doi: 10.1161/hq0801.093507
© 2001 American Heart Association, Inc.
Vascular Biology |
Electron Paramagnetic Resonance Investigation on Modulatory Effect of 17ß-Estradiol on Membrane Fluidity of Erythrocytes in Postmenopausal Women
From the Division of Cardiology (K.T., Y.K., K.K., I.N.), Department of Medicine, Wakayama Medical University, Wakayama, Japan, and Tokyo Rosai Hospital (Y.M.), Tokyo, Japan.
Correspondence to Kazushi Tsuda, MD, Division of Cardiology, Department of Medicine, Wakayama Medical University, Kimiidera 811-1, Wakayama 641-8509, Japan. E-mail tsudak{at}mail.wakayama-med.ac.jp
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Abstract |
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Abstract—— Many studies have shown that estrogen may exert cardioprotective effects and reduce the risk of hypertension and coronary events. On the other hand, it has been proposed that cell membrane abnormalities play a role in the pathophysiology of hypertension, although it is not clear whether estrogen would influence membrane function in essential hypertension. The present study was performed to investigate the effects of 17ß-estradiol (E2) on membrane fluidity of erythrocytes in normotensive and hypertensive postmenopausal women. We determined the membrane fluidity of erythrocytes by means of an electron paramagnetic resonance and spin-labeling method. In an in vitro study, E2 significantly decreased the order parameter for 5-nitroxide stearate and the peak height ratio for 16-nitroxide stearate obtained from electron paramagnetic resonance spectra of erythrocyte membranes in normotensive postmenopausal women. The finding indicates that E2 might increase the membrane fluidity of erythrocytes. The effect of E2 was significantly potentiated by the NO donor, S-nitroso-N-acetylpenicillamine, and a cGMP analogue, 8-bromo-cGMP. In contrast, the change in the membrane fluidity evoked by E2 was attenuated in the presence of the NO synthase inhibitor, NG-nitro-L-arginine methyl ester, and asymmetric dimethyl-L-arginine. In hypertensive postmenopausal women, the membrane fluidity of erythrocytes was significantly lower than that in normotensive postmenopausal women. The effect of E2 on membrane fluidity was significantly more pronounced in the erythrocytes of hypertensive postmenopausal women than in the erythrocytes of normotensive postmenopausal women. The results of the present study showed that E2 significantly increased the membrane fluidity and improved the microviscosity of erythrocyte membranes, partially mediated by an NO- and cGMP-dependent pathway. Furthermore, the greater action of E2 in hypertension might be consistent with the hypothesis that E2 could have a beneficial effect in regulating rheological behavior of erythrocytes and could have a crucial role in the improvement of the microcirculation in hypertension.
Key Words: 17ß-estradiol • nitric oxide • membrane fluidity • erythrocytes • postmenoapusal women
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Introduction |
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There is a strong link between menopause and an increased incidence of cardiovascular diseases, and recent studies have demonstrated that estrogen replacement therapy reduced morbidity or mortality due to coronary heart diseases in postmenopausal women.1–4 It has also been shown that estrogen supplementation induced a significant fall in blood pressure in postmenopausal women.5 These findings propose the idea that estrogen may exert protective effects against arteriosclerosis and hypertension. However, fundamental mechanisms of cellular and molecular events by estrogen are still unclear.
It has been proposed that cell membrane abnormalities are an etiological factor in hypertension, including functional abnormalities, such as transmembrane cation fluxes.6–8 An electron paramagnetic resonance (EPR) and spin-labeling method have been developed to elucidate the membrane fluidity and perturbations of the membrane function by external agents.9 The membrane fluidity is a physicochemical feature of biomembranes and is an important factor in modulating the cell rheological behavior.10 We have shown previously that the membrane fluidity of erythrocytes is significantly lower in spontaneously hypertensive rats and in patients with essential hypertension than in the normotensive controls,11–14 and we have proposed that abnormal microviscosity of the cell membranes might contribute to blood pressure elevation. However, it is not clear whether estrogen influences the membrane fluidity of erythrocytes. In the present study, to assess the modulatory action of estrogen on the membrane function, we investigated the effects of 17ß-estradiol (E2) on the membrane fluidity of erythrocytes in normotensive and hypertensive postmenopausal women by means of the EPR and spin-labeling method.
