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Estriol (E3) Replacement Improves Endothelial Function and Bone Mineral Density in Very Elderly Women

^a Department of Geriatrics, Nagoya University School of Medicine, Japan.
^b Department of Internal Medicine, National Chubu Hospital, Oobu, Japan.

Toshio Hayashi, Department of Geriatrics, Nagoya University School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466-8550, Japan E-mail: hayashi{at}med.nagoya-u.ac.jp.

Decision Editor: Jay Roberts, PhD

	Abstract

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We investigated the effects of estriol (E3) on endothelial function and bone mineral density (BMD) in very elderly women. Twenty-four very elderly women (80 ± 3.5 years old) were administered CaCl₂ with or without estriol treatment (2 mg/day) for 30 weeks (hormone replacement treatment [HRT] group vs control group). Endothelium-dependent flow-mediated dilatation (FMD), endothelium-independent dilatation by nitroglycerin of the brachial artery, and BMD were assessed. Levels of plasma lipids and apoproteins were not changed; however, both plasma E3 and E2 were substantially increased (E2, 4.6 to 31.3 ± 8.1; E3, <5 to 45.3 ± 7.9 pg/mL) by HRT. The FMD value was also increased by HRT, as were the plasma nitrite/nitrate and cGMP values. The response to nitroglycerin was not changed. The BMD was increased by HRT, but decreased in the control group. There were significant differences between the HRT group and control group after 30 weeks' treatment in the levels of osteocalcin, P1CP, and urinary deoxypiridinoridine. E3 significantly improved BMD by inhibiting bone resorption. Endothelial function was improved in line with the antiatherosclerotic effects. E3 might be effective for use in HRT in elderly patients.

IT is well known that hormone replacement therapy (HRT) using estradiol (17ß-estradiol or conjugated estrogen) decreases the risk of coronary events and osteoporosis in postmenopausal women ⁽¹⁾. An abundance of epidemiological data confirms this atheroprotective effect of estradiol and has prompted recommendations for its widespread use in postmenopausal estrogen replacement therapy ⁽²⁾. The antiatherosclerotic effect of estradiol has traditionally been thought to be attributable to changes in plasma lipid levels (i.e., the increase in high-density lipoprotein [HDL] cholesterol and decrease in low-density lipoprotein [LDL] cholesterol) ⁽³⁾. Recently, estrogen receptors have been found in vascular endothelium and smooth muscle cells, as well as in blood cells such as monocytes ⁽⁴⁾⁽⁵⁾. The direct action of estrogen on the vessel wall has been studied vigorously over the last 10 years ⁽⁶⁾⁽⁷⁾. Estradiol also prevents fatty streak formation in the arterial wall of various atherosclerotic animal models, confirming the direct action of estradiol on cells of the vascular wall ⁽⁷⁾. However, the mechanism mediating this protection has remained obscure, although several candidate mechanisms have been proposed, including the inhibition of smooth muscle cell proliferation or migration and such antioxidizing effects as the inhibition of LDL oxidation ⁽⁸⁾⁽⁹⁾. In fact, it has been suggested that 50% of the antiatherosclerotic effects of estrogen are due to this direct action on the vessel wall ⁽¹⁰⁾.

On the other hand, the beneficial effects of HRT in the treatment of very elderly women are controversial, and the concomitant use of progesterone is known to result in complications between the two hormones ⁽¹¹⁾. The primary reason that HRT is ineffective in elderly women is that estrogen cannot sufficiently inhibit bone resorption because of the already low bone turnover ⁽¹²⁾. In addition, uterine bleeding often troubles elderly patients. We are interested in the application of estriol (E3), which is considered to have almost the same beneficial extrauteral effects as estradiol, but a much weaker uteral influence ⁽¹³⁾. Estriol has been identified to be safe even over periods of several years in terms of such adverse effects as breast cancer or endometrial cancer when administered without progesterone ⁽¹³⁾. In this study we assessed vascular endothelial function and bone mineral density as well as osteometabolism markers in elderly women.

