Vitamin D Does Not Increase Calcium Absorption in Young Women: A Randomized Clinical Trial

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1 ORIGINAL ARTICLE JBMR Vitamin D Does Not Increase Calcium Absorption in Young Women: A Randomized Clinical Trial J Christopher Gallagher, 1 Prachi S Jindal, 1 and Lynette M Smith 2 1 Bone Metabolism Unit, Creighton University Medical Center, Omaha, NE, USA 2 Department of Biostatistics, University of Nebraska Medical Center, Omaha, NE, USA ABSTRACT It is commonly said that vitamin D should be used to increase calcium absorption. We tested this statement in a dose response study of vitamin D on calcium absorption. A total of 198 white and African American women, aged 25 to 45 years, with vitamin D insufficiency, serum 25 hydroxyvitamin D (25OHD) <20 ng/ml, were randomized in a double blind study to vitamin D3 400, 800, 1600, 2400 IU, or placebo. A calcium supplement was given to increase mean calcium intake at baseline from 706 mg/d to 1031 mg/d. Calcium absorption was measured at baseline and after 12 months using a single isotope method with radiocalcium45 and 100 mg of calcium. Mean baseline serum 25OHD was 13.4 ng/ml (33.5 nmol/l) and increased to 40 ng/ml (100 nmol/l) on the highest dose of 2400 IU. Using a multivariate regression analysis with significant predictors, baseline absorption, calcium intake, and weight, there was no increase in 12 month calcium absorption compared with baseline on any dose of vitamin D in either whites or African Americans. There was no significant relationship between 12 month calcium absorption and final serum 25OHD. In an analysis of calcium absorption and serum 25OHD at baseline, serum 25OHD levels were divided into groups: 0 to 5, 6 to 10, 11 to 15, or 16 to 20 ng/ml. There was no evidence of a threshold decrease in calcium absorption or serum 1,25 dihydroxyvitamin D (1,25(OH) 2 D) amongst the lowest groups. Vitamin D doses up to 2400 IU daily did not increase calcium absorption. No threshold level of serum 25OHD for calcium absorption was found at baseline or in the longitudinal study, suggesting that active transport of calcium is saturated at very low serum 25OHD levels <5 ng/ml. There is no need to recommend vitamin D for increasing calcium absorption in normal subjects. Very efficient calcium absorption at very low levels of serum 25OHD explains why people do not develop osteomalacia provided that dietary intakes of calcium and phosphorus are adequate American Society for Bone and Mineral Research. KEY WORDS: VITAMIN D; CALCIUM ABSORPTION; 25OHD; 1,25(OH) 2 D Introduction Adequate calcium and vitamin D nutrition is important in people of all ages. (1) The major sources of vitamin D are food (mainly fatty fish), supplemented products, and sunlight. Vitamin D itself is biologically inactive and has to be converted to the metabolites 25 hydroxyvitamin D (25OHD) and 1,25 dihydroxyvitamin D (1,25(OH) 2 D) in the liver and kidney, respectively. 1,25 (OH) 2 D is the most potent form of vitamin D and is the only metabolite that stimulates intestinal calcium absorption. 1,25 (OH) 2 D production is stimulated by various factors, including parathyroid hormone (PTH), (2) which is stimulated or suppressed by changes in serum calcium levels and hypophosphatemia. Recently it was shown that osteocytes produce fibroblast growth factor (FGF 23), which suppresses 1,25(OH) 2 D production, another feedback loop controlling 1,25(OH) 2 D metabolism. (3) Calcium absorption occurs mainly in the duodenum and jejunum through an active energy dependent transcellular process that is stimulated by 1,25(OH) 2 D. (4,5) There is also a passive absorption process along a paracellular pathway that is gradient dependent and occurs throughout the intestine, especially in the ileum and colon. (6) Studies suggest that the tight junction proteins claudins 2 and 12 are involved in this paracellular pathway and are upregulated by 1,25(OH)2D3 through the vitamin D receptor. (7) 1,25(OH) 2 D regulates the active transport of calcium by binding to the vitamin D receptors (VDR) in the intestine. In an animal that has the VDR knocked out and develops osteomalacia, these bone effects can be overcome by a rescue diet of high calcium. (8) One can consider that the active transport system, the VDR and action of 1,25(OH) 2 D 3, represents the most efficient way to absorb calcium and adapt to a low calcium diet. In a study of normal women and osteoporotics, low calcium intake is associated with higher serum 1,25(OH) 2 D and higher fractional calcium absorption, (9) highlighting the role of 1,25 (OH) 2 D in the process of adaptation, first noted and described during long term calcium balance studies. (10) The amount of dietary calcium absorbed is best estimated by a metabolic calcium balance technique, but because of their effort and cost, calcium absorption is now measured by double or Received in original form August 21, 2013; revised form September 27, 2013; accepted October 14, Accepted manuscript online October 28, Address correspondence to: Christopher J Gallagher, MD, Creighton University Medical Center, 601 North 30th Street, Suite 6718, Omaha, NE 68131, USA. E mail: jcg@creighton.edu Additional Supporting Information may be found in the online version of this article. Journal of Bone and Mineral Research, Vol. 29, No. 5, May 2014, pp DOI: /jbmr American Society for Bone and Mineral Research 1081

