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Lack of Effect of Calcium Intake on the 25-Hydroxyvitamin D Response to Oral Vitamin D₃

Division of Endocrinology, Tufts-New England Medical Center (R.G.); and Bone Metabolism Laboratory (L.S., B.D.-H.), and Biostatistics Department (G.E.D.), Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts 02111

Address all correspondence and requests for reprints to: Dr. Bess Dawson-Hughes, Bone Metabolism Laboratory at the Jean Mayer, U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, 711 Washington Street, Boston, Massachusetts 02111. E-mail: bess.dawson-hughes{at}tufts.edu.

	Abstract

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This study was conducted to examine the effect of calcium intake on the rise in serum 25-hydroxyvitamin D [25(OH)D] levels in response to supplemental vitamin D₃. Fifty-two healthy older men and women were randomly assigned to take calcium (500 mg twice daily with meals) or placebo tablets for 90 d between October 1 and the end of March. All participants were placed on 800 IU/d (20 µg/d) vitamin D₃. Serum 25(OH)D measurements were made at baseline and on d 30, 60, and 90. The mean baseline 25(OH)D values were 19.2 ± 6.4 ng/ml (47.9 ± 15.9 nmol/liter) in the calcium group and 19.6 ± 6.7 ng/ml (49.1 ± 16.7 nmol/liter) in the control group (P = 0.808). The difference in pattern of change in 25(OH)D was not statistically significant (group by time interaction, P = 0.651); the calcium group increased 6.5 ± 5.9 ng/ml (16.2 ± 14.8 nmol/liter; P < 0.001), and the control group increased 6.6 ± 7.0 ng/ml (16.6 ± 17.4 nmol/liter; P < 0.001). The 95% confidence interval for difference in mean increase, calcium vs. control, was –3.8 ± 3.5 ng/ml (–9.6, 8.7) nmol/liter. In older men and women, the level of calcium intake, within the range of 500-1500 mg/d, does not have an important effect on the rise in serum 25(OH)D that occurs in response to 800 IU (20 µg)/d vitamin D₃.

	Introduction

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CALCIUM INTAKE MAY be one of the many factors that affect 25-hydroxyvitamin D [25(OH)D] levels in the blood. Bell found that 2000 mg/d calcium inhibited the rise in 25(OH)D in response to 100,000 IU vitamin D₃ given daily for 4 d (1). In contrast, Berlin and Bjorkhem (2) found that an increase in calcium intake caused an increase in serum 25(OH)D levels in rats on constant vitamin D intakes. Consistent with this latter finding, 2000 mg/d (50 mmol/d) supplemental calcium increased serum 25(OH)D levels significantly over a 6- to 7-wk period in 28 healthy young men studied during the winter months in Sweden (3).

There are several potential mechanisms by which calcium may promote an enhanced 25(OH)D response to oral vitamin D. Increased calcium intake causes a subtle increase in the circulating ionized calcium concentration and a decline in the serum PTH level. Both the higher ionized calcium and the lower PTH concentrations suppress the production of 1,25-dihydroxyvitamin D [1,25(OH)₂D] (4, 5). The lower 1,25(OH)₂D levels may increase 25(OH)D levels by increasing the production of 25(OH)D as a result of release of negative feedback inhibition of 1,25(OH)₂D on 25-hydroxylase (2, 6, 7). Alternatively, lower 1,25(OH)₂D levels may increase 25(OH)D levels because of a modestly decreased utilization of 25(OH)D as substrate or by delaying the metabolic clearance of 25(OH)D in the liver (8, 9). In support of the latter possibility, Clements et al. (10) examined the impact of increased PTH [and 1,25(OH)₂D] levels on 25(OH)D metabolism in seven patients with primary hyperparathyroidism before and after surgical removal of their parathyroid adenomas (10). In these patients the hepatic clearance of 25(OH)D was 1.4 times faster before parathyroid surgery than afterward.

