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From the Clinical Research Centers |
University of Connecticut Health Center, Farmington, Connecticut 06030; and University of Heidelberg (M.J.S.), Heidelberg, Germany
Address all correspondence and requests for reprints to: Karen M. Prestwood, M.D., Center on Aging, MC-5215, University of Connecticut Health Center, Farmington, Connecticut 06030-5215.
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Abstract |
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Thirty-one healthy women over 70 yr of age were randomized to 12 weeks of treatment with either micronized 17ß-estradiol [0.5 mg/day Estrace (E2)] or 1500 mg/day elemental calcium, given as carbonate plus vitamin D (800 IU/day; Ca+D). At the end of the initial 12-week treatment period, both groups received both Ca+D and E2 for an additional 12 weeks. Eleven older women were followed for 36 weeks without any treatment and served as a control group.
Serum and urine were collected at baseline, at 12 and 24 weeks on treatment, and at 12 weeks after treatment was terminated for measurement of biochemical markers of bone turnover. Markers of bone formation were bone alkaline phosphatase, osteocalcin, and type I procollagen peptide; markers of bone resorption were urinary cross-linked C-telopeptides and N-telopeptides of type I collagen, serum cross-linked N-telopeptides of type I collagen, urinary free deoxypyridinoline cross-links, and serum bone sialoprotein. Repeated measures ANOVA was used to determine changes in bone turnover measures over time by group.
All markers of bone resorption decreased with initial treatment and decreased further with combination therapy (P < 0.001). Markers of bone formation decreased with Ca+D treatment, but not with E2 alone; there was no additional effect of combination therapy on formation markers compared to Ca+D alone. Neither markers of formation nor resorption changed in the control group.
These results suggest that there is an additive effect of low dose estrogen and calcium on bone resorption, but not on bone formation, in older women. Thus, the combination of low dose estrogen plus calcium is likely to be more effective in older women than either treatment alone.
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Introduction |
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Calcium supplementation has also been shown to reduce bone loss in postmenopausal women with low dietary calcium intake (6), to slow bone loss in healthy women at least 3 yr past menopause (7), and to reduce the risk of fractures in older women when given with vitamin D (8, 9). Compared to ERT, however, calcium is not as effective in decreasing the activation frequency of the bone remodeling cycle or preventing bone loss (10, 11).
Few studies have directly examined the effects of combined ERT and calcium on bone, and these studies have included only early postmenopausal women. In a nonrandomized, unblinded study, Ettinger et al. compared the effects of different levels of dietary calcium intake and low dose estrogen on spinal bone loss and demonstrated that low dose estrogen and calcium preserved bone mass more effectively than no treatment or calcium supplements alone (12). A subsequent randomized, double blind study compared three doses of 17ß-estradiol (0.5, 1.0, and 2.0 mg) given with calcium supplements (1500 mg/day total calcium intake) and found that all three doses reduced spinal bone loss compared to placebo; higher daily calcium intake was associated with a better bone density response to estrogen (13). In women receiving long term ERT with low serum calcium, administration of 600–800 mg/day calcium lactate significantly increased lumbar spine bone mineral density (14). On the other hand, Riis et al. did not find an additive effect of calcium supplementation and 17ß-estradiol on bone in a population of early postmenopausal women (15). Studies examining the effect of a combined regimen of ERT and calcium supplementation on bone in healthy, community-dwelling older women have not been reported. We undertook this study to examine the combined effects of estrogen and calcium treatment on bone turnover in older women. Our hypothesis was that low dose ERT and calcium would have an additive or synergistic effect on bone turnover in healthy older women.
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Materials and Methods |
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Of the 50 women recruited, 5 women were excluded and 31 women were randomized to 1 of 2 treatment groups: 1) 12 weeks of 1500 mg/day elemental calcium as carbonate with 800 IU/day vitamin D (Ca+D) followed by 12 weeks of Ca+D plus 0.5 mg/day micronized 17ß-estradiol (E2; Estrace, Bristol-Myers-Squibb; Princeton, NJ), or 2) 12 weeks of E2 followed by 12 weeks of E2 plus Ca+D. The remaining 14 women were enrolled to serve as a control group. Fasting blood and urine were collected at baseline and at 12, 24, and 36 weeks for measurement of biochemical markers of bone turnover, PTH, 25-hydroxyvitamin D (25OHD), 1,25-dihydroxyvitamin D [1,25-(OH)2D], ionized calcium, phosphorus, and urinary calcium. The bone mineral densities of the proximal femur, lumbar spine, and total body were measured by dual energy x-ray absorptiometry at baseline only. Each participant completed a 4-day food record at each visit to provide an estimate of nutritional calcium intake. Compliance was assessed by pill count, and side-effects were ascertained by questionnaire at 12-week intervals.
Sample collection, processing, and storage
Samples were collected between 0700–0930 h after a 10- to 12-h fast. Serum and urine samples were divided into 0.5-mL aliquots and placed immediately into a freezer for storage at -70 C. Bone marker assays were performed in duplicate after thawing once; all samples for an individual were assayed using the same kit. All bone marker assays except bone sialoprotein (BSP) and serum cross-linked N-telopeptides of type I collagen (SNTx) were performed in the Core Laboratory of the General Clinical Research Center at the University of Connecticut Health Center. Off-site assays were shipped on dry ice by overnight mail.