2 single isotope methods. Fractional calcium absorption varies according to the amount of calcium used in the test; if small amounts of calcium are used, such as 20 mg, the average absorption is 80% of the dose, whereas with a larger calcium load of 200 mg, the average amount absorbed is 25%. (11) All of these tests as well as metabolic balance studies show a decrease in absorption with age. (12) Reduced levels of serum 1,25(OH) 2 D with age due to decreased production by the aging kidney, (2,13) and increased intestinal resistance to circulating 1,25(OH) 2 D contribute to decreased absorption. (14,15) The effect of vitamin D supplementation on calcium absorption in younger adults has not been studied previously. The main purpose of this study was to determine the dose of vitamin D that increases calcium absorption by measuring calcium absorption before and after increasing doses of vitamin D in young white and African American women aged 25 to 45 years. Materials and Methods Study design and subject population The primary outcome of this trial was to establish the dose of vitamin D 3 required to increase serum 25OHD levels and normalize serum PTH. Measurement of calcium absorption was a predetermined secondary outcome of this trial. The essential details of the trial are summarized in the following paragraphs, but a more detailed methodology can be obtained from the primary article. (16) A total of 198 healthy young women, 119 white and 79 African American, who met the inclusion and exclusion criteria were entered into the study. The main inclusion criteria were serum 25OHD levels 20 ng/ml (50 nmo/l) and aged 25 to 45 years. The exclusion criteria were significant comorbidities; pregnancy; uncontrolled type I diabetes or significant proteinuria or fasting blood sugar >140 mg in type II diabetes; history of cancer except skin cancer within the last 10 years; chronic renal failure (serum creatinine >1.4 mg/dl); active kidney stone disease or kidney stones more than two times in lifetime; physical conditions severe enough to prevent reasonable physical activity; evidence of chronic liver disease including alcoholism; severe vitamin D deficiency that is a serum 25OHD <5 ng/ml (<12.5 nmol/l); serum calcium 10.3 mg/dl (>2.575 mmol/l); 24 hour urine calcium >290 mg/dl (7.25 mmol) on two baseline tests; and a T score < 3 on spine or hip specific to race. Additional exclusion criteria were treatment with fluoride; body mass index >45 kg/m 2 ; parathyroid hormone or derivatives, calcitonin, or estrogen during the last 6 months; treatment with bisphosphonates for more than 3 months in the past; anticonvulsants; chronic high dose corticosteroid therapy (>10 mg/d); and high dose thiazide therapy (>37.5 mg/d). They were recruited from the local population by means of advertising the study in local newspapers, hair salons, social media, and church bulletins. The Institutional Review Board at Creighton University, Omaha, Nebraska, approved the study protocol. All participants signed and received a copy of an informed consent form. Before the study was to begin, a data safety and monitoring board was established. Randomization and treatment White and African American women were randomly assigned to one of four vitamin D dose groups (400, 800, 1600, 2400 IU/d) or placebo. Because this was a double blind study design, all the study participants and staff involved in assessing primary outcomes were blinded to the treatment, and only the statistician had access to the treatment code. The randomization method was randomized blocks with block sizes of 4 and 8, stratified by race and screening serum 25OHD level (<15 versus 15 ng/ml for white and <12 versus 12 ng/ml for African Americans). SAS software (SAS Institute Inc., Cary, NC, USA) was used to generate the randomization list. Screening took place throughout the year from January 2008 to January The drug company (Douglas Labs, Pittsburgh, PA, USA) manufactured vitamin D 3, 400 IU, 800 IU, 1600 IU, and 2400 IU capsules and matching placebo capsules for