0021-972X/90/7105-1288$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society
Vol. 71, No. 5 Printed in U.S.A.
Estrogen Preserves a Normal Intestinal Responsiveness to 1,25-Dihydroxyvitamin D 3 in Oophorectomized Women* CARLO GENNARI, DONATO AGNUSDEI, PAOLO NARDI, AND ROBERTO CIVITELLI Institute of Medical Semeiotics, University of Siena School of Medicine, Siena, Italy; and the Division of Bone and Mineral Disease, The Jewish Hospital of St. Louis, Washington University Medical Center (R.C.), St. Louis, Missouri 63110
estrogen-treated women before and at the end of the study period (45.8 ± 6.9% vs. 42.9%± 14.9% from basal, respectively; P = 0.142), but a blunted response to 1,25-(OH)2D3 was observed in the placebo group at 6 months (27.9 ± 17.7%) compared to the result obtained before surgery (36.7 ± 9.1%; P = 0.032). Multifactor analysis of variance revealed that the effects of estrogen and 1,25-(OH)2D3 on 47Ca FA were independent of basal serum 1,25-(OH)2D3 levels. On the other hand, calcitriol administration increased serum 1,25-(OH)2D3 to a similar extent before and 6 months after surgery in the placebo group (24.2 ± 18.3% vs. 34.7 ± 16.7% from basal, respectively; P = 0.484) as well as in the estrogen-treated women (34.2 ± 17.2% vs. 26.6 ± 15.45%; P = 0.302). The significant impairment of 1,25-(OH)2D3 stimulation of 47Ca FA in spite of increased levels of circulating 1,25-(OH)2D3 in the untreated women is suggestive of an end-organ resistance to the vitamin D metabolite in a hypoestrogenic condition, which can be prevented by hormone replacement, and supports the hypothesis of a vitamin D-independent action of estrogen on intestinal calcium absorption. (J Clin Endocrinol Metab 7 1 : 1288-1293,1990)
ABSTRACT. Estrogen treatment improves calcium malabsorption induced by surgical or natural menopause, but the mechanisms involved are still under debate, with both increased production of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and improved peripheral responsiveness to the steroid having been proposed. To address this issue, we studied the effect of short term administration of 1,25-(OH)2D3 (1 Mg/day for 7 days) on intestinal fractional absorption of 47Ca (47Ca FA) and vertebral bone density, measured by dual photon absorptiometry, in 14 premenopausal women (aged 31-50 yr) before and 6 months after oophorectomy. After surgery, patients were randomly allocated to a 6-month treatment with either conjugated estrogens (0.625 mg/day; n = 7) or placebo (n = 7). Oophorectomy caused a decrease in both basal 47Ca FA (-40.8 ± 23.4%; P = 0.004) and vertebral bone density (-7.21 ± 1.20%; P < 0.001) in the placebo group. Estrogen replacement prevented these changes and increased basal serum 1,25-(OH)2D3 (+10.3 ± 10.9%; P = 0.047), whereas a detectable but not significant decrease was observed in the control group (-8.8 ± 10.5%; P = 0.07). Assessment of 47Ca FA before and after 1,25-(OH)2D3 administration revealed a similar degree of responsiveness to the steroid in the
S
EVERAL studies have demonstrated that intestinal calcium absorption is decreased in postmenopausal osteoporosis (1-4). This is thought to be an important factor leading to the bone loss occurring after ovarian failure (5). Estrogen replacement therapy corrects this calcium malabsorption, as previously demonstrated by our (6, 7) and other groups (8, 9), but the mechanisms of this estrogen action have not yet been established. Since serum levels of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] decline after the menopause (4, 10, 11), and estrogen treatment increases 1,25-(OH)2D3 (8,12, 13), it
Received March 19, 1990. Address all correspondence to: Roberto Civitelli, M.D., Division of Bone and Mineral Metabolism, The Jewish Hospital of St. Louis, 216 South Kingshighway Boulevard, St. Louis, Missouri 63110. Address requests for reprints to: Carlo Gennari, M.D., Istituto di Semeiotica Medica, Universita di Siena, 1-53100 Siena, Italy. * Part of this work was presented in abstract form to the First Joint Meeting of the ICCRH/ASBMR, Montreal, Quebec, Canada, September 1989 (Abstract 225).
