effects. The theory that the antioxidant potential of estrogen molecules is due to the presence of a phenol ring that scavenge oxygen free radicals is based only on observations made at high estrogen concentrations [7]. We have previously observed that estrogen up-regulates GSH content and G6PDH activity in dog myocardium [8]. GSH acts directly as a free radical scavenger by neutralizing H2O2, restores damaged molecules by hydrogen donation, reduces peroxides, and maintains protein thiols in the reduced state. The resistance of endothelial cells to oxidative stress is associated with high intracellular levels of GSH [9]. The reduction of GSSG (oxidized form of GSH) to GSH requires NADPH, produced in the hexose monophosphate shunt (HMPS) by the activity of G6PDH. On this basis, we hypothesized that estradiol treatment may prevent or reduce vascular injury resulting from H2O2 generation during hyperhomocysteinemia. To test this hypothesis, we assessed the effect of estradiol on the integrity of the endothelial lining of the aortic intima and endothelial function during experimentally induced hyperhomocysteinemia in male rats. To determine whether the beneficial effects of estradiol on hyperhomocysteinemia are related to its action on ROS and/or the antioxidant system, myocardial GSH content and G6PDH activity, as well as H2O2 blood levels were measured.


   1. Methods
Top
 Abstract
 1. Methods
2. Results
 3. Discussion
 Acknowledgments
 References
 
1.1. Animals and diet
Young male albino rats of the Wistar strain (Charles River Lab., Wilmington, MA; n=36) were used in all experiments. All rats weighed between 250 and 300 g. They were housed in groups of three in suspended screen-bottomed stainless steel cages at 23°C in a room with a 12-h light–dark cycle and fed with a commercial rodent diet. All protocols and procedures of this investigation were approved by the Animal Care and Use Committee of Georgetown University, conformed to the Guiding Principles of the American Physiological Society and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Rats were divided into six groups. Group P was the control (male rats without estradiol), Group 1E2 and 2E2 received subcutaneous implants of 1 and 2 mg 17β-estradiol administered in the form of 60-day slow release pellets (Innovative Research of America Inc.). Another three groups (P/Hcy, 1E2/Hcy and 2E2/Hcy) were treated with subcutaneously implanted estradiol in the same manner as the above groups, and moderate hyperhomocysteinemia was induced by adding Hcy (100 mg/kg/day) in the drinking water for 60 days.

After the 60-day period, animals were anesthetized with sodium pentobarbital (50 mg/kg) and sacrificed by exsanguination. Blood, plasma and serum were collected and immediately stored for the measurement of H2O2, Hcy and estradiol.

1.2. Estradiol concentration
Radioimmunoassay with Coat-A-Count Estradiol-6 kit (Diagnostic Products Co., CA) was used for the quantitative measurement of estradiol in serum. The sensitivity of the assay was 7.4 pg/ml.

1.3. Hcy concentration
For determination of Hcy levels in serum we used the HPLC method described by Jacobsen et al. [14]. Total serum Hcy concentration was analyzed by HPLC on a Varian Model 5000 liquid chromatograph with a Varian UV-50 detector, by using: (a) borohydride reduction of disulfide bonds and derivitization of thiols with monobromobimane; (b) perchloric acid precipitation of protein; and (c) HPLC analysis of thiols in the supernatant. Separation was performed on a RP C8 ultrasphere column (4.6x250 mm, Waters Co, MA) with a Brawnlee RP18 New Guard column. The excitation wavelength was set at 256 nm and the flow rate was 2 ml/min. The standard samples contained a mixture of known Hcy concentration and serum from control rats. The retention time for Hcy was estimated at 8.5 min.

1.4. Histological evaluation
At sacrifice the thoracic aortas were dissected and stored in Bouin's fixative and processed for light microscopy. The thickness of the post-mortem aortic sections was 1–3 mm (n=5 per aorta). The integrity of the endothelial lining around the aortic intima was determined from hematoxylin-eosin stained thin sections (5 µm, n=3 per section).