If calcium intake does modulate serum 25(OH)D levels, then it should be considered when determining the vitamin D intake requirement in clinical studies and in clinical practice. We conducted this study to test the impact of calcium intake, in the amounts usually recommended, on the rise in serum 25(OH)D that occurs in response to a daily oral dose of 800 IU (20 µg) vitamin D₃. We also describe 25(OH)D levels achieved as a result of taking oral vitamin D supplements over a 3-month period in the winter.

	Subjects and Methods

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Subjects

The subjects were healthy, ambulatory men and postmenopausal women, aged 50 yr and older. They were recruited through direct mailings and local newspapers advertisements. Telephone prescreening was used to identify subjects with usual calcium intakes of 600 mg/d (15 mmol/d) or less and to determine general eligibility. Exclusion criteria included any history or disorder known to alter calcium or vitamin D metabolism; therapy with a bisphosphonate, selective estrogen receptor modulators, estrogen, testosterone, glucocorticoids, anticonvulsants, or thiazide diuretics; travel to latitudes less than 35°N, or use of tanning salons 3 months before enrollment or during the study. Screening evaluation included blood and urine tests. Subjects were excluded if their screening 25(OH)D levels were outside the range of 8.9–25.2 ng/ml (22.3–63.0 nmol/liter) or their 24-h urinary calcium levels were above 300 mg/d (7.5 mmol/d). Ninety-one subjects were screened; 55 were found to be eligible and were enrolled. A couple withdrew from the study because the spouse was diagnosed with a serious medical condition, leaving 53 subjects who completed the study. One subject was excluded from the analysis because of an unexplained large increase in 25(OH)D level between screening and enrollment from 25 to 39 ng/ml (62.5 to 97.5 nmol/liter). Eight other subjects with 25(OH)D levels in the required range at screening, but with levels up to 31.2 ng/ml (78 nmol/liter) at enrollment, were included in the analysis. All subjects were studied between October 1, 2003, and March 31, 2004. The investigation review board at Tufts University approved the study, and written informed consent was obtained from each subject.

Experimental methods

Of the subjects enrolled, 44% reported current use of a multivitamin (frequency not specified). All subjects were asked to stop taking their own calcium and vitamin D supplements from 1 wk before the screening visit to the end of the study. Subjects were randomly assigned to high or low calcium intake groups throughout the 90-d study. Subjects in the high intake group took 1000 mg (25 mmol) calcium/d (500 mg chewable calcium carbonate twice a day with meals) on d 1–90; subjects in the low intake group took matching placebo supplements twice daily with meals on these days. All participants were placed on 800 IU (20 µg) vitamin D₃ daily on d 1–90. The calcium and placebo tablets were provided by GlaxoSmithKline (Pittsburgh, PA). The vitamin tablets were purchased from a local pharmacy (Nature’s Bounty brand, Nature’s Bounty, Inc., Bohemia, NY). Fasting blood was drawn at baseline and on d 30, 60, and 90, and 24-h urine collections were returned on d 1 and 90. Compliance was assessed at all visits by pill count and diary checks as recorded by the subjects.

Dietary assessments

Dietary intake of calcium and vitamin D over the preceding 3 months was assessed at baseline and on d 90 with use of the Fred Hutchinson Food Frequency Questionnaire (11). The questionnaires were self-administered on site and reviewed for completeness.

Biochemical measurements

Serum 25(OH)D and 1,25(OH)₂D levels were measured with RIA kits from Diasorin (Stillwater, MN), and serum PTH was determined by immunoradiometric assay (Nichols Institute Diagnostics, San Juan Capistrano, CA). The coefficients of variation of these assays ranged from 5.6–10%. Serum calcium and phosphorous and urinary creatinine were measured with a Cobas Mira chemistry Analyzer (Roche, Indianapolis, IN), and 24-h urinary calcium was determined with a Nucleus Chemistry Analyzer (Nova Biochemical, Waltham, MA) with a coefficient of variation below 3%.

Statistical analysis

Subjects were randomized in blocks of 10. Three subjects missed one visit each, and their previous 25(OH)D values were carried forward. Baseline characteristics of the two groups were compared using ² tests (categorical variables) and two-sample t tests (continuous variables). Variables with more than two time points [serum 25(OH)D and PTH] were examined using repeated measures ANOVA, and variables with only two time points were examined using two-sample t tests. The main statistical analyses were performed with SPSS 11.5 for Windows (version 11.5.2.1, SPSS, Inc., Chicago, IL). All results are the mean ± SD unless otherwise stated. All P values were two-sided, and P < 0.05 was considered to indicate statistical significance.