	Materials and Methods

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Patients
We assessed endothelial function and bone mineral density in 24 elderly women (80 ± 3.5 years old, mean ± SD): 12 receiving hormone replacement therapy (HRT) of 2 mg/day estriol for 6 months and 12 controls. All subjects were at least 25 years postmenopause with bilateral ovaries, no history of hysterectomy, and no history of prior HRT. They all had bone mineral densities (BMD) lower than -3 SD for young mean BMD (-3.5 ± 0.3 SD), and all were diagnosed with osteoporosis. They were randomly divided into two groups (an HRT group and a control group) in order to maintain the same value of mean BMD in either group. Although they were in geriatric nursing homes, the patients were ambulatory and active, and were within 25% of normal weight and height values as determined by an investigator based on standard weight tables. All patients provided informed consent, agreed to the protocols, and were willing to participate in the study. Both groups were administered CaCl₂ (1g/day). They had no history of cardiovascular diseases, and none of the patients were either hypertensive or hypercholesterolemic. Ineligible patients included those who had taken any bisphosphonate, thyroid hormones, glucocorticoids, anabolic agents, calcitonin, vitamin D (as a drug >0.5 µg/day), high-dose calcium (>1000 mg per day), diuretics, or anticonvulsants for more than 2 weeks in the year prior to the study, or estrogen and/or progestogens for more than 2 weeks at any point. No patients had a history of any generalized bone disease, including hyperparathyroidism, Paget's disease of the bone, renal osteodystrophy, or any other acquired or congenital bone disease except osteoporosis, a documented history of alcohol or drug abuse, or evidence of significant organic or psychiatric disease. Eight healthy premenopausal women, aged 42 to 54 years, were examined as premenopausal control. They had a history of regular vaginal bleeding and were not taking any medication, including oral contraceptives, known to influence sex hormone metabolism. Estriol was obtained from Mochida Pharmaceutical Co. Ltd. (Tokyo, Japan).

Vascular Function
The study was carried out with subjects in a supine position in an air-conditioned room with a room temperature approximately 25 to 26°C. Flow-mediated dilatation (FMD) and nitroglycerin-induced dilatation (NTG-D) were performed according to the method described by Celermajer and colleagues ⁽¹⁴⁾. The diameter of the artery was measured from high-resolution, two-dimensional ultrasound images obtained by an SSA-270A ultrasound machine (Toshiba Co. Ltd. Tokyo) ⁽¹⁵⁾. Machine-operating parameters were kept constant during each study. A subject reclined on the examination bed 15 minutes before the initial ultrasound scanning of the brachial artery. The right brachial artery was scanned over a longitudinal section 3 to 5 cm above the right elbow, where the clearest image was obtained. Blood pressure was monitored in the left arm every 2 minutes during the study by an automated blood pressure recorder. The changes in diameter of the right brachial artery were measured at rest, during reactive hyperemia, again at rest, and after infusion of NTG spray, which causes endothelium-independent vasodilatation. To produce reactive hyperemia, blood flow to the forearm was prevented by inflation of the cuff on the arm to suprasystolic pressure (250 mm Hg). The duration of arterial occlusion was 5 minutes. It has been shown that maximal vasodilatation of forearm resistance vessels is achieved during peak reactive hyperemia. Interobserver and intraobserver variability was less than 0.2% of diameter. Then, 15 minutes later, a further resting scan was recorded to confirm the vessel recovery. Sublingual NTG spray (300 µg; Myocol Spray, Toa Eiyo Co., Tokyo) was then administered, and 3 to 4 minutes later the last scan (maximal dilatation after NTG spray) was performed. The ultrasound images were recorded on S-VHS videotape with an SLV-RS7 videocassette recorder (Sony Co., Ltd., Tokyo). The diameter of the brachial artery was measured from the anterior to the posterior interface between the media and adventitia at a fixed distance. The mean diameter was calculated from three cardiac cycles synchronized with the R-wave peaks on the electrocardiogram (ECG). All measurements were made at end-diastole to avoid possible errors resulting from variable arterial compliance. Measurement of the diameter of the brachial artery at 60 seconds after the cuff release, as previously reported, showed the maximal dilatation. The diameter change caused by FMD was expressed as the percentage of change relative to that at initial resting scan (%FMD). The diameter change caused by NTG administration was expressed in a similar manner, as the percentage of change relative to that at the recovery scan (%NTG-D).