1.5. Assessment of endothelial function
Half of the aortas were kept in oxygenated Krebs-bicarbonate buffer, cut into 5 mm ring segments and used for bioassay. The aortic rings were suspended in 10-ml organ chambers. The tension was monitored using an isometric force transducer (Model 363, Harvard Apparatus, Dover, MA). Rings were maintained at 37°C in Krebs-bicarbonate buffer solution and bubbled continuously with a mixture of 95% O2 and 5% CO2. Rings were equilibrated at an initial tension determined from the passive tension-force relationship measured individually for each ring at the beginning of the experiment. After equilibration, rings were pre-contracted with KCl (80 mM) and the relaxation response to the endothelium-dependent vasodilator ACh (10–8–10–5) and endothelium-independent vasodilator, sodium nitroprusside (SNP) (10–10–10–7 M) were determined.

1.6. Glutathione concentration
Rat hearts were isolated, rapidly cleaned of connective tissue and immediately frozen in liquid nitrogen and kept frozen at –70°C until analysis. GSH-400'' colorimetric assay for glutathione (OXIS, International Inc) was used for the quantitative measurement of the myocardial content of GSH and its oxidized form GSSG. Myocardial homogenates were prepared by washing the tissue in ice-cold 0.9% NaCl solution, mincing in ice-cold 5% metaphosphoric acid (MPA) and centrifuging the homogenate at 3000 g, 4°C for 10 min. The upper clear aqueous layer was collected and kept at 0–4°C for assay (within 1 h); the samples were filtered through 0.2 µm filters [10]. The total GSH was determined spectrophotometrically at 412 nm. Commercially available GSH was used as the standard.

1.7. Protein concentration
Protein concentration of the samples was measured by the method described by Bradford [11]. The results are expressed as nmol/mg protein.

1.8. Hydrogen peroxide assays
H2O2 levels were determined in red blood cell samples, prepared by the method of Giulivi and Hochstein [12]. BIOXYTECH H2O2-560 kit (OXIS International, Inc.) was used for quantitative H2O2 assay, which is based on the oxidation of ferrous ions (Fe2+) to ferric ions (Fe3+) by hydrogen peroxide under acidic conditions. The ferric ion binds with the indicator dye xylenol orange to form a stable colored complex which was measured at 560 nm.

1.9. G6PDH activity
The myocardium samples were placed in a –4°C refrigerator and then in a Potter glass homogenizer in 8 vol. of ice-cold isotonic (0.15 M) KCl containing KHCO3 (8 ml of 0.02 M KHCO3) to maintain the pH at 7.0. The supernatants were centrifuged at 4000 g for 60 min at 4°C and dialyzed overnight against the same extracting medium at 2°C. G6PD activity was determined spectrophotometrically by following the rate of reduction of NADPH at 340 AU at room temperature. The reaction mixture consisted of 100 µl dialyzed myocardial supernatant, 500 µl 0.1 M MgCl2, 500 µl 0.25 M glycylglycine buffer (pH 7.6) and 0.2 mg NADPH in a total volume of 2.4 ml. The reaction was started by the addition of 100 µl of 0.05 M G6PDH [13].

1.10. Statistical methods
The results are presented as mean±S.E.M. Analysis was performed with StatView statistical software. ANOVA was used to compare the differences between groups of the mean values of GSH, G6PDH and H2O2. Two-tail Student's t tests were also performed to compare mean differences between estrogen-treated and estrogen-free groups for the total Hcy concentrations, estradiol levels, glutathione content, G6PDH activity, and hydrogen peroxide levels. A stepwise logistic regression analysis was preformed to evaluate the estradiol dose–response relationships. For purposes of this report, a value of P<0.05 was considered statistically significant.


   2. Results
Top
 Abstract
 1. Methods
 2. Results
3. Discussion
 Acknowledgments
 References
 
2.1. Estrogen levels
Hyperhomocysteinemia did not induce any significant changes in estradiol level in the animals on high Hcy diet compared to those on normal diet (P>0.5). Serum levels of 17 β-estradiol in P (±Hcy) were 24±14 pg/ml. Rats in 1E2 and 1E2/Hcy, receiving 1 mg of 17 β-estradiol had an estradiol level 155±30 pg/ml; rats in 2E2 and 2E2/Hcy had 2-fold higher level: 302±45 pg/ml (Table 1).


View this table:
[in this window]
[in a new window]

  		Table 1 Estradiol and homocysteine levels

 
2.2. Hcy levels
Hcy administration in the drinking water resulted in significantly elevated plasma Hcy concentrations, as compared to animals that did not received Hcy. Estradiol-treated rats had lower total Hcy concentrations (P<0.001) than those in non-estradiol treated groups (see Table 1).