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Methods |
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Study I
Subjects
To investigate the effects of E2 on the membrane fluidity in vitro, erythrocytes were obtained from postmenopausal women who were normotensive volunteers (n=78, aged 61±1 [mean±SEM] years, blood pressure 130.0±2.4/79.1±1.5 mm Hg, heart rate 76.8±1.4 bpm, body mass index 22.8±0.3 kg/m2, and plasma E2 concentration 4.5±0.7 pg/mL). Written informed consent was obtained from all volunteers.
Effects of E2 Alone on Membrane Fluidity of Erythrocytes in Normotensive Postmenopausal Women In Vitro
Blood samples were obtained in patients by venipuncture after a minimum of 30 minutes of bed rest while fasting. After plasma and buffy coat were carefully removed by centrifugation at 155g for 10 minutes at 4°C, washed erythrocytes were resuspended in the isotonic buffer (140 mmol/L NaCl and 20 mmol/L Tris-HCl, pH 7.4) at a hematocrit of 50%. The erythrocyte suspension (100 µL erythrocytes and 100 µL Tris-HCl buffer, 200 µL total) was incubated for 2 hours at 37°C in the NaCl-Tris buffer (100 µL) containing E2 (1x10-9 to 1x10-6 mol/L) alone or 17
-estradiol (1x10-7 to 1x10-5 mol/L) alone, because the preliminary examination demonstrated that the maximal effect of E2 on the membrane fluidity of erythrocytes was obtained after a 2-hour incubation at 37°C. After incubation with E2, 100 µL of the solution containing fatty acid spin-label agents (5-nitroxide stearate [5-NS] and 16-nitroxide stearate [16-NS], 5x10-5 mol/L) was added to the erythrocyte suspension (300 µL). The mixed solution was then incubated for 2 hours at 37°C with gentle shaking, and the EPR measurements were performed.
Effects of E2 in Combination With SNAP and 8-Bromo-cGMP on Membrane Fluidity of Erythrocytes in Normotensive Postmenopausal Women In Vitro
To examine the effects of E2 in combination with an NO donor and cGMP, erythrocytes (100 µL) were pretreated with the same volume of Tris-HCl solution containing S-nitroso-N-acetylpenicillamine (SNAP) or a cGMP analogue (8-bromo-cGMP) before the application of E2. After a 2-hour incubation with 100 µL E2 (1x10-7 to 1x10-6 mol/L) at 37°C, 100 µL of the solution containing fatty acid spin-label agents (5-NS and16-NS, 5x10-5 mol/L) was added to the erythrocyte suspension (300 µL). The mixed solution was then incubated for 2 hours at 37°C with gentle shaking, and the EPR measurements were performed.
Effects of E2 in Combination With L-NAME and ADMA on Membrane Fluidity of Erythrocytes in Normotensive Postmenopausal Women
To examine the effects of NG-nitro-L-arginine methyl ester (L-NAME) and asymmetric dimethyl-L-arginine (ADMA), erythrocytes (100 µL) were pretreated with the same volume of Tris-HCl solution containing L-NAME (1x10-5 mol/L) or ADMA (1x10-4 mol/L) before the application of E2 (1x10-7 to 1x10-6 mol/L). The mixed solution was incubated at 37°C for 2 hours. Then the spin-label agents (5-NS and 16-NS in 100 µL of Tris-HCl buffer, 5x10-5 mol/L) were added to the erythrocyte suspension. After a 2-hour incubation at 37°C with gentle shaking, the EPR measurements were performed.