Serum Lipid, Sex Steroid Hormones, Bone Metabolism Markers, Etc.
Serum lipids and sex steroid hormones were measured before and 8 and 30 weeks after treatment in both groups. Blood sampling was performed on the morning of the ultrasound examination after a 14-hour overnight fast; serum concentrations of estrone, estradiol, estriol, lipid, apoprotein, bone metabolism markers, and cGMP; plasma concentration of NO₂^-/NO₃^-; and other biochemical parameters were measured. Blood (15 mL) was drawn from the individuals at the times designated above and then centrifuged at 1,000 g for 20 minutes. After the centrifugation, 3 mL of the serum was kept at 4°C, and the remaining serum was kept at -20°C until the time of measurement. All specimens were measured within 48 hours of the blood sampling. Serum total cholesterol and triglyceride concentrations were measured enzymatically, and serum HDL cholesterol concentrations were determined as described in the Manual of Laboratory Operations of the Lipid Research Clinics Program ⁽¹⁶⁾. Serum steroid hormone concentrations were measured by sensitive radioimmunoassay. Bone formation was assessed by measuring serum osteocalcin (OC), bone specific serum alkaline phosphatase (B-ALP), and serum carboxyl terminal propeptide of type I procollagen (PICP). Bone resorption was assessed by measuring the urinary excretion of pyridinoline crosslinked peptides (u-deoxypyridinoline), plasma concentration of pyridinoline crosslinked teropeptide domain of type I collagen (1-CTP), and plasma concentration of tartrate-resistant acid phosphatase (TRACP). u-Deoxypyridinoline was assessed by means of urine samples taken at 2 hours after the first morning urination ⁽¹⁷⁾. Serum intact parathyroid hormone (PTH) and calcitonin were also measured. Samples were kept frozen at -70°C until the assay for osteocalcin and PICP. Serum intact osteocalcin was measured with a human immunoradiometric assay (IRMA) using two monoclonal antibodies and purified intact human bone OC as a standard (ELSA-OST-NAT; CIS Biointernational, Gifs/Yvette, France). Serum ALP was measured with a human-specific IRMA. PICP was measured with a two-site enzyme-linked immunosorbent assay (ELISA) that uses a monoclonal and a polyclonal antibody raised against human PICP (Prolagen-C, Metra Biosystems, Inc., Palo Alto, CA). Serum intact PTH and calcitonin were measured with a two-site immunochemiluminometric assay (MagicLit PTH; Ciba-corning, Mannheim, Germany). Serum calcium and phosphorus concentrations were also measured. The intra- and interassay coefficient of variation (Cvs) of all measurements described above were less than 5%.

Plasma Nitrite/Nitrate and cGMP Measurement
Concentrations of nitrite/nitrate (NO₂^-/NO₃^-) in plasma were measured with an automated NO detector–HPLC system (ENO10: Eicom Co., Kyoto, Japan) as previously reported ⁽¹⁸⁾. In brief, samples were collected in an automated sample injector connected to an automated NO detector. NO₂^- and NO₃^- in the dialysates were separated by a reverse-phase separation column packed with polystyrene polymer (NO-PAK, 4.6 x 50 mm; Eicom), and NO₃^- was reduced to NO₂^- in a reduction column packed with copper-plated cadmium fillings (NO-RED; Eicom). NO₂^- was mixed with a Griess reagent to form a purple azo dye in a reaction coil. The absorbance of the color of the product dye at 540 nm was measured by a flow-through spectrophotometer (NOD-10; Eicom). The mobile phase, which was delivered by pump at a rate of 0.33 mL/min, consisted of 10% methanol containing 0.15 M NaCl/NH₄Cl and 0.5 g/L Na-EDTA. The Griess reagent (0.5% sulfonamide, 0.025% N-naphthylethylethylenediamine dihydrochloride, and 1.25% HCl) was employed to form a purple azo dye. The plasma concentration of cGMP was determined by a specific radioimmunoassay. In brief, plasma samples were promptly frozen in liquid nitrogen and stored at -80°C. To determine the cGMP basal levels, plasma samples were washed four times with 4 mL water-saturated ethyl ether. Liquid samples were then frozen at -80°C and lyophilized overnight. The lyophylate was resolubilized in 1 mL 0.05 M sodium acetate buffer, and 50-µL aliquots were placed in test tubes. Samples were acetylated and assayed using a cGMP radioimmunoassay kit ⁽¹⁹⁾.

Bone Mineral Density (BMD)
BMD was estimated by the measurement of right hand bone mass (GS/D) using a digital image processing (DIP) method (Chugai Med Coop., Ltd., Tokyo, Japan) and x-ray film. The Cvs has been reported as 2.4% ⁽²⁰⁾. In postmenopausal subjects, significant linear correlations were observed between the BMD values estimated from GS/D by this method and the BMD values measured by DEXA in vertebrae ^(20)(21).