2.3. Endothelial changes
In P/Hcy rats, hematoxylin-eosin-stained histological sections showed significant endothelial denudation and cell detachment affecting 20±8% of the aortic surface in all H&E thin sections, respectively (Fig. 1b). In 1E2/Hcy and 2E2/Hcy, the thoracic aortic segments revealed endothelial structure similar to those of controls (Fig. 1a and c).


Figure 1
View larger version (107K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Hematoxylin-eosin-stained histological sections (5 µm) of aortic segments (1 and 3 mm) from male rats on normal diet (1a); placebo rats on Hcy diet (1b); and rats on Hcy diet treated with 1 mg and 2 mg 17 β estradiol for 60 days (1c). Arrows point the endothelial layer. Magnification E.10x0.25.

 
2.4. Vascular reactivity responses to ACh and SNP
Evaluation of endothelium-dependent relaxation in P showed maximum relaxation response to ACh of 96.3±7%. Significantly depressed relaxation to ACh was observed in aortic rings from P/Hcy (15±4%), despite a normal relaxation to the endothelium-independent vasodilator SNP, suggesting endothelial dysfunction. Estrogen treatment had no effect on control rings (1E2 and 2E2), but the aortic segment from 1E2/Hcy and 2E2/Hcy rats showed significantly improved relaxation response to ACh (86.8±9.3%, P<0.001) compared to P/Hcy (see Fig. 2). The responses to SNP were not different between all groups studied (data not shown).


Figure 2
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Endothelium-dependent relaxation response to increasing concentrations of ACh in isolated rat aortic segments from placebo, and from Hcy-fed rats treated with placebo (Hcy) or with 1 and 2 mg 17 β estradiol (1E2/Hcy and 2E2/Hcy). Each point represents mean±S.E.M. of six observations. Endothelial-dependent relaxation responses to ACh are expressed as a percent of KCl induced contraction. *P<0.0001 vs. placebo treated rats on Hcy diet.

 
2.5. GSH content
Hcy caused significant depletion of intracellular myocardial GSH content (P vs. P/Hcy, P=0.02). Estradiol treatment dose-dependently attenuated Hcy-induced decreases in GSH levels (see Fig. 3). Myocardial GSH content was significantly increased in all estrogen-treated rats (1E1=4.16±0.9 nmol/mg protein; 2E2=5.38±0.79 nmol/mg protein; 1E2/Hcy=2.5±0.3 nmol/mg protein and 2E2/Hcy=3.11±1.1 nmol/mg protein) compared to placebo±Hcy rats (P=1.87±0.5 nmol/mg protein and P/Hcy=1.5±0.01 nmol/mg protein; P<0.001).


Figure 3
View larger version (25K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Myocardial GSH levels in rats on normal diet (white) and on Hcy diet (black); divided in placebo (P); estradiol 1 mg treated rats (1E2); estradiol 2 mg treated rats (2E2). *P=0.02 vs. P/Hcy; **P<0.001 vs. P/Hcy; ***P<0.001 vs. P.

 
2.6. H2O2levels
We found that in the RBC of P/Hcy, H2O2 levels were approximately 4-fold higher (7.96±1.5 nM/ml, P<0.0001) than in the other groups. In estradiol/Hcy treated rats (1E2/Hcy 1.5±0.2 nM and 2E2/Hcy 1.5±0.3 nM) estradiol attenuated H2O2 accumulation (see Fig. 4); in these groups, the H2O2 level was close to the level found in the non-Hcy treated rats (P=1.7±0.8 nM, 1E2 1.4±0.8 nM, 2E2 1.0±0.5 nM, P>0.05).


Figure 4
View larger version (64K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 H2O2 level in RBC from rats on a normal diet (white) and on a Hcy diet (black); divided in placebo (P); estradiol 1 mg treated rats (1E2); estradiol 2 mg treated rats (2E2). *P<0.0001 vs. all other groups; **P>0.05 vs. estradiol and placebo treated groups on a normal diet.