EPR Measurements of Erythrocytes
The EPR measurements were performed by using an EPR spectrometer (model Jeol JES-FE2XG, Nihon Denshi) with a microwave unit (model Jeol ES-SCXA, Nihon Denshi).11–14 The microwave power was 5 mW, and the modulation frequency was 100 KHz, with a modulation amplitude of 2.0 G. The temperature of the measurement was controlled at 30°C. The receiver scan width was 3280±50 G, with a sweep time of 8 minutes, and receiver gain was 4.0x103 to 7.9x103, with a response time of 1.0 second.
The fatty acid spin-label agents (5-NS and 16-NS) are believed to be anchored at the lipid-aqueous interface of the cell membranes by their carboxyl ends, whereas the nitroxide group moves rapidly through a restricted angle around the point of attachment.11–15 Therefore, the EPR spectra of the fatty acid spin-label agents are used to detect an alteration in the freedom of motion in biological membranes and to provide an indication of membrane fluidity.11–15 In addition, 5-NS could serve as an example of the properties of superficial membrane layers, whereas 16-NS could be an indicator referring to more hydrophobic core of the lipid membranes. For indexes of membrane fluidity, we have evaluated the values of outer and inner hyperfine splitting (2T’|| and 2T’
in gauss, respectively) in the EPR spectra for 5-NS and calculated the order parameter from 2T’|| and 2T’.11–15 In the EPR spectra for 16-NS, we used the peak height ratio (ho/h-1) for an index of the membrane fluidity.14,15 The greater the values of the order parameter and ho/h-1, the lesser is the freedom of motion of the spin labels in the biomembrane bilayers, indicating lower membrane fluidity.11–15
Study II
Subjects and Protocol
Twenty-eight postmenopausal women with mild essential hypertension were studied and compared with 33 age-matched normotensive postmenopausal women. The characteristics of the hypertensive patients and normotensive subjects are given in the Table. Written informed consent was obtained from all participants after they were informed about the nature and objective of the study. All hypertensive patients had no cardiovascular complications and had no mediation at least 4 weeks before the EPR study. In addition, they had no other diseases, such as hematological or hepatic disorders. The effect of E2 (1x10-7 and 1x10-6 mol/L) on membrane fluidity of erythrocytes in vitro was compared between hypertensive and normotensive subjects by means of EPR and the spin-labeling method.
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Measurement of Plasma E2 Concentration
Plasma E2 concentration was measured with a radioimmunoassay kit (Shionogi Co, Ltd).
Drugs
E2 was obtained from Biomedical Technologies Inc, and its stereoisomer, 17-estradiol, was obtained from Sigma Chemical Co. The spin label agents, 5-NS and 16-NS, were purchased from Aldrich Co, Ltd. SNAP, 8-bromo-cGMP, L-NAME, and ADMA were obtained from Funakoshi Co, Ltd. All other drugs were standard laboratory reagents of analytical grade.
Statistical Analysis
Values are expressed as mean±SEM. The differences between the means of the drug treatment and their corresponding controls were tested with a 1-way ANOVA. To compare the means of the different study groups, the Wilcoxon signed rank sum test was used. The differences between hypertensive and normotensive postmenopausal women were analyzed with a 2-way ANOVA, followed by the Mann-Whitney U test. A value of P<0.05 was accepted as the level of significance.