was proposed that deficient calcitriol production is the major cause of calcium malabsorption in postmenopausal osteoporosis (8). In support of this hypothesis, Riggs and Nelson (14) observed a normalization of calcium absorption after long term treatment with small doses of 1,25(OH)2D3 in postmenopausal women. On the other hand, an English group reported resistance of calcium malabsorption to therapy with 25-hydroxyvitamin D in elderly subjects (15), pointing to a primary intestinal defect in these cases, a hypothesis also supported by other groups (16, 17). In a previous study we found that estrogen improved calcium absorption and raised serum 1,25-(OH)2D3 levels in a group of postmenopausal osteoporotic women, but we could not demonstrate an improvement of PTHstimulated 1,25-(OH)2D3 secretion after treatment with estrogen (6). That study, therefore, was not conclusive as to whether the estrogen effect on calcium absorption is mediated by increased basal production of 1,25-
1288
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 September 2015. at 07:32 For personal use only. No other uses without permission. . All rights reserved.
ESTROGEN AND CALCIUM ABSORPTION (OH)2D3 or by an improvement of the intestinal responsiveness to the steroid. To address this issue more directly, we reasoned that if a peripheral resistance to 1,25-(OH)2D3 does exist in a hypoestrogenic state, then we should expect an abnormal response to exogenous 1,25-(OH)2D3. Therefore, the present study was designed to analyze the changes induced by short term treatment with high doses of 1,25(OH)2D3 on 47Ca fractional absorption in oophorectomized women before and after estrogen therapy.
Materials and Methods Study design Fourteen women undergoing hysterectomy and bilateral oophorectomy for various reasons (mostly ovarian cysts or uterine fibroids) were enrolled in the study. All subjects gave informed consent to participate in the study, which was performed in the out-patient clinic of the Institute of Medical Semeiotics at the University of Siena, Italy. About 2 weeks before the surgery, each patient underwent pretreatment testing. This consisted of an early morning (0800 h) blood sample, drawn after an overnight fast, for measurement of serum 1,25(OH)2D3, followed by an intestinal calcium absorption test, as detailed below. Each patient was then started on 1,25-(OH)2D3 (1 jug daily) for 7 days. On day 2, vertebral bone density (VBD) was measured. On day 8, the patients returned to the clinic for a repeat calcium absorption test, and serum 1,25-(OH)2D3 determination. After surgery, the subjects were randomly allocated to a 6-month treatment with either conjugated estrogens (Premarin, Ayerst, Milan, Italy; 0.625 mg daily) or placebo. During the treatment period, the dietary calcium intake was maintained between 1000-1200 mg daily, and no vitamin D supplementation was given. After the treatment period, the entire testing procedure was repeated, as explained above, including measurement of bone density. Methods The methods employed in this study have been previously described (6, 18). VBD was measured by dual photon absorptiometry (BMCLAB 22a, Novo Diagnostic, Bagsvaerd, Denmark) on vertebrae L2-L4, employing 153Gd as radioactive source. The coefficient of variation of this technique obtained from repeated measures on normal volunteers within periods of 2-18 months is 3.18% at the vertebral site. Intestinal absorption of calcium was estimated as the fractional absorption of 47Ca (47Ca FA) after oral administration of the radioisotope. Briefly, fasting patients were given 10 /*Ci 47 Ca, orally (Radiochemical Centre, Amersham, United Kingdom), as calcium chloride in 70 mg calcium carrier. Blood samples were drawn just before the administration of 47Ca and 30, 60, 90, 120, and 240 min thereafter. After counting the radioactivity of 2-mL plasma samples, the circulating fraction of the absorbed dose (fx) was calculated according to the method of Marshall and Nordin (19). Plasma radioactivity was expressed as a fraction of the dose per L and multiplied by 15%
1289
of the body weight. Serum 1,25-(OH)2D3 was measured in high pressure liquid chromatography purified samples by RIA based on a goat anti1,25-(OH)2D3 antibody (Calcitriol kit, Buhlman Laboratory, Basel, Switzerland). Intra- and interassay coefficients of variations were 9.5% and 12.8%, respectively. Statistical analysis Data were analyzed using a statistical software package (Statgraphics, vers. 3.0, STSC, Rockville, MD). Group means were compared by Student's two-sided t test. Multifactor analysis of variance (ANOVA) was employed to analyze the effect of oophorectomy and estrogen treatment on 1,25-(OH)2D3 levels and 47Ca FA, before and after 1,25-(OH)2D3 administration. When the F value was found to be significant, comparisons between groups were performed by Tukey's HSD multiple range test. Data are expressed as the mean ± SD unless otherwise indicated.