	Results

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The baseline clinical characteristics of the 52 subjects in the high and low calcium intake groups are shown in Table 1. The groups were similar in age, ethnicity, sex distribution, weight, and dietary calcium and vitamin D intakes. Compliance with vitamin D pills was 96.7 ± 7.9% in the calcium group (n = 22; data missing in one subject) and 97.1 ± 4.5% in the control group (P = 0.83). Compliance with the calcium (n = 22; data missing in one subject) and placebo pills was similar in the two groups (97.6 ± 2.6% and 98.1 ± 2.7%, respectively; P = 0.533).

View larger version (15K): [in this window] [in a new window]   FIG. 1. Mean serum 25(OH)D levels (±SEM) in the calcium (dashed line) and control (solid line) groups on d 1, 30, 60, and 90 in the 52 subjects. The pattern of change in the two groups did not differ significantly (by ANOVA, P = 0.700). The 25(OH)D levels increased significantly over time in both groups (P < 0.001). To convert values for serum 25(OH)D to nanograms per milliliter, divide by 2.5.

The 24-h urinary calcium/creatinine ratio was higher in the calcium group than in the control group at baseline and increased more in the calcium group than in the control group over 90 d (49 ± 146 vs. –51 ± 174 mmol/mol, respectively; P = 0.035; 95% confidence interval for difference in mean increase, calcium vs. control: 7, 192; Table 2). There was no significant group difference in the mean baseline values or in changes during the study in serum 1,25(OH)₂D, calcium, or phosphorous levels. In all subjects, serum 25(OH)D and 1,25(OH)₂D were significantly correlated at baseline (r = 0.362; P = 0.010; n = 50), but changes in these two parameters were not significantly correlated.

The 25(OH)D values of the individual subjects on d 1, 30, 60, and 90 are shown in Fig. 2. By d 90, 44% of the subjects had reached a 25(OH)D level of 28 ng/ml (70 nmol/liter), and only 21% of the subjects had reached 32 ng/ml (80 nmol/liter).

View larger version (19K): [in this window] [in a new window]   FIG. 2. Serum 25(OH)D values of the individual subjects on d 1, 30, 60, and 90 in response to supplementation with 800 IU vitamin D3 daily for 3 months. To convert values for serum 25(OH)D to nanograms per milliliter, divide by 2.5.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

Another design difference that could account for the different findings is that the Berlin study did not actually employ vitamin D supplementation and did not estimate the dietary vitamin D intake of the study subjects. Although the subjects in that study were randomized to the calcium or control group, the study was small (14 subjects/group), so there is no certainty that the vitamin D intakes of the two groups were similar during the study. There is also no certainty that dietary calcium intakes were balanced in the two groups, because dietary calcium intake was not estimated, and urinary calcium excretion at baseline was about 50% greater in the control than in the calcium supplement group [248 ± 16 (±SEM) vs. 168 ± 16 mg/24 h (6.2 ± 0.4 vs. 4.2 ± 0.4 mmol/24 h)]. Finally, the Berlin study was carried out for 6–7 wk, a period that is only half that needed to reach a steady state 25(OH)D level after changing vitamin D intake (12) and presumably also the rate of 25(OH)D metabolism. Both studies were conducted at high latitudes (Sweden and Boston) in the winter, when the contribution of sunshine to vitamin D synthesis is minimal (13).

Our results also differ from those of Bell et al. (1), who found that 2,000 mg/d calcium blunted the rise in 25(OH)D in response to 100,000 IU (2,500 µg) vitamin D daily for 4 d. In that study the calcium supplement increased serum Ca²⁺ and lowered serum 1,25(OH)₂D levels, suggesting that calcium inhibited the production of 25(OH)D (1). The different calcium (and vitamin D) doses and durations in the two studies probably account for the different results.