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Results |
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Effects of E2 Alone on Membrane Fluidity of Erythrocytes in Normotensive Postmenopausal Women In Vitro
E2 (1x10-9 to 1x10-6 mol/L) decreased the order parameter for 5-NS and peak height ratio (ho/h-1) for 16-NS obtained from erythrocyte membranes in normotensive volunteers in a dose-dependent manner (order parameter was as follows: control, 0.716±0.001 [n=52]; 1x10-9 mol/L E2, 0.696±0.001 [n=52], P<0.01; 1x10-8 mol/L E2, 0.688±0.002 [n=52], P<0.01; 1x10-7 mol/L E2, 0.683±0.001 [n=52], P<0.01; and 1x10-6 mol/L E2, 0.679±0.002 [n=52], P<0.01; ho/h-1 was as follows: control, 5.10±0.02 [n=52]; 1x10-9 mol/L E2, 4.93±0.02 [n=52], P<0.01; 1x10-8 mol/L E2, 4.85±0.02 [n=52], P<0.01; 1x10-7 mol/L E2, 4.78±0.02 [n=52], P<0.01; and 1x10-6 mol/L E2, 4.74±0.02 [n=52], P<0.01). This finding shows that E2 increased the membrane fluidity of erythrocytes. On the other hand, 17
-estradiol, the stereoisomer of E2, showed no significant effects of membrane fluidity of erythrocytes (order parameter was as follows: control, 0.711±0.004 [n=5]; 1x10-7 mol/L 17-estradiol, 0.711±0.006 [n=5]; 1x10-6 mol/L 17-estradiol, 0.715±0.008 [n=5]; and 1x10-5 mol/L 17-estradiol, 0.715±0.009 [n=5]; ho/h-1 was as follows: control, 5.24±0.08 [n=5]; 1x10-7 mol/L 17-estradiol, 5.30±0.12 [n=5]; 1x10-6 mol/L 17-estradiol, 5.30±0.11 [n=5]; and 1x10-5 mol/L 17-estradiol, 5.29±0.10 [n=5]).
Effects of E2 in Combination With SNAP or 8-Bromo-cGMP on Membrane Fluidity of Erythrocytes
A preliminary study showed that SNAP alone reduced the values of the order parameter and ho/h-1 of erythrocyte membranes (order parameter was as follows: control, 0.716±0.004 [n=6]; 5x10-6 mol/L SNAP, 0.712±0.007 [n=6]; and 5x10-5 mol/L SNAP, 0.688±0.007 [n=6], P<0.05; ho/h-1 was as follows: control, 5.15±0.03 [n=6]; 5x10-6 mol/L SNAP, 5.11±0.06 [n=6]; and 5x10-5 mol/L SNAP, 4.76±0.09 [n=6], P<0.05). In the present experiment, it was clearly demonstrated that the effect of E2 on the fluidity was significantly potentiated by a low concentration of SNAP (5x10-6 mol/L), which showed no effects of its own (Figure 1).
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Similarly, the cGMP analogue, 8-bromo-cGMP, reduced the values of the order parameter and ho/h-1 of erythrocyte membranes (order parameter was as follows: control, 0.704±0.004 [n=6]; 1x10-6 mol/L 8-bromo-cGMP, 0.703±0.002 [n=6]; and 1x10-5 mol/L 8-bromo-cGMP, 0.686±0.004 [n=6], P<0.05; ho/h-1 was as follows: control, 5.31±0.05 [n=6]; 1x10-6 mol/L 8-bromo-cGMP, 5.30±0.05 [n=6]; and 1x10-5 mol/L 8-bromo-cGMP, 5.08±0.06 [n=6], P<0.05). The effect of E2 on the fluidity was significantly enhanced in the presence of a low concentration (1x10-6 mol/L) of 8-bromo-cGMP (Figure 2), although this concentration of 8-bromo-cGMP alone showed no significant effects on the membrane fluidity of its own.
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Effects of E2 in Combination With L-NAME and ADMA on Membrane Fluidity of Erythrocytes
Figure 3 shows the effects of E2 on the membrane fluidity of erythrocytes in the presence of L-NAME (1x10-5 mol/L). The effect of E2 was significantly attenuated in the presence of L-NAME. Similarly, ADMA (1x10-4 mol/L) significantly counteracted the E2-induced changes in membrane fluidity of erythrocytes (Figure 4).
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Membrane Fluidity of Erythrocytes in Postmenopausal Women With Essential Hypertension and Normotensive Postmenopausal Women
The values of the order parameter and ho/h-1 of the EPR spectra were significantly greater in postmenopausal women with essential hypertension than in age-matched normotensive postmenopausal women (order parameter was as follows: hypertensive group, 0.722±0.002 [n=28]; normotensive group, 0.712±0.002 [n=33], P<0.01; ho/h-1 was as follows: hypertensive group, 5.25±0.03 [n=28]; normotensive group, 5.10±0.02 [n=33], P<0.01). The finding indicated that the erythrocyte membrane fluidity was decreased in postmenopausal women with essential hypertension compared with normotensive postmenopausal women.