Results As shown in Table 1, the patients assigned to the two treatment groups (placebo and estrogen) were matched for age, weight, and height and had similar baseline values of 47Ca FA, serum 1,25-(OH)2D3, and bone density. Oophorectomy was followed by a significant decrease in VBD in the placebo group (-7.21 ± 1.20%; P < 0.001), but not in the subjects treated with estrogen (—0.45 ± 2.20%; P = 0.689; Fig. 1A). A 6-month treatment with conjugated estrogen increased serum levels of 1,25-(OH)2D3 in oophorectomized women compared to the basal presurgery values (+10.3 ± 10.9%; P = 0.047), whereas a detectable but not significant decrease was observed in the placebo-treated group (-8.8 ± 10.5%; P = 0.07; Fig. IB). Although after 6 months the basal levels of 1,25-(OH)2D3 were lower in the control patients than in the estrogen-treated group (177.9 ± 27.8 us. 213.4 ± 42.2 pmol/L, respectively), the difference did not reach significance (t = 1.84; P = 0.09). On the other hand, oophorectomy caused an important decrease in intestinal calcium absorption at 6 months in the control women (-40.8 ± 23.4%; P = 0.004), a change not observed in the subjects treated with estrogen (—4.0 ± 14.9%; P = 0.509; Fig. 1C). As shown in Fig. 2, the serum levels of 1,25-(OH)2D3 TABLE 1. Characteristics of patients
Age (yr) Wt (kg) Ht (cm) 47 Ca FA (fx) 1,25-(OH)2D3 (pmol/L) VBD (g/cm2)
Placebo
Estrogen
42.1 ± 6.1 60.0 ± 5.2 155.9 ± 9.9 0.200 ± 0.015 195.3 ± 34.6 0.833 ± 0.073
44.1 ± 4.5 60.3 ± 5.5 159.4 ± 7.6 0.195 ± 0.015 193.6 ± 36.8 0.817 ± 0.067
' Determined by Student's t test for unpaired data.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 September 2015. at 07:32 For personal use only. No other uses without permission. . All rights reserved.
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FIG. 1. Changes in VBD (A), serum 1,25-(OH)2D3 levels (B), and intestinal calcium absorption (C) in women treated with placebo (D) or estrogen (•), before and 6 months after oophorectomy (oox). *, Significantly different from presurgery values, by Student's t test for paired data (see text for details).
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Vol. 71, No. 5 Printed in U.S.A.
Estrogen Preserves a Normal Intestinal Responsiveness to 1,25-Dihydroxyvitamin D 3 in Oophorectomized Women* CARLO GENNARI, DONATO AGNUSDEI, PAOLO NARDI, AND ROBERTO CIVITELLI Institute of Medical Semeiotics, University of Siena School of Medicine, Siena, Italy; and the Division of Bone and Mineral Disease, The Jewish Hospital of St. Louis, Washington University Medical Center (R.C.), St. Louis, Missouri 63110
estrogen-treated women before and at the end of the study period (45.8 ± 6.9% vs. 42.9%± 14.9% from basal, respectively; P = 0.142), but a blunted response to 1,25-(OH)2D3 was observed in the placebo group at 6 months (27.9 ± 17.7%) compared to the result obtained before surgery (36.7 ± 9.1%; P = 0.032). Multifactor analysis of variance revealed that the effects of estrogen and 1,25-(OH)2D3 on 47Ca FA were independent of basal serum 1,25-(OH)2D3 levels. On the other hand, calcitriol administration increased serum 1,25-(OH)2D3 to a similar extent before and 6 months after surgery in the placebo group (24.2 ± 18.3% vs. 34.7 ± 16.7% from basal, respectively; P = 0.484) as well as in the estrogen-treated women (34.2 ± 17.2% vs. 26.6 ± 15.45%; P = 0.302). The significant impairment of 1,25-(OH)2D3 stimulation of 47Ca FA in spite of increased levels of circulating 1,25-(OH)2D3 in the untreated women is suggestive of an end-organ resistance to the vitamin D metabolite in a hypoestrogenic condition, which can be prevented by hormone replacement, and supports the hypothesis of a vitamin D-independent action of estrogen on intestinal calcium absorption. (J Clin Endocrinol Metab 7 1 : 1288-1293,1990)