Our negative findings are not likely to have resulted from ineffective use of calcium or vitamin D supplements. Compliance with the calcium and vitamin D supplements, to the extent that self-reports and pill counts are reliable, was good at 98% and 97%, respectively. The calcium tablets were chewable, and so were known to disintegrate. The calcium supplements were taken with meals (14, 15), and the dosage was split at 500 mg, twice daily (16), to optimize absorption. The rise in urinary calcium excretion in the calcium group is additional evidence that the supplements were consumed. The vitamin D tablets were not assayed independently, but the increment in 25(OH)D levels in both groups is within the range predicted by Heaney’s dose-response study in men [0.28 ng/ml (0.7 nmol/liter)/40 IU (1 µg) input of vitamin D₃] (17), suggesting that the vitamin D tablets were consumed and that they contained the expected amount of vitamin D. Finally, our two study groups had similar baseline 25(OH)D levels because of the restricted range imposed as an entry criterion. This is important, because the starting level of 25(OH)D is a significant determinant of the change in 25(OH)D in response to vitamin supplementation.

It is somewhat surprising that the supplements did not induce significant changes in serum PTH and perhaps in 1,25(OH)₂D. However, changes in these hormones are variable. In our calcium and vitamin D supplement trial [700 IU (17.5 µg)/d vitamin D₃ and 500 mg (12.5 mmol)/d calcium vs. placebo] in older men and women, for instance, there was a significant effect of supplements on serum PTH, but not on serum 1,25(OH)₂D (18). Barger-Lux et al. (19) found that supplementation with 1,000 IU (25 µg)/d vitamin D₃ did not significantly alter serum PTH or 1,25(OH)₂D levels in men, but higher daily doses of vitamin D (5,000 and 10,000 IU or 125 and 250 µg) did. In another study of men treated with 800 IU (20 µg) vitamin D₃ daily for 2 months, we observed an increase in serum 1,25(OH)₂D, but not in serum PTH (20). In neither of the latter two studies did subjects receive supplemental calcium, and their self-selected calcium intakes were not reported (19, 20).

Although 25(OH)D levels in both the calcium and control groups increased significantly over time, relatively few subjects reached the level considered by many to be optimal for bone health (21), i.e. 32 ng/ml (80 nmol/liter), after taking 800 IU (20 µg) vitamin D daily for 3 months. This dose is twice that recommended by the National Academy of Sciences for men and women the age of those in this study (22).

In conclusion, in healthy older men and women, a calcium intake within the range usually consumed and recommended does not appear to have an important effect on the rise in serum 25(OH)D levels that occurs in response to daily doses of 800 IU (20 µg) vitamin D₃. Moreover, only 21% of these subjects reached a 25(OH)D level of 32 ng/ml (80 nmol/liter) in the winter after 3 months of supplementation.

	Acknowledgments

 
We are grateful to the staff of Metabolic Research Unit at Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University for assistance in carrying out this study. We also appreciate the support of the Division of Endocrinology at Tufts-New England Medical Center, where Rula Goussous is a fellow. Finally, we thank GlaxoSmithKline for providing the calcium and placebo tablets used in this study.

	Footnotes

 
This work was supported by the U.S. Department of Agriculture under Agreement 59-1950-9001. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the NIH or the U.S. Department of Agriculture.

First Published Online November 23, 2004

Abbreviations: 1,25(OH)₂D, 1,25-Dihydroxyvitamin D; 25(OH)D, 25-hydroxyvitamin D.

Received July 14, 2004.

Accepted November 10, 2004.

	References

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This Article Abstract Full Text (PDF) All Versions of this Article: 90/2/707    most recent Author Manuscript (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Goussous, R. Articles by Dawson-Hughes, B. Search for Related Content PubMed PubMed Citation Articles by Goussous, R. Articles by Dawson-Hughes, B. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB CADMIUM COMPOUNDS CADMIUM, ELEMENTAL CALCIUM COMPOUNDS CALCIUM, ELEMENTAL CHOLECALCIFEROL PARATHYROID HORMONE Related Collections Calcium and Bone Metabolism