this study. Every 6 months over 3 years, the actual concentration of vitamin D 3 in the capsules was measured in Dr. Hector DeLuca s laboratory at the University of Wisconsin (Madison, WI, USA). No significant change in potency was noted over the time period. The exact amount of vitamin D 3 was 503 IU in the 400 IU tablet, 910 IU in the 800 IU tablet, 1532 IU in the 1600 IU tablet, and 2592 IU in the 2400 IU tablet. Manufacturer s stated concentrations were used in the calculations, but we found no difference if we used the analyzed doses. Every participant was directed to take one vitamin D 3 tablet in the morning after breakfast. To maintain approximately a total calcium intake of 1000 mg/d (the recommended daily allowance for young women), each subject was advised to take calcium supplements 200 mg elemental (Citracal; Bayer Health Care, Morristown, NJ, USA) in divided doses after determining the usual calcium intake from a 7 day food diary. A central medication log was maintained of all study drugs dispensed to the subjects. Compliance was measured at 3, 6, 9, and 12 month visits by counting the number of pills returned at each visit, and new bottles of these pills were provided at the same visit. Information regarding any subject taking any concomitant medications and over the counter supplements was also gathered at each visit. Subjects were not permitted to take any other vitamin D supplements during the study, and the ones who wanted to take multivitamins were given multivitamins without vitamin D (Kirkman multivitamin without vitamins A&D, Lake Oswego, OR, USA) free of cost. At the baseline visit, proper medical history was acquired from all the subjects. Validated questionnaires were used to obtain history of smoking, alcohol use, caffeine intake, depression scale, exposure to sun, physical activity, and fall and fracture incidence. Biochemical measurements At all visits, fasting blood samples were collected and the serum stored frozen at 70 C. Serum 25OHD and 1,25(OH)D were measured by radioimmunoassay (RIA) after an acetonitrile extraction in the Bone Metabolism laboratory using kits manufactured by Diasorin, Inc. (Stillwater, MN, USA). The minimum detection range of 25OHD in this assay is 5 ng/ml. The intra assay variation was 9.2%, and interassay variation is 9.8%. The Bone Metabolism Laboratory participates in the vitamin D External Quality Assessment Scheme (DEQAS) of serum 25OHD assays; (17) our results were within 1 standard deviation (SD) of the all laboratory trimmed mean. The National Institute of Standards and Technology standards (NIST) were also used in the assays. (18) The interassay variations for NIST standards were as follows: level 1, 8.4%; level 2, 9%; level 3, 7.7%; and level 4, Seven day food diaries were used to estimate dietary intake of calcium and vitamin D at baseline and 12 months using Food 1082 GALLAGHER ET AL. Journal of Bone and Mineral Research

3 Processor II Plus nutrition and diet analysis system (version 5.1, ESHA Research, Salem, OR, USA). Calcium absorption measurement A single isotope method was used to measure calcium absorption at baseline and 12 months. After an overnight fast, subjects were asked to drink a 5 microcurie (mci) dose of radioactive calcium ( 45 Ca) together with 100 mg of elemental calcium (calcium chloride) in 100 ml of distilled water. (9) Two hours later, 10 ml of venous blood was collected for analysis of 45 Ca. Duplicate samples of 2 ml of serum in 18 ml of scintillation liquid were measured for 45 Ca using a 1900 CA Tri Carb Liquid Scintillation Counter (Packard Instrument, Meriden, CT, USA) at Creighton University Bone Metabolism Laboratory. On the morning of the test, a 50l aliquot was taken from each dose, dissolved in 20 ml of scintillation liquid, and counted at the same time as the serum samples of the subject. Samples are counted to 2 sigma %. Serum 45 Ca absorption is expressed as percentage of the actual dose per liter (% AD/L). Because the subjects differ on body weight that affects the calcium absorption (lower in overweight and higher in thin people), the value is corrected for 15% of the body weight for inter subject comparisons. The single isotope value at 2 hours in % AD/L is converted into the percent of calcium absorbed using a formula derived from a previous double isotope study. (9) The 2 hour value for serum 45 Ca after the oral single isotope test is highly correlated with the percent of calcium absorbed in the double isotope test. (9,19) Statistical methods For the univariate comparisons of calcium absorption, the % AD/L was corrected for 15% of body weight based on previously published data. (19) Subject characteristics at baseline were compared between races using