ABSTRACT. Estrogen treatment improves calcium malabsorption induced by surgical or natural menopause, but the mechanisms involved are still under debate, with both increased production of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and improved peripheral responsiveness to the steroid having been proposed. To address this issue, we studied the effect of short term administration of 1,25-(OH)2D3 (1 Mg/day for 7 days) on intestinal fractional absorption of 47Ca (47Ca FA) and vertebral bone density, measured by dual photon absorptiometry, in 14 premenopausal women (aged 31-50 yr) before and 6 months after oophorectomy. After surgery, patients were randomly allocated to a 6-month treatment with either conjugated estrogens (0.625 mg/day; n = 7) or placebo (n = 7). Oophorectomy caused a decrease in both basal 47Ca FA (-40.8 ± 23.4%; P = 0.004) and vertebral bone density (-7.21 ± 1.20%; P < 0.001) in the placebo group. Estrogen replacement prevented these changes and increased basal serum 1,25-(OH)2D3 (+10.3 ± 10.9%; P = 0.047), whereas a detectable but not significant decrease was observed in the control group (-8.8 ± 10.5%; P = 0.07). Assessment of 47Ca FA before and after 1,25-(OH)2D3 administration revealed a similar degree of responsiveness to the steroid in the
S
EVERAL studies have demonstrated that intestinal calcium absorption is decreased in postmenopausal osteoporosis (1-4). This is thought to be an important factor leading to the bone loss occurring after ovarian failure (5). Estrogen replacement therapy corrects this calcium malabsorption, as previously demonstrated by our (6, 7) and other groups (8, 9), but the mechanisms of this estrogen action have not yet been established. Since serum levels of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] decline after the menopause (4, 10, 11), and estrogen treatment increases 1,25-(OH)2D3 (8,12, 13), it
Received March 19, 1990. Address all correspondence to: Roberto Civitelli, M.D., Division of Bone and Mineral Metabolism, The Jewish Hospital of St. Louis, 216 South Kingshighway Boulevard, St. Louis, Missouri 63110. Address requests for reprints to: Carlo Gennari, M.D., Istituto di Semeiotica Medica, Universita di Siena, 1-53100 Siena, Italy. * Part of this work was presented in abstract form to the First Joint Meeting of the ICCRH/ASBMR, Montreal, Quebec, Canada, September 1989 (Abstract 225).
was proposed that deficient calcitriol production is the major cause of calcium malabsorption in postmenopausal osteoporosis (8). In support of this hypothesis, Riggs and Nelson (14) observed a normalization of calcium absorption after long term treatment with small doses of 1,25(OH)2D3 in postmenopausal women. On the other hand, an English group reported resistance of calcium malabsorption to therapy with 25-hydroxyvitamin D in elderly subjects (15), pointing to a primary intestinal defect in these cases, a hypothesis also supported by other groups (16, 17). In a previous study we found that estrogen improved calcium absorption and raised serum 1,25-(OH)2D3 levels in a group of postmenopausal osteoporotic women, but we could not demonstrate an improvement of PTHstimulated 1,25-(OH)2D3 secretion after treatment with estrogen (6). That study, therefore, was not conclusive as to whether the estrogen effect on calcium absorption is mediated by increased basal production of 1,25-
1288
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 September 2015. at 07:32 For personal use only. No other uses without permission. . All rights reserved.
ESTROGEN AND CALCIUM ABSORPTION (OH)2D3 or by an improvement of the intestinal responsiveness to the steroid. To address this issue more directly, we reasoned that if a peripheral resistance to 1,25-(OH)2D3 does exist in a hypoestrogenic state, then we should expect an abnormal response to exogenous 1,25-(OH)2D3. Therefore, the present study was designed to analyze the changes induced by short term treatment with high doses of 1,25(OH)2D3 on 47Ca fractional absorption in oophorectomized women before and after estrogen therapy.