Safety Measures
All adverse events, regardless of severity, were recorded at each examination, including those observed by the investigators and those reported by patients, their families, or their nurses. Vital sign assessments, physical examinations, hematology, and serum and urine chemistry assays (liver and renal function, bone metabolism) were conducted throughout the study. Radiographs of the thoracic and lumbar spine were taken before and after the study. Vertebral deformities were defined as a 25% or greater decrease in anterior, mid, and/or posterior height of a vertebral body compared to baseline.

Statistical Analysis
Values are expressed as the mean ± SD. For statistical analysis, we used two-way analysis of variance. The Scheffé-F post hoc test was used when a significant F value was found ^(22)(23). When significant interaction was detected between the effect of E3 treatment and the effect of treatment term (30 weeks), all combinations (four) classified by treatment and term were analyzed by one-way analysis of variance and examined by the Scheffé-F post hoc test. Significance was accepted at p < .05.

	Results

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Serum lipid concentrations (total cholesterol, triglyceride, and HDL cholesterol) and apoprotein concentrations (A1, B100, C2, and E) were unchanged in all patients in response to HRT (Table 1 ). In contrast, levels of HDL cholesterol and apo A1 protein tended to increase, although statistical significance was not achieved by HRT. Serum Ca and P concentrations did not change significantly in either group. Intact PTH and calcitonin levels were not changed between treatment groups during the 30-week investigation. Plasma E3, E2, and E1 levels were significantly and absolutely increased by estriol treatment in the E3 group (E3, <5 to 45.3 ± 7.9, p < .001; E2, 4.6 ± 0.8 to 31.3 ± 8.1, p < .001; E1, <5 to 10.8 ± 4.9 pg/mL, p < .05; interaction, p < .001). Plasma female hormone levels did not change in the control group during the 30-week study period. No abnormal findings were noted in the other biochemical data, including those for liver or renal function, throughout the treatment term in either group. No subject showed any ultrasound evidence of arterial narrowing in the vessels studied, and there were no significant blood pressure changes during the ultrasound examination. The %FMD in women receiving HRT increased during the 30-week HRT period and became significantly larger than the control value (Fig. 1). The change of %FMD in the HRT group was significantly larger than that in the control group (p < .01; interaction, p < .01). To our knowledge, this is the first study to show an improvement of endothelial function in elderly women. No difference in response to NTG infusion was demonstrated between the two groups before or after treatment (%NTG-D, Fig. 2; interaction, ). The %FMD and %NTG-D were 108.3 ± 1.1 and 112.3 ± 0.7% in premenopausal control women (follicle phase, 3 to 5 days after menstruation). The %FMD in the premenopausal control women was not different from that in the E3 group after 30 weeks of treatment and was significantly larger than that in the control group (p < .01; interaction, p < .01). The %NTG-D was not different among these three groups (control, E3, and premenopausal group). Plasma nitrite/nitrate and cGMP levels also became higher in patients receiving HRT (Fig. 3, p < .01; interaction, p < .01). No adverse effects were observed except for uterine bleeding in one treated case. BMD was significantly increased by estriol treatment; although it tended to decrease in the control group, and there were significant differences between the two groups after 30 weeks' treatment (Fig. 4, p < .01). BMD (GS/D) in the premenopausal control group was 0.354 ± 0.021. This was significantly larger than the BMD value found in the data of E3 and control groups before treatment (0 week, 0.289 ± 0.012, p < .01) and also significantly larger than that after E3 treatment for 30 weeks (0.302 ± 0.022, p < .05). No bone fractures were observed in the estriol-treatment group, but one patient in the control group suffered a bone deformity (compression fracture) of the lumbar vertebrae. Variable markers of osteometabolism were also evaluated (Table 2 ). Osteocalcin and P1CP, the bone formation–related markers, decreased in both groups by treatment, and there was a significant difference between E3 group and control group at 30 weeks of treatment (osteocalcin, interaction, p < .001; and P1CP, interaction, ). Urinary deoxypiridinoridine and TRACP, the bone resorption–related markers, tended to increase respectably in the control group, but tended to decrease in the estriol group, and for both markers there was a significant difference between the estriol and control groups after 30 weeks' treatment (deoxypiridinoridine, interaction, p < .001; and TRACP, interaction, p < .05). 1-CTP tended to decrease respectably in the control groups, and it decreased significantly in the estriol group (interaction, ). These tendencies were observed at 8 weeks of treatment, although they did not achieve statistical significance (data not shown). Estriol treatment had no adverse effects on any of the human tissues examined, including the breasts and endometrium.