t tests. Toseeif therewasathresholdchangein calciumabsorption and serum 1,25(OH) 2 D according to the baseline serum 25OHD values, we divided baseline serum 25OHD into four groups (0 to 5, 6 to 10, 11 to 15, and 16 to 20 ng/ml) and compared mean calcium absorption and serum 1,25(OH) 2 D levels between the four groups with ANOVA models. The effect of vitamin D dose on 12 month calcium absorption was analyzed utilizing multiple linear regression. Baseline values for serum 25OHD, serum 1,25(OH) 2 D, calcium absorption, weight, total calcium intake, tertiles of dietary calcium intake, and age and race were used as covariates. A similar model examining the effect of 12 month serum 25OHD level with 12 month calcium absorption was also studied by using multiple linear regression analysis, adjusting for the same baseline characteristic. To improve model fit, the vitamin D dose was divided by Model fit was assessed with residual plots. Any p values <0.05 are considered to be statistically significant. SAS software was used for data analysis (SAS Institute Inc., Cary, NC, USA). R software was used to create the figures. Results A total of 198 subjects (white ¼ 119, African American ¼ 79) were randomized in the study, 70 subjects (white ¼ 30, African American ¼ 40) withdrew after randomization, and 128 subjects completed the study. Details of the study subjects are shown in Supplemental Fig. S1. The baseline characteristics for the complete study population separated into dose groups are in Table 1 and by race group in Table 2. Mean ( SD) age of African American women was 35 ( 6) years and for white women was 38 ( 6) years. The range was 25 to 45 years. There were significant differences between white and African American women for some variables (Table 2). African American women had significantly higher mean weight, body mass index (BMI), and serum 1,25(OH) 2 D levels. African American women had significantly lower calcium intake, 24 hour urine calcium, and serum 25OHD levels. Body weight was a significant predictor of calcium absorption (p ¼ ). Calcium absorption was compared amongst individuals by correcting the absorption value for 15% of body weight. There was no significant race difference in weight corrected calcium absorption (Table 2). We looked for any threshold change in calcium absorption or serum 1,25(OH2)D according to the baseline serum 25OHD values. When baseline serum 25OHD was divided into four groups of 0 5, 6 10, 11 15, and ng/ml, there was no difference in calcium absorption or in serum 1,25(OH) 2 D levels amongst groups (p ¼ 0.86 and p ¼ 0.12, respectively). Table 1. Baseline Characteristics by Dose Group All (N ¼ 198) Placebo (n ¼ 38) 400 IU (n ¼ 37) 800 IU (n ¼ 42) 1600 IU (n ¼ 41) 2400 IU (n ¼ 40) N Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Age (years) BMI (kg/m 2 ) Weight (kg) Serum calcium (mg/dl) hour urine calcium Diet calcium (mg) Diet vitamin intake (IU) Serum 25OHD(ng/mL) Serum 1,25OHD(pg/mL) Calcium absorption (% AD/L) Calcium absorption (% weight correctedad/l) Calcium (% absorbed) Serum PTH(pg/mL) Journal of Bone and Mineral Research VITAMIN D AND CALCIUM ABSORPTION 1083

4 Table 2. Baseline Characteristics by Race African American White n Mean SD n Mean SD p Value Age (years) BMI (kg/m 2 ) < Weight (kg) Serum calcium (mg/dl) hour urine calcium (mg) Diet calcium (mg) < Vitamin D intake (IU) Serum 25OHD (ng/ml) < Serum 1,25OHD (pg/ml) Calcium (% AD/L) Calcium (%weight correctedad/l) Calcium (% absorbed) Serum PTH (pg/ml) Overall adherence for vitamin D was 84% in African Americans and 88% in whites and for calcium was 82% in African Americans and 91% in white women. For the 128 women who completed the study, mean baseline calcium intake was 706 mg, 12 month diet intake was 671 mg, and the average calcium supplement was 400 mg or 360 mg adjusted for compliance, leading to a mean 12 month calcium intake of 1031 mg. Effect of vitamin D dose on calcium absorption (percent of calcium absorbed) at 12 months A multivariate analysis of the effect of vitamin D on 12 month calcium absorption was conducted (Table 3). Vitamin D dose was not predictive of the final 12 month calcium absorption (Fig. 1). Race was marginally predictive (p ¼ 0.053) with African American women tending to have lower 12 month calcium absorption than whites. Total calcium intake (p < 0.009), baseline weight (p ¼ 0.019), and baseline calcium absorption (p ¼ 0.012) were predictive of 12 month absorption. Baseline serum 25OHD and 1,25(OH) 2 D were not predictive. The adjusted R 2 for this model is Effect of serum 25OHD on 12 month calcium absorption (percent calcium absorbed) A multivariate analysis of 12 month calcium absorption was conducted (Table 4). The final 12 