Effects of E2 on Membrane Fluidity of Erythrocytes in Postmenopausal Women With Essential Hypertension and Normotensive Postmenopausal Women
The preliminary study showed that the effect of E2 on membrane fluidity of erythrocytes was also reversed in the presence of L-NAME in hypertensive postmenopausal women (order parameter was as follows: control, 0.723±0.002 [n=7]; 10-7 mol/L E2, 0.688±0.003 [n=7], P<0.05 versus control; 10-6 mol/L E2, 0.685±0.004 [n=7], P<0.05 versus control; 10-5 mol/L L-NAME alone, 0.724±0.003 [n=7]; 10-7 mol/L E2 plus 10-5 mol/L L-NAME, 0.725±0.003 [n=7], P<0.05 versus the same concentration of E2 alone; and 10-6 mol/L E2 plus 10-5 mol/L L-NAME, 0.722±0.004 [n=7], P<0.05 versus the same concentration of E2 alone; ho/h-1 was as follows: control, 5.38±0.05 [n=7]; 10-7 mol/L E2, 5.11±0.06 [n=7], P<0.05 versus control; 10-6 mol/L E2, 4.97±0.07 [n=7], P<0.05 versus control; 10-5 mol/L L-NAME alone, 5.39±0.05 [n=7]; 10-7 mol/L E2 plus 10-5 mol/L L-NAME, 5.37±0.06 [n=7], P<0.05 versus the same concentration of E2 alone; and 10-6 mol/L E2 plus 10-5 mol/L L-NAME, 5.37±0.04 [n=7], P<0.05 versus the same concentration of E2 alone).
E2 (1x10-7 and 1x10-6 mol/L) decreased the order parameter (increased the membrane fluidity) to a greater extent in hypertensive postmenopausal women than in normotensive postmenopausal women (percent change in order parameter was as follows: for 1x10-7 mol/L E2, -5.4±0.2% in hypertensive group [n=28] and -3.2±0.2% in normotensive group [n=33], P<0.01; for 1x10-6 mol/L E2, -5.9±0.3% in hypertensive group [n=28] and -3.7±0.2% in normotensive group [n=33], P<0.01). Similarly, the effect of E2 on ho/h-1 was also more pronounced in the erythrocytes of hypertensive postmenopausal women than in the erythrocytes of normotensive postmenopausal women (percent change in ho/h-1 was as follows: for 1x10-7 mol/L E2, -6.4±0.3% in hypertensive group [n=28] and -3.9±0.3% in normotensive group [n=33], P<0.01; and for 1x10-6 mol/L E2, -8.0±0.3% in hypertensive group [n=28] and -4.7±0.3% in normotensive group [n=33], P<0.01).
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Discussion |
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Estrogen replacement therapy may provide beneficial effects on blood pressure and other cardiovascular regulations. However, the precise mechanisms underlying estrogen effects are unclear. In the present study, we investigated the effects of E2 on the membrane fluidity of erythrocytes in postmenopausal women by means of EPR and the spin-labeling method. We showed that E2 dose-dependently decreased the order parameter for 5-NS and the peak height ratio (ho/h-1) for 16-NS obtained from EPR spectra of erythrocyte membranes. On the other hand, 17
-estradiol, an inactive stereoisomer of E2, showed no significant effects on membrane fluidity. These findings indicated that E2 significantly increased the membrane fluidity of erythrocytes in postmenopausal women. Because membrane fluidity is inversely correlated with membrane microviscosity, it would be possible that the membrane action of E2 could be one of the mechanisms responsible for its beneficial effects in improving the rheological behavior of erythrocyte membranes. In the present study, we used a concentration range of 10-9 to 10-6 mol/L for E2. The concentrations might be higher than those expected by the endogenous E2 content in human plasma.16 However, in an in vitro preparation, higher dosages were necessary because the compound could be gradually inactivated by degradation. It is well recognized that signal transduction induced by estrogen is mediated through intranucleus estrogen receptors (genomic receptors). Recently, it has also been shown that nongenomic estrogen receptors are present on the membranes.17–19 It was reported that when erythrocytes were incubated with estrogen in vitro, two thirds was bound to the membrane, whereas one third was in the soluble fraction.20 Puca and Sica21 provided evidence for the existence of specific and high-affinity binding components to estrogen in the cytoskeletal matrix of the erythrocyte membranes. However, it is still uncertain whether erythrocytes might bear the specific receptors for estrogen, and the nonspecific action of estrogen cannot be fully excluded.