 
2.7. G6PDH activity
Both estrogen and Hcy were found to alter the activity of G6PDH. Hyperhomocysteinemia led to significant lowering of G6PDH activity (P/Hcy 3.21±1.2), compared to placebo rats (P 8.27±1.2 U/mg protein, P<0.001). Estradiol treatment dose dependently enhanced G6PDH activity in placebo rats (1E2 16.87±2 U/mg protein and 2E2 23.71±2 U/mg protein) and significantly moderated the negative effect of Hcy on the enzyme activity in hyperhomocysteinemic rats (1E2/Hcy 12.81±1.5 U/mg and 2E2/Hcy 15.66±4 U/mg, P<0.001 for both), (see Fig. 5).


Figure 5
View larger version (63K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Myocardial G6PDH activity in rats on normal diet (white) and on Hcy diet (black); divided in placebo (P); estradiol 1 mg treated rats (1E2); estradiol 2 mg treated rats (2E2). *P<0.001 vs. placebo on normal diet; **P<0.001 vs. placebo on Hcy diet.

 

   3. Discussion
Top
 Abstract
 1. Methods
 2. Results
 3. Discussion
Acknowledgments
 References
 
The unique finding of our study is that estradiol prevents Hcy-induced endothelial damage in male rats, extending our previous findings of the beneficial effects of estrogens on the cardiovascular system [15]. The observed ultrastructural damage and functional impairment of the endothelium associated with elevated Hcy levels were related primarily to the generation of H2O2, depletion of myocardial GSH content and diminished G6PDH activity. Our results demonstrate that estrogen mediates its protective effect against Hcy-induced oxidative stress by increasing, dose-dependently, myocardial GSH level and G6PDH activity. A number of studies have suggested that estradiol is responsible for the sex-linked differences in the glutathione peroxidase (GSHPx) activity, and the rate of GSH oxidation and lipid peroxidation, as well as the enhanced hepatic and myocardial G6PDH activity [8,16–18]. Our results are consistent with those observations. Furthermore, we determined that estradiol/Hcy-treated rats had considerably higher GSH levels and G6PDH activity (P<0.001) despite being on a Hcy-enhanced diet. Additionally, we found that the Hcy-mediated impact on the endothelial function and integrity correlates not only with an intensified H2O2 formation, as reported previously [19], but also with suppressed G6PDH activity and decreased GSH availability in the myocardial tissue. G6PDH regulates formation of NADPH+, which is the principal H+ donor for plasma aminothiols and GSH. Under normal conditions the balance of the redox-reaction is in the direction of maintaining cellular glutathione in its reduced form (GSHí99%) [20,21]. The antioxidant effects of estradiol described in this study may be based on driving the cellular thiol status towards this state. Hyperhomocysteinemia is associated with a decrease in the ratio of reduced to total plasma aminothiols and an increased pro-oxidant activity [22], augmented lipid oxidation [23], higher VLDL and cholesterol levels [24], decreased NO concentrations [25], and diminished vasodilator response [26]. The most important beneficial effects of estradiol on CVD are opposite of those harmful effects induced by Hcy. We also observed that the beneficial effects of estradiol on endothelium were related closely to lower serum Hcy concentrations. This finding is consistent with recently published statement from the Third National Health and Nutrition Examination Survey [27], "...higher estrogen status is associated with a decreased mean serum total Hcy concentration, independently of nutritional status and muscle mass, and that estrogen may explain the previously reported male-female difference in total homocysteine concentration". To our knowledge, this is the first report to show that estradiol-mediated decreases in Hcy serum levels are associated with a very significant reduction of Hcy-mediated atherogenetic effects on the endothelium. In this sense, one question that remains unanswered is whether the anti-atherogenic effect of estradiol is due simply to the consequences of anti-oxidant system modification or is part of a more sophisticated mechanism regulating Hcy levels. This may be an additional mechanism underling the cardioprotective effects of hormonal replacement therapy, and requires further studies.

Estrogen replacement therapy (ERT) in postmenopausal women has shown to reduce the risk of myocardial infarction in users compared to non-users by 44–50% [28,29]. On the other hand, the first two randomized double blind, placebo-controlled studies (HERS) did not support these findings [30,31]. Recently, Post et al. [32] proposed that a higher degree of ER methylation in a subpopulation of atherosclerotic tissue cells, when compared to normal aortas, might cause a lack of ER gene expression. This lack of expression may result in an inability to respond to estrogen's protective effects. The methylated and inactivated ER gene in diseased vessels may inhibit the potential protective effects of estrogens in women with advanced atherosclerotic disease and therefore, account for the lack of benefit observed in the HERS study. The results from our study support the theory that estradiol has a beneficial effect on preserving the endothelial integrity and function in condition of Hcy-induced oxidative stress.