month serum 25OHD was not a significant predictor of 12 month calcium absorption (Fig. 2). Predictive factors in the model were race (p ¼ 0.05), total calcium intake (p ¼ 0.01), baseline weight (p ¼ 0.018), and baseline calcium absorption (p ¼ 0.011). Baseline serum 25OHD and 1,25(OH) 2 D were not predictive. The adjusted R 2 for this model is We compared calcium absorption in younger women with that for older women from our previous absorption study. (17) Older women have lower calcium absorption at every level of serum 25OHD (Fig. 3). Discussion This is the first randomized placebo controlled trial of vitamin D 3 supplementation on calcium absorption in younger white and African American women. There was no significant effect of vitamin D dose on calcium absorption in either whites or African Table 3. Multiple Regression Model of 12 Month Calcium Absorption With Vitamin D Dose as a Predictor Effect Estimate SE p Value Intercept < Race African American versuswhite Age (years) 1 year increase Tertile of dietary calcium (mg) 216 to to >700 Ref. Total calcium intake(mg) 1 mg increase Baseline weight (kg) 1 kg increase Baseline calcium(% absorbed) 1% increase Baseline serum 1,25OHD(pg/mL) 1 pg/ml increase Baseline serum 25OHD (ng/ml) 1 ng/ml increase Dose 1000 unit increase Vitamin D dose was divided by 1000 to fit the models. To estimate the outcome variable, use dose 0 in the models above to correspond to placebo; 0.4 for vitamin D 400IU/d; 0.8 for vitamin D 800 IU/d; 1.6 for vitamin D 1600 IU/d;and 2.4 for vitamin D 2400 IU/d GALLAGHER ET AL. Journal of Bone and Mineral Research

5 Fig. 1. Dose response of 12 month calcium absorption expressed as percent of calcium absorbed. These are the results of the multivariate model. Average values were used for the covariates in the model to plot the response of dose. We used white, age 37 years, dietary calcium >700 mg, total calcium 970 mg, weight 82 kg, baseline calcium absorption 57.5%, baseline serum 1,25(OH) 2 D 47.6 pg/ml, and baseline serum 25OHD 13.4 ng/ml to plot the model. Fig. 2. Twelve month serum 25OHD as a predictor of 12 month calcium absorption expressed as percent calcium absorbed. These are the results of the multivariate model. Average values were used for the covariates in the model to plot the response of dose. We used white, age 37 years, dietary calcium >700 mg, total calcium intake 970 mg, weight 82 kg, baseline calcium absorption 57.5%, baseline serum 1,25OHD 47.6 pg/ml, and baseline serum 25OHD 13.4 ng/ml to plot the model. Americans. The women started with vitamin D insufficiency, mean serum 25OHD of 13.6 ng/ml, and reached a state of vitamin D sufficiency with a mean serum 25OHD of 40 ng/ml on the highest dose of 2400 IU, yet there was no increase in calcium absorption. In a study of calcium absorption in older women using similar doses as well as three higher doses, there was no increase in calcium absorption on doses up to 2400 IU. (20) On higher doses of vitamin D (3000, 4000, 4800 IU), there was a greater increase in serum 25OHD and a small but significant increase in calcium absorption presumably resulting from increased serum 1,25 (OH) 2 D. However, over a serum 25OHD range of 20 to 60 ng/ml, the increase in absorbed calcium was 6% (from 53% to 59%), which is relatively small probably because the active transport system for calcium is saturated at very low levels of serum 25OHD. In a study conducted on adolescent girls aged 12 to 14 years with low serum 25OHD levels, daily supplementation with vitamin D IU/d for 4 weeks increased serum 25OHD but did not increase calcium absorption, (21) and similar results were reported recently in children aged 4 to 8 years. (22) We did not find any threshold for calcium absorption at low serum 25OHD levels. There are now two cross sectional studies of approximately 1000 women who do not show a threshold change in calcium absorption over the serum 25OHD range of 10 to 50 ng/dl (23,24,25) and one study of subjects that shows a small decrease in calcium absorption when serum 25OHD is <4 ng/ml. (25) This is in agreement with our previous results from older women that active transport of calcium is saturated at very low levels of serum 25OHD. (20) This threshold level has not been defined exactly; measurement of very low serum 25OHD from 0 to 5 ng/ml may be insensitive, and an assay that measures Table 4. Multiple Regression Model of 12 Month Calcium Absorption With Serum 25OHD as a Predictor Effect Estimate SE p Value Intercept < Race African American versuswhite Age(years) 1 year increase Tertile of dietary calcium (mg) 216 to to >700 Ref. Total calcium (mg) 1 mg increase Baseline weight (kg) 1 kg increase Baseline calcium(% absorbed) 1% increase Baseline serum 1,25OHD(pg/mL) 1 pg/ml increase Baseline serum 25OHD (ng/ml) 1 ng/ml increase month serum 25OHD (ng/ml) 1 ng/mlincrease Journal of Bone and Mineral Research VITAMIN D AND CALCIUM ABSORPTION 1085