View larger version (61K): [in this window] [in a new window]   Figure 1. Flow-mediated dilatation. Endothelial function was assessed by measuring dilatation of the brachial artery using high-resolution external vascular ultrasound in response to reactive hyperemia (flow-mediated endothelial-dependent dilatation, FMD). Bar graphs showing effects of estriol treatment for very elderly women. The percent increase in vessel diameter induced by FMD (% diameter) is shown. Data are expressed as mean ± SD. *p < .01. , before (week 0); , after (week 30).

View larger version (84K): [in this window] [in a new window]   Figure 2. Nitroglycerin (NTG)-induced dilatation. Endothelial-independent function was assessed by measuring dilatation of the brachial artery using high-resolution external vascular ultrasound in response to sublingual NTG infusion. Bar graphs showing effects of estriol treatment for very elderly women. The percent increase in vessel diameter induced by NTG (% diameter) is shown. Data are expressed as mean ± SD. No significant differences in % diameter is observed with or without estriol treatment. , before (week 0); , after (week 30).

View larger version (54K): [in this window] [in a new window]   Figure 3. Plasma nitrite/nitrate (left), spontaneous oxidation products of NO, and cGMP (right) levels before and after estriol treatment for 30 weeks. Data are expressed as mean ± SD. *p < .01. Estriol treatment for 30 weeks significantly increased plasma nitrite/nitrate and cGMP levels, whereas control groups were unchanged. , before (week 0); , after (week 30).

View larger version (12K): [in this window] [in a new window]   Figure 4. Changes in bone mineral density (BMD) from baseline to week 30, expressed as mean percentage change (estriol treatment group and control group). BMD was measured by DIP levels. Data are expressed as mean ± SD. . Estriol treatment for 30 weeks resulted in a significant increase in BMD. ——, E3 + Ca group; ......, Ca group.

	Discussion

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Estrogen is known to have various effects, some beneficial and some deleterious, on numerous human tissues. Various mechanisms have been proposed to explain the antiatherosclerotic effect of estrogen, including antioxidant activity, increases in prostaglandin levels, calcium antagonistic activity, and enhanced nitric oxide activity ^(8)(9)(24). Estrogen has been reported to increase endothelial nitric oxide synthase (eNOS) activity in vitro ⁽²⁴⁾. Because NO released from eNOS in the vascular endothelium shows many antiatherosclerotic effects, such as inhibition of the migration of monocytes, inhibition of smooth muscle cell proliferation, and inhibition of platelet aggregation, the increase in the activity of eNOS and increased NO release may partially, though not completely, explain the antiatherosclerotic effects of estrogen. Flow-mediated vascular endothelium-dependent dilatation has been studied extensively in recent years and is believed to constitute the NO function in vessels ^(14)(22)(25). The impairment of FMD has been reported to precede coronary artery disease ⁽²⁶⁾. In this study, the endothelial function measured by FMD was elevated in elderly women receiving HRT. The increase in plasma nitrite/nitrate and cGMP levels in response to HRT supports the increase in NO activity in arteries. This might suggest that estriol treatment in elderly females can retard atherosclerotic changes, including coronary atherosclerosis, via the improvement of endothelial function.

Bone loss is naturally accelerated after menopause at around age 52 ⁽²⁷⁾. Estrogen therapy begun soon after menopause has been shown to prevent the loss of bone mass ⁽²⁸⁾. In the very elderly women studied here, bone turnover was relatively fast in the Ca group, based on the finding that the bone resorption related markers, u-deoxypyridinolin and 1-CTP, were not decreased in this group at 30 weeks of treatment, but it became slower in the E3 group. This is consistent with the finding that increased bone turnover in late postmenopausal women is a major determinant of osteoporosis ⁽²⁸⁾. There is now other clear evidence that bone loss persists in elderly women ⁽¹²⁾. Although the mechanisms responsible for bone loss are still controversial, bone loss occurring in late postmenopause is usually thought to be attributable to an age-related decrease in bone formation, based on histological studies indicating a decline in osteoblast function with age ⁽²⁹⁾. However, a decrease in bone formation at the cellular level does not preclude an increase in the rate of bone formation and resorption at the skeletal level ⁽³⁰⁾. This may partially explain the changes of bone metabolism related markers in this study (osteocalcin and P1CP). Once-daily therapy with 2 mg estriol increased bone mass in elderly women with osteoporosis as shown by the changes in the levels BMD, osteocalcin, and u-deoxypyridinolin. Estriol treatment resulted in decreases in urinary deoxypyridinoline in as little as 8 weeks in this patient population, although this effect did not achieve statistical significance (data not shown). We speculate that this decrease would become statistically significant if a larger study group were used. Importantly, there was no evidence of the oversuppression of bone turnover. By the end of the follow-up, BMD values were higher relative to the control group, clearly indicating a persistent overall benefit. Bone mineral density has recently been measured by dual-energy x-ray absorptiometry (DEXA); in this study, however, digital image processing (DIP) was used for its cost-effectiveness and its benefit in elderly women, whose lumbar vertebrae often suffer bone fractures that can effect DEXA measurements ^(31)(32). We also measured bone metabolism markers.