Time for primary review 26 days.


   Acknowledgments
Top
 Abstract
 1. Methods
 2. Results
 3. Discussion
 Acknowledgments
References
 
This study was supported by Grant In Aid 9960347U from American Heart Association. The authors wish to thank Dr Lester D.R. Thompson from Army Forced Institute of Pathology (AFIP) for the preparation and scoring of the aortic slides and photos.


   References
Top
 Abstract
 1. Methods
 2. Results
 3. Discussion
 Acknowledgments
 References
 

  1. McCully K.S. Vascular pathology of homocysteinemia: implications for the pathogenesis of atherosclerosis. Am J Pathol (1969) 56:111–128.[Web of Science][Medline]
  2. Kanani P., Sinkey C., Browning R., et al. Role of oxidant stress in endothelial dysfunction produced by experimental hyperhomocysteinemia in humans. Circulation (1999) 100:1161–1168.[Abstract/Free Full Text]
  3. Lentz S., Sobey C., Piegors D., et al. Vascular dysfunction in monkeys with diet-induced hyperhomocysteinemia. J Clin Invest (1996) 98:24–29.[Web of Science][Medline]
  4. Stampfer M., Malinow M., Willett W., et al. A prospective study of plasma homocysteine and risk factor of myocardial infarction in US physicians. J Am Med Assoc (1992) 268:877–881.[Abstract/Free Full Text]
  5. Loscalzo J. The oxidant stress of hyperhomocysteinemia. J Clin Invest (1996) 98:5–7.[Web of Science][Medline]
  6. Bush T., Barrett-Connor E., Cowan L., et al. Cardiovascular mortality and non-contraceptive use of estrogen in women: result from the Lipid Research Clinics Program Follow-up Study. Circulation (1987) 75:1102–1109.[Abstract/Free Full Text]
  7. Romer W., Oettel M., Droescher P., Schwarz S. Novel ‘scavestrogens’ and their radical scavenging effects, iron-chelating, and total antioxidative activities: delta 8,9-dehydro derivatives of 17 alpha-estradiol and 17 beta-estradiol. Steroids (1997) 62:304–310.[CrossRef][Web of Science][Medline]
  8. Kim Y., Farhat M., Myers A., et al. 17-Beta estradiol regulation of myocardial glutathione and its role in protection against myocardial stunning in dogs. J Cardiovasc Pharmacol (1998) 32:457–465.[CrossRef][Web of Science][Medline]
  9. Lappena D., De Gioia S., Ciofani G., et al. Glutathione-related antioxidant defenses in human atherosclerotic plaques. Circulation (1998) 97:1930–1934.[Abstract/Free Full Text]
  10. Anderson M. Determination of glutathione and glutathione disulfide in biological samples. Methods Enzymol (1985) 113:548–551.[Web of Science][Medline]
  11. Bradford M. A rapid and sensitive method for the quantitation of micrograms quantities of protein utilizing the principle of protein-day binding. Anal Biochem (1976) 72:248.[CrossRef][Web of Science][Medline]
  12. Giulivi C., Hochstein P., Davis K. Hydrogen peroxide production by red blood cells. Free Radic Biol Med (1994) 16:123–129.[CrossRef][Web of Science][Medline]
  13. Glock G., McLean P. Further studies on the properties and assay of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase of rat liver. Biochemistry (1953) 55:400–408.
  14. Jacobsen D., Gatautis V., Green R., et al. Rapid HPLC determination of total homocysteine and other thiols in serum and plasma: sex differences and correlation with cobalamin and folate concentrations in healthy subjects. Clin Chem (1994) 40:873–881.[Abstract/Free Full Text]
  15. Kim Y., Chen B., Beaurgard T., Kouretas P., Thomas G., Farhat M.Y., Myers A.K., Lees D.E. 17 β-estradiol prevents dysfunction of canine coronary endothelium and myocardium, and reperfusion arrhythmias following brief ischemia-reperfusion. Circulation (1996) 94:2901–2908.[Abstract/Free Full Text]
  16. Pinto R., Bartley W. The nature of the sex-linked differences in glutathione peroxidase activity and aerobic oxidation of glutathione in male and female rat liver. Biochem J (1969) 115:449–456.[Web of Science][Medline]