6 an intravenous isotope should be more accurate than estimating pool size using a body weight correction, although results from single isotope tests are highly correlated with those of double isotope studies. (19) In summary, there is no effect of vitamin D using doses from 400 to 2400 IU on calcium absorption in young women even though they reach an average serum 25OHD level of 40 ng/ml on the highest dose. We suggest that there is no need in healthy women to give vitamin D to increase absorption because saturation of active transport occurs at very low levels of serum 25OHD below 4 ng/ml. To ensure adequate mineralization of the skeleton and prevent osteomalacia when vitamin D levels are low, it is important to provide adequate calcium and phosphorus intake. Fig. 3. A comparison of the multivariate regression lines of 12 month calcium absorption expressed as percent AD/liter corrected for 15% of body weight in young and older women. In the multivariate model, average values were used to estimate the fitted line: white, age 37 years, dietary calcium >700 mg, total calcium intake 970 mg, baseline weightcorrected calcium absorbed 63%, baseline serum 1,25OHD 47.6 pg/ml, and baseline serum 25OHD 13.4 ng/ml in the young women. Average estimates to fit the line for the older women were age 67 years, dietary calcium >735 mg, total calcium intake 1248 mg, baseline weightcorrected calcium absorbed 56%, baseline serum 1,25OHD 43.3 pg/ml, and baseline serum 25OHD 15.3 ng/ml. (17) The slope in the young women was b ¼ 0.15 (p ¼ 0.32), and the slope in the older women was b ¼ 0.24 (p ¼ 0.004). pg/ml rather than ng/ml may more precisely detect the threshold for low calcium absorption. With regard to ethnic differences in absorption, a previous study of calcium absorption in 30 healthy African American and white women using the method of fractional 47 calcium retention showed similar absorption responses to low and high calcium intake in both groups, but African American women had significantly higher serum 1,25(OH )2 D levels compared with white women, and it was suggested that African Americans might have intestinal resistance to 1,25(OH) 2 D. (26) Our results were similar. Baseline weight adjusted calcium absorption was not significantly different between African American and white women, but serum 1,25(OH) 2 D was higher in African American women, supporting the previous finding of a difference in the intestinal absorption response to 1,25(OH)2D in African American women. When the regression lines for calcium absorption and serum 25OHD at 12 months are compared between young and older women, it is notable that absorption is lower in the older women at all levels of serum 25OHD. In several cross sectional studies, an age related decrease in calcium absorption has been reported previously, but this is the first longitudinal comparison of young and older women matched for serum 25OHD levels (Fig. 3). There are various strengths of this study. We used a 7 day food diary to record nutritional intake, adjustments of calcium absorption were made for known covariates, and there was an excellent compliance from our study groups. There were some limitations. Calcium absorption was not measured using a double isotope method, which is probably a more accurate technique because correcting for the extracellular pool size using Disclosures JCG received calcium tablets from Bayer Pharmaceuticals at no cost. PJ and LS state that they have no conflicts of interest. Acknowledgments This study was supported by a grant from the Department of Defense (DOD) W81XWH DOD was not involved in the study design or protocol development except for review of the safety aspects of the trial. DOD was not involved in data collection, data analysis, data management or interpretation or preparation of the manuscript. The Clinical Trial number is NCT We thank all VITADAS participants and the Bone Metabolism Unit Research staff for their hard work and contribution to the study. Thanks to Corinna Suiter for performing the assays. Thanks to Jane Meza, PhD, professor and chair, Department of Biostatistics, and director, Center for Collaboration on Research, Design, and Analysis at College of Public Health University of Nebraska Medical Center, for statistical advice. Authors roles: Study design: JCG and LMS. Study conduct: JCG. Data collection: JCG and LMS. Data analysis: LMS and JCG. Data interpretation: JCG, LMS, and PSJ. Drafting manuscript: JCG, LMS, and PSJ. 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