Materials and Methods Study design Fourteen women undergoing hysterectomy and bilateral oophorectomy for various reasons (mostly ovarian cysts or uterine fibroids) were enrolled in the study. All subjects gave informed consent to participate in the study, which was performed in the out-patient clinic of the Institute of Medical Semeiotics at the University of Siena, Italy. About 2 weeks before the surgery, each patient underwent pretreatment testing. This consisted of an early morning (0800 h) blood sample, drawn after an overnight fast, for measurement of serum 1,25(OH)2D3, followed by an intestinal calcium absorption test, as detailed below. Each patient was then started on 1,25-(OH)2D3 (1 jug daily) for 7 days. On day 2, vertebral bone density (VBD) was measured. On day 8, the patients returned to the clinic for a repeat calcium absorption test, and serum 1,25-(OH)2D3 determination. After surgery, the subjects were randomly allocated to a 6-month treatment with either conjugated estrogens (Premarin, Ayerst, Milan, Italy; 0.625 mg daily) or placebo. During the treatment period, the dietary calcium intake was maintained between 1000-1200 mg daily, and no vitamin D supplementation was given. After the treatment period, the entire testing procedure was repeated, as explained above, including measurement of bone density. Methods The methods employed in this study have been previously described (6, 18). VBD was measured by dual photon absorptiometry (BMCLAB 22a, Novo Diagnostic, Bagsvaerd, Denmark) on vertebrae L2-L4, employing 153Gd as radioactive source. The coefficient of variation of this technique obtained from repeated measures on normal volunteers within periods of 2-18 months is 3.18% at the vertebral site. Intestinal absorption of calcium was estimated as the fractional absorption of 47Ca (47Ca FA) after oral administration of the radioisotope. Briefly, fasting patients were given 10 /*Ci 47 Ca, orally (Radiochemical Centre, Amersham, United Kingdom), as calcium chloride in 70 mg calcium carrier. Blood samples were drawn just before the administration of 47Ca and 30, 60, 90, 120, and 240 min thereafter. After counting the radioactivity of 2-mL plasma samples, the circulating fraction of the absorbed dose (fx) was calculated according to the method of Marshall and Nordin (19). Plasma radioactivity was expressed as a fraction of the dose per L and multiplied by 15%
1289
of the body weight. Serum 1,25-(OH)2D3 was measured in high pressure liquid chromatography purified samples by RIA based on a goat anti1,25-(OH)2D3 antibody (Calcitriol kit, Buhlman Laboratory, Basel, Switzerland). Intra- and interassay coefficients of variations were 9.5% and 12.8%, respectively. Statistical analysis Data were analyzed using a statistical software package (Statgraphics, vers. 3.0, STSC, Rockville, MD). Group means were compared by Student's two-sided t test. Multifactor analysis of variance (ANOVA) was employed to analyze the effect of oophorectomy and estrogen treatment on 1,25-(OH)2D3 levels and 47Ca FA, before and after 1,25-(OH)2D3 administration. When the F value was found to be significant, comparisons between groups were performed by Tukey's HSD multiple range test. Data are expressed as the mean ± SD unless otherwise indicated.
Results As shown in Table 1, the patients assigned to the two treatment groups (placebo and estrogen) were matched for age, weight, and height and had similar baseline values of 47Ca FA, serum 1,25-(OH)2D3, and bone density. Oophorectomy was followed by a significant decrease in VBD in the placebo group (-7.21 ± 1.20%; P < 0.001), but not in the subjects treated with estrogen (—0.45 ± 2.20%; P = 0.689; Fig. 1A). A 6-month treatment with conjugated estrogen increased serum levels of 1,25-(OH)2D3 in oophorectomized women compared to the basal presurgery values (+10.3 ± 10.9%; P = 0.047), whereas a detectable but not significant decrease was observed in the placebo-treated group (-8.8 ± 10.5%; P = 0.07; Fig. IB). Although after 6 months the basal levels of 1,25-(OH)2D3 were lower in the control patients than in the estrogen-treated group (177.9 ± 27.8 us. 213.4 ± 42.2 pmol/L, respectively), the difference did not reach significance (t = 1.84; P = 0.09). On the other hand, oophorectomy caused an important decrease in intestinal calcium absorption at 6 months in the control women (-40.8 ± 23.4%; P = 0.004), a change not observed in the subjects treated with estrogen (—4.0 ± 14.9%; P = 0.509; Fig. 1C). As shown in Fig. 2, the serum levels of 1,25-(OH)2D3 TABLE 1. Characteristics of patients
Age (yr) Wt (kg) Ht (cm) 47 Ca FA (fx) 1,25-(OH)2D3 (pmol/L) VBD (g/cm2)
Placebo
Estrogen
42.1 ± 6.1 60.0 ± 5.2 155.9 ± 9.9 0.200 ± 0.015 195.3 ± 34.6 0.833 ± 0.073
44.1 ± 4.5 60.3 ± 5.5 159.4 ± 7.6 0.195 ± 0.015 193.6 ± 36.8 0.817 ± 0.067
' Determined by Student's t test for unpaired data.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 September 2015. at 07:32 For personal use only. No other uses without permission. . All rights reserved.
NS NS NS NS NS NS
GENNARI ETAL.
1290
JCE & M • 1990 Vol 71 • No 5
300
1.000
0.400
• B
"5 250 E
FIG. 1. Changes in VBD (A), serum 1,25-(OH)2D3 levels (B), and intestinal calcium absorption (C) in women treated with placebo (D) or estrogen (•), before and 6 months after oophorectomy (oox). *, Significantly different from presurgery values, by Student's t test for paired data (see text for details).
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