Bone turnover and hormone measurements
Markers of bone formation included osteocalcin (OC), bone alkaline phosphatase (BAP), and type I procollagen peptide (CICP). BAP and CICP were measured by enzyme-linked immunosorbent assay (ELISA; Metra Biosystems, Inc., Mountain View, CA), and OC was determined by immunoradiometric assay (Nichols Institute Diagnostics, San Juan Capistrano, CA). The intraassay variability was less than 5% for BAP, less than 4.6% for OC, and less than 6.3% for CICP.
Markers of bone resorption were urinary cross-linked N-telopeptides (UNTx), C-telopeptides (CTx), free deoxypyridinoline cross-links (Dpyr), BSP, and SNTx. UNTx, CTx, and Dpyr were measured by ELISA (Ostex International, Inc., Seattle, WA; Osteometer A/S, Copenhagen, Denmark; and Metra Biosystems, Mountain View, CA, respectively). SNTx was measured by ELISA at Ostex International, Inc. (16). The intraassay variability was less than 7.6% for UNTx, less than 6.1% for Dpyr, and less than 4.4% for CTx. BSP was measured by RIA in the laboratory of Dr. Markus Seibel, University of Heidelberg (Heidelberg, Germany), with an intraassay variability of less than 10% (17).
Assays for PTH, 25OHD, and 1,25-(OH)2D were performed at Endocrine Sciences, Inc. (Calabras Hills, CA), on unbatched serum. Intact PTH was measured by immuno chemiluminometric assay (ICMA), with an interassay variability of 1.1–1.9% for the range 15–91 pg/mL. 25OHD was measured by competitive protein binding, with an interassay variability of 6.5–9.6%, and 1,25-(OH)2D was measured by RRA, with an interassay variability of 6.6–17%. Ionized calcium, urinary calcium, and serum phosphorus were measured at University of Connecticut Health Center Clinical Laboratories.
Statistical analysis
One-way ANOVA was performed to compare the characteristics of the three groups at baseline. Multivariate repeated measures ANOVA was used to compare changes in marker of bone turnover, the various hormones, ionized calcium, phosphorus, and urinary calcium over time within each treatment group and between groups. Post-hoc analysis using the Bonferroni method and paired t tests were also used to examine within- and between-group differences. The level of significance was taken as P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Forty-two of the 45 women who entered the study completed it; the
only women to drop out of the study were in the control group. Baseline
characteristics are shown in Table 1
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other than a higher mean body mass index in the control group compared
to the treatment groups, there were no baseline differences. All women
who participated in the study were Caucasian, except for one
African-American woman in group 2, and all nonhysterectomized women had
an endometrial thickness less than 4 mm at baseline. Mean dietary
calcium intake at baseline was similar between groups, but individual
values ranged widely from 127-1656 mg/day. Mean dietary calcium intake
in each group did not change during the study. Total compliance with
the treatment regimen was greater than 95% for all volunteers.
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In group 1, 7 women reported constipation while taking Ca+D, and 8 reported breast fullness or tenderness when E2 was added. In group 2, 11 subjects experienced breast fullness or tenderness with E2, and 4 experienced constipation with the addition of Ca+D. Among women who received E2 during the study, endometrial thickening occurred more frequently in women who received estrogen for the longest period of time (group 2). However, we found no significant difference in the mean increase in endometrial thickening between the 2 treatment groups (group 1, 2 ± 4 mm; group 2, 2 ± 2 mm). By the end of treatment, endometrial thickness increased to more than 5 mm in 2 women from group 1; one woman had an increase in thickness from 2 to 15 mm, and the other from 2 to 8 mm. The former woman experienced some bleeding and received medroxyprogesterone (10 mg for 2 weeks); a repeat ultrasound performed 12 weeks later showed an endometrial thickness of 2 mm. The latter woman had withdrawal bleeding, and repeat ultrasound revealed an endometrial thickness of 3 mm. Four women in group 2 had an increase in endometrial thickness to greater than 5 mm during the study (range, 6–8 mm). Only 2 of these women had withdrawal bleeding. One woman had bleeding near the end of the treatment period, and the other experienced no bleeding. Repeat ultrasound in all women 12 weeks posttreatment revealed an endometrial thickness less than 5 mm. Endometrial thickness did not change in the control group.
Markers of bone resorption and bone formation
Mean values of bone turnover markers by group are shown in Table 2, and changes in CTx by group are
pictured in Fig. 1. At baseline, Dpyr was
higher in group 1 than in the control group, and CTx was higher in
group 1 than in group 2. In group 1, mean values of bone resorption
markers decreased by 13–35%, and mean values of bone formation
markers decreased by 13–21% after 12 weeks of Ca+D (Table 2). The
addition of E2 to Ca+D resulted in a further mean reduction
(11–22%) in all resorption markers except UNTx (total decrease,
25–57%) and a small mean reduction in OC and BAP (total decrease,
15–25%). All markers except SNTx, BSP, and BAP returned to baseline
levels 12 weeks after cessation of therapy.