In the present study, it has also been clearly shown that the effect of E2 is significantly potentiated by a low concentration of SNAP, an NO donor, and a cGMP-analogue, 8-bromo-cGMP, which have no effects by themselves. These synergistic effects suggest that the action of E2 might be, at least in part, mediated by the NO- and cGMP-related pathway. The hypothesis was confirmed by the finding that the effects of E2 were blocked by L-NAME and ADMA, the NO synthase inhibitors. NO is a potent stimulator of guanylate cyclase activity and is produced by different isoforms of NO synthase.22 Jubelin and Gierman23 have shown that erythrocytes of rats and humans are positive for NO synthase, which indicates that erythrocytes possess all the cellular machinery to synthesize their own NO. They proposed that erythrocytes would synthesize and use NO to modulate their own physiology. We also reported that NO might have a crucial role in the regulation of the membrane fluidity of erythrocytes.24 In other tissues, it has been demonstrated that the effects of estrogen might, at least in part, be mediated by the production of NO.25–28 These previous findings coupled with our present results suggest that NO might play a role in estrogen-induced alterations in membrane properties, although further studies should be conducted to assess more thoroughly the relationship between NO and estrogen effects on the membrane function.
The values of the order parameter and ho/h-1 obtained from the erythrocyte EPR spectra were significantly greater in postmenopausal women with essential hypertension than in normotensive postmenopausal women. The results suggest that the membrane fluidity of erythrocytes was lower in hypertensive postmenopausal women than in normotensive postmenopausal women and confirm our previous reports showing that the cell membranes were stiffer and less fluid in primary hypertension.11–15 If the deformability of erythrocytes is highly dependent on the membrane fluidity,10,29 the reduction in membrane fluidity could cause a disturbance in the blood rheological behavior and in the microcirculation, which might contribute to the pathophysiology of hypertension. The present study also demonstrated that E2 increased the membrane fluidity of erythrocytes to a greater extent in hypertensive postmenopausal women than in normotensive postmenopausal women. The finding might be consistent with our previous report showing that the effect of the NO donor, SNAP, on the erythrocyte membrane fluidity was more pronounced in patients with essential hypertension than in normotensive subjects.24 Although the precise role of estrogen in the regulation of membrane fluidity in hypertension is still unclear, one hypothesis is that estrogen may improve membrane fluidity and contribute to the defense against a further increase in microviscosity in hypertension.
In summary, the results of the present study showed that E2 increased membrane fluidity of erythrocytes in postmenopausal women. The effects were mediated, at least to some extent, by the NO- and cGMP-dependent pathway. Our data also suggest that estrogen may have a crucial modulatory action on erythrocyte membrane fluidity that may also be of considerable biological and clinical significance in determining rheological properties of the membranes. Furthermore, the greater action of E2 in hypertension might be consistent with the hypothesis that estrogen could have a beneficial effect on erythrocyte membrane function and the microcirculation in hypertensive postmenopausal women.