Estrogen therapy is associated with a number of adverse effects, e.g., bleeding, breast tenderness, and weight gain, that often result in discontinuation of therapy. In this study, estriol was well tolerated throughout treatment. The uterine bleeding that resulted in a discontinuance of therapy in one dropped patient may have been related to this patient's having the highest plasma levels of estriol (80 pg/mL) and estradiol (53 pg/mL) among all patients. Although the detailed mechanism of such uterine bleeding is not known, the monitoring of plasma female hormone levels may be helpful in avoiding this effect.

We were unable to determine, based on the present results, which hormone (estriol or estrogen) was responsible for the beneficial effects on bone metabolism and vascular endothelial function. Plasma estradiol levels were increased to nearly the same levels as with conventional HRT. It may be possible that estriol itself is responsible for the above-mentioned effects. Estriol is the only one of the three human estrogens that has little mitogenic activity, but it protects the reproductive organs by means of its receptor competition with the endogenous estrogens ^(13)(33). Estriol is sometimes described as a "weak hormone," a term originating from experimental tests and from bioassays currently used in pharmacology ⁽¹³⁾. Plasma gonadotropin levels decrease when high estriol doses are administered, but the decrease is not always statistically significant. However, estriol shows a similar or sometimes higher efficacy in relation to some other parameters, such as vascularization of target organs, polysaccharide accumulation in the stromal tissue, and various isoforms of nitric oxide synthases ⁽³⁴⁾. Estriol may not change the lipid profile when administered at doses less than 5 mg/day ^(34)(35). In addition, estriol does not affect the plasma coagulation factors, plasminogen concentrations, or platelet sensitivity to aggregation induced by ADP and collagen, in contrast with the effects of estradiol ⁽³⁶⁾. Estrogen has also been reported to upregulate endothelial constitutive nitric oxide synthase expression in human osteoblast-like cells ⁽³⁷⁾. Because estriol acts on the same estrogen receptors as estradiol, this report may suggest a new mechanism for estrogen in osteoporosis, as a bone formation–related factor and not just an inhibitory factor in bone resorption. Estriol has also been defined as a "blocking hormone" because it decreases the uterotrophic activity of other estrogens by 30–35% ⁽³⁸⁾. Estriol binds to its receptors in target cells, crossreacting with estradiol receptors as well and thereby reducing the estradiol stimulating activity. Based on this report, the effect of HRT in this study might be partly due to its own action and partly to an increase in plasma estradiol concentrations. Although we found no evidence of a conversion from estriol to estradiol, we suppose that the mechanism responsible for the increase in estradiol concentrations in response to estriol treatment may have been the stimulation of intrinsic estrogen synthesis or a relative increase in estradiol concentration in response to the occupation of common estrogen receptors by estriol. Further studies are necessary to elucidate this phenomenon.

Several estrogen derivatives have recently been studied as HRT agent candidates. Raloxifen (LY 139481 HCl) is one such derivative, and it has been shown to have estrogen agonist effects on bone and cholesterol metabolism, as well as estrogen antagonist effects on the mammary gland and uterus ⁽³⁹⁾. Although this study may not indicate that estriol may become a more appropriate candidate as an HRT agent in elderly women than these other derivatives, past studies showing that estriol can be used for several years without adverse effects when progestin is not prescribed may suggest the safety of estriol. Because estriol is a natural and cost-effective hormone, it may provide a new direction for the future of HRT in elderly women ⁽⁴⁰⁾.

In conclusion, in very elderly women with age-matched bone mass and osteoporosis at baseline, estriol therapy may have the potential to significantly increase BMD, as well as to improve endothelial function without significant adverse effects. Estriol seems to be an appropriate drug for HRT in elderly women.

Received November 30, 1998

Accepted August 26, 1999

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