  17. Ibim S., Randall R., Han P., et al. Modulation of hepatic glucose-6-phosphate dehydrogenase activity in male and female rats by estrogen. Life Sci (1994) 45:1559–1565.
  18. Sugioka K., Shimosegawa Y., Nakano M. Estrogens as natural antioxidants of membrane phopholipid peroxidation. FEBS Lett (1987) 210:37–39.[CrossRef][Web of Science][Medline]
  19. Starkebaum G., Harlan J. Endothelial cell injury due to copper catalyzed hydrogen peroxide generation from homocysteine. J Clin Invest (1993) 77:1370–1376.[CrossRef]
  20. Ueland P., Mansoor M., Guttormsen A., et al. Reduced, oxidized and protein-bound forms of homocysteine and other aminothiols in plasma comprise the redox thiol status-a possible element of the extracellular antioxidant defense system. J Nutr (1996) 126:1281S–1284S.[Abstract/Free Full Text]
  21. Winterbourn C., Metodieva D. The reaction of superoxide with reduced glutathione. Arch Biochem Biophys (1994) 314:284–290.[CrossRef][Web of Science][Medline]
  22. Mansoor A., Bergmark C., Svardal A., et al. Redox status and protein-binding of plasma homocysteine and other aminothiols in patients with early-onset peripheral vascular disease. Arterioscler Thromb Vasc Biol (1995) 15:232–240.[Abstract/Free Full Text]
  23. Loscalzo J. The oxidant stress of hyperhomocysteinemia. J Clin Invest (1996) 98:5–7.[Web of Science][Medline]
  24. Foody J., Milberg J., Robinson K., Pearce G., Jacobsen D.W., Sprecher D.L. Homocysteine and lipoprotein(a) interact to increase CAD risk in young men and women. Arterioscler Thromb Vasc Biol (2000) 20:493–499.[Abstract/Free Full Text]
  25. Pruefer D., Scalia R., Lefer A. Homocysteine provokes leukocyte-endothelium interaction by downregulation of nitric oxide. Gen Pharmacol (1999) 33:487–498.[CrossRef][Web of Science][Medline]
  26. Lentz S., Malinow M., Piegors D., Bhopatkar-Teredesai M., Faraci F., Heistad D. Consequences of hyperhomocyst(e)inemia on vascular function in atherosclerotic monkeys. Arterioscler Thromb Vasc Biol (1997) 17:2930–2934.[Abstract/Free Full Text]
  27. Morris M.S., Jacques P.E., Selhub J., Rosenberg I.H. Total homocysteine and estrogen status indicators in the Third National Health and Nutritional Examination Survey. Am J Epidemiol (2000) 152:140–148.[Abstract/Free Full Text]
  28. Wenger N., Speroff L., Packard M. Cardiovascular health and disease in women. N Engl J Med (1993) 329:247–256.[Free Full Text]
  29. Barrett-Connor E., Bush T.L. Estrogen and coronary heart disease in women. Clin Cardiol (1991) 265:1861–1867.
  30. Hulley S., Grady D., Bush T., Furberg C., Herrington D., Riggs B., Vittinghoff E. Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. J Am Med Assoc (1998) 280:605–613.[Abstract/Free Full Text]
  31. Herrington D., Reboussin D., Brosnihan B., Sharp P., Shumaker S., Snyder T., Furberg C., Kowalchuk G., Stukey T., Rogers W., Givens D., Waters D. Effects of estrogen replacement on the progression of coronary-artery atherosclerosis. N Engl J Med (2000) 343:522–529.[Abstract/Free Full Text]
  32. Post W., Goldschmidt-Clermont P., Wilhide C., et al. Methylation of estrogen receptor gene is associated with aging and atherosclerosis in the cardiovascular system. Cardiovasc Res (1999) 43:985–991.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Eur J EndocrinolHome page
H. R. N. Pour, D. E Grobbee, M. Muller, M. Emmelot-Vonk, and Y. T van der Schouw
Serum sex hormone and plasma homocysteine levels in middle-aged and elderly men
Eur. J. Endocrinol., December 1, 2006; 155(6): 887 - 893.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Dimitrova, K. R.
Right arrow Articles by Kim, Y. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dimitrova, K. R.
Right arrow Articles by Kim, Y. D.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?