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Acknowledgments |
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This study was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan (05670631, 08670819, and 10670674), the Japan Clinical Pharmacology Foundation (1992 and 1999), the Naito Foundation (1993), the Uehara Memorial Foundation (1994 and 1999), the Kimura Foundation for Cardiovascular Diseases (1996), and the Mitsui Foundation(1999).
Received March 26, 2001; accepted May 10, 2001.
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 K. Tsuda A New Hormone Therapy With Drospirenone and NO Production in Postmenopausal Women Hypertension, November 1, 2006; 48(5): e106 - e106. [Full Text] [PDF]
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K. Tsuda Surgical Menopause, Salt Sensitivity, and NO Bioavailability in Women Hypertension, September 1, 2006; 48(3): E12 - E12. [Full Text] [PDF]
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K. Tsuda Sex Hormones and Carotid Atherosclerosis in the Metabolic Syndrome Stroke, December 1, 2005; 36(12): 2528 - 2528. [Full Text] [PDF]
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 K. Tsuda, I. Nishio, E. Ros, I. Nunez, A. Perez-Heras, M. Serra, R. Gilabert, E. Casals, and R. Deulofeu Modulation of Endothelial Function by Walnuts and Sex Hormones * Response Circulation, August 17, 2004; 110(7): e73 - e73. [Full Text] [PDF]
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K. Tsuda, I. Nishio, P. J. Kelly, and K. L. Furie Homocysteine, Vitamin B6, and Endothelial Dysfunction in Circulatory Disorders * Response Stroke, August 1, 2004; 35(8): 1785 - 1785. [Full Text] [PDF]
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K. Tsuda and I. Nishio Serum Homocysteine and Endothelial Dysfunction in Circulatory Disorders in Women Circulation, July 27, 2004; 110(4): e37 - e37. [Full Text] [PDF]
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K. Tsuda, I. Nishio, M. Weis, J. P. Cooke, K. Y. Lin, S. N. Panchal, S.Z. Gao, H. A. Valantine, T. N. Kledal, and E. S. Mocarski Sex Hormones and Asymmetric Dimethylarginine in Transplant Arteriosclerosis * Response Circulation, July 20, 2004; 110(3): e21 - e22. [Full Text] [PDF]
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K. Tsuda and I. Nishio Hyperinsulinemia and Estrogen-Deficiency as Risk Factors for Stroke in Women Stroke, June 1, 2004; 35(6): e134 - e134. [Full Text] [PDF]
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K. Tsuda, I. Nishio, V. Vaccarino, Z. Q. Lin, J. A. Mattera, S. A. Roumanis, S. V. Kasl, J. L. Abramson, and H. M. Krumholz Sex Hormones and Membrane Microviscosity in Elderly Women * Response Circulation, May 11, 2004; 109(18): e218 - e218. [Full Text] [PDF]
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K. Tsuda, I. Nishio, T. Kurth, and J. E. Buring Smoking, Estrogen, and Membrane Microviscosity in Women * Response Stroke, April 1, 2004; 35(4): e71 - e71. [Full Text] [PDF]
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K. Tsuda, I. Nishio, D. P. Holden, J. E. Cartwright, G. St.J. Whitley, and S. S. Nussey Estrogen and Asymmetric Dimethylarginine in Postmenopausal Women * Response Circulation, March 30, 2004; 109(12): e174 - e174. [Full Text] [PDF]
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K. Tsuda, I. Nishio, J. P. Cooke, and M. Stuhlinger Asymmetric Dimethylarginine and Circulatory Disorders in Postmenopausal Women * Response Circulation, February 10, 2004; 109 (5): e35 - e35. [Full Text] [PDF]
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 K. Tsuda and I. Nishio Role of Estrogens in the Regulation of Membrane Microviscosity Circ. Res., February 6, 2004; 94 (2): e17 - e17. [Full Text] [PDF]
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K. Tsuda, M. E. Mussolino, J. H. Madans, and R.F. Gillum Bone Mineral Density, Blood Pressure, and Stroke in Elderly Women * Response Stroke, November 1, 2003; 34 (11): e210 - e211. [Full Text] [PDF]
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