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Calcium and calcitropic hormones
Mean values of calcium and calcitropic hormone levels by group are
shown in Table 3. Ionized calcium levels
did not change with either E2 or Ca+D alone, but fell in
both treatment groups with combined therapy. Twelve weeks after
treatment ended, ionized calcium returned to baseline in group 1 but
remained below baseline in group 2. Ionized calcium did not change in
the control group. Urinary calcium and serum phosphorus did not change
in any of the groups during the study.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One limitation of the study is that we did not follow women receiving either E2 or Ca+D alone for the entire course of the study. Therefore, it is possible that the additional reductions in bone resorption seen with combined therapy represent time-related increases in the effectiveness of the initial therapies rather than an additive effect of E2 and Ca+D. However, previous work in early postmenopausal women demonstrated a plateau in the reduction of resorption markers 3 weeks to 3 months after ERT (5, 20). Further, recent data in older women suggest that a reduction in bone resorption markers with calcium alone or calcium and vitamin D supplementation plateaus after 4–6 weeks of therapy (21, 22). The decrease in bone formation seen in both groups most likely represents the response of bone formation to decreased bone resorption, given the known coupling of the two processes. Alternatively, Ca+D may have induced transient decreases in PTH levels that could have mediated the effects of Ca+D on bone formation as well as bone resorption. As we measured PTH only in the early morning, we might not have been able to detect these transient changes.
Ionized calcium levels did not change with either E2 or Ca+D alone, but decreased in both groups with combined therapy, consistent with the additional decrease in bone resorption seen in both groups during that time. The decrease in ionized calcium in group 2 probably persisted after treatment ended because bone turnover remained diminished from the baseline value. Although E2 alone was not associated with a change in ionized calcium, an increase in 1,25-(OH)2D levels was observed. Increases in 1,25-(OH)2D levels in response to estrogen replacement have been reported previously, and these increases have been associated with increased intestinal calcium absorption. The increased 1,25-(OH)2D levels may be a response to maintain serum calcium in the face of the estrogen-mediated decrease in bone resorption. Alternatively, the increase in measured 1,25-(OH)2D levels may be due to an estrogen-related increase in vitamin D-binding protein (DBP) (23). The addition of Ca+D to E2 resulted in decreased 1,25-(OH)2D levels and may be due to a transient decrease in PTH after the large increase in calcium intake.
The decrease in mean 25OHD levels in group 1 despite calcium and vitamin D supplementation is puzzling. The decrease in 25OHD levels may be due to seasonal changes reported previously, as 93% of the baseline samples from this group were collected between June and September, week 12 and 24 samples were obtained between October and March, and final samples were obtained in the late spring and early summer. However, similar mean seasonal changes were not seen in the control group or group 2. On closer evaluation of the data, two women in group 1 had an increase in 25OHD levels with calcium supplementation. One woman had baseline sampling completed in March, with the 12 and 24 week samples in summer and the final sample in December, and her levels followed a seasonal pattern. The other woman had very low levels of 25OHD at baseline, which increased with supplementation. In group 2 and the control group, more women (31% and 38%, respectively) had baseline serum samples collected after the first of October, when 25OHD levels may be lower due to decreased sun exposure. Women in these groups who underwent baseline sampling after October 1 had 25OHD levels that either rose slightly or did not change with Ca+D. However, in women whose baseline samples were collected between June and September, the 25OHD levels fell almost uniformly by the second visit. Thus, it appears that the changes in 25OHD in the study are primarily due to seasonal effects, although changes in circulating vitamin D-binding protein, which we did not measure, or the interassay variability of the 25OHD assay may also explain these changes.
Treatment was tolerated well by most women in the study. Reports of breast tenderness, constipation, and fluid retention did not differ between the treatment groups, but no women from the control group reported the new onset of such symptoms during the treatment period. Significant endometrial thickening did occur in 6 of the 22 nonhysterectomized women who received low dose estrogen during the study. Four of these 6 women were from group 2, which received estrogen for a total of 24 weeks. Endometrial thickness decreased in all of these women 12 weeks after treatment ended, although 1 woman was given a course of medroxyprogesterone for particularly marked thickening. Given the well established link between unopposed estrogen and endometrial hyperplasia and cancer, it appears that the addition of progesterone to low dose estrogen will be required in women receiving long term replacement therapy (24).
Our results suggest that low dose E2 combined with Ca+D have an additive effect on bone resorption, but not bone formation, in older women. The combination of low dose estrogen and calcium may be useful for preventing further bone loss in this population while causing fewer side-effects than traditional estrogen replacement therapy, although long term studies are required to determine whether this regimen will protect older women from bone loss and fractures.
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Footnotes |
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Received May 21, 1998.
Revised October 7, 1998.
Accepted October 13, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, Group 1 (Ca+D alone for 12 weeks, then Ca+D and
E2 for 12 weeks). •, Group 2 (E2 alone for 12
weeks, then E2 and Ca+D for 12 weeks). 
, Group 3
(control). x, P < 0.05 compared to baseline for
groups 1 and 2. Values reported are the mean ± SE.