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1 From the Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel (RPD-G, GR, and SI-S); the Department of Clinical Nutrition (GSR) and the Metabolic Bone Disease Unit (SI-S), Rambam Medical Center, Haifa, Israel; and the Department of Community Medicine and Epidemiology, Carmel Medical Center, Haifa, Israel (GR and HSR)
2 Supported by grants from the Chief Scientist of The Israel Ministry of Health and the Rambam Medical Center Research Foundation. 3 Reprints not available. Address reprint requests to S Ish-Shalom, Metabolic Bone Disease Unit, Rambam Medical Center, PO Box 9602, Haifa 31096, Israel. E-mail: s_ish_shalom{at}rambam.health.gov.il.
ABSTRACT
Background: The effect of short-term calcium supplementation on peak bone mass in adolescent girls is not completely defined. In our previous double-blind, placebo-controlled, calcium-supplementation study (1000 mg calcium carbonate/d), we showed that calcium supplementation of postmenarcheal girls with low calcium intakes enhances bone mineral acquisition.
Objective: The objective of this follow-up study, conducted 3.5 y after the end of calcium supplementation, was to investigate the sustained effect of calcium supplementation on bone mineral mass.
Design: Anthropometric data, nutrient intakes, and bone variables were reassessed in 96 of the 100 adolescent girls whose data had been studied at the end of the supplementation period. Bone mineral content and bone mineral density (BMD) of the total body, lumbar spine, and femoral neck were determined by dual-energy X-ray absorptiometry.
Results: The calcium-supplemented group tended to have a greater accretion of total-body BMD (TBBMD) than did the control group 3.5 y after the end of supplementation. The finding was statistically significant in the active-treatment cohort (n = 17 in the calcium-supplemented group and 28 in the placebo group), who had a compliance rate of 75% during the intervention study. In a multivariate linear-regression analysis, TBBMD accretion from the beginning of the intervention study to the follow-up study in the active-treatment cohort was attributed to calcium supplementation and to the time since inclusion in the initial study.
Conclusion: Calcium supplementation for 1 y in postmenarcheal girls with low calcium intakes may provide a sustained effect on the basis of TBBMD measurements in participants with compliance rates of 75%.
Key Words: Low calcium intake • calcium supplementation • bone density • adolescents • postmenarcheal girls • follow-up study
INTRODUCTION
Osteoporotic fractures are a common cause of morbidity in the Western world (1-4). At present there are 2 important approaches to the prevention of this disease: increasing bone mass acquisition at skeletal maturity and reducing the rate of bone loss after menopause (5-14). A maximal peak bone mass at skeletal maturity is considered the best protection against age-related bone loss and subsequent fracture risk (1). Calcium intake seems to be an important determinant of peak bone mass. Recent calciumsupplementation trials in children and adolescents have confirmed a positive effect of calcium intake on bone mineral accretion (15-23). However, an increase in bone mineral density (BMD) in childhood will not prevent osteoporosis in later life unless it is sustained. Several follow-up studies were conducted to confirm whether the positive effect of calcium supplementation on bone mass during growth would be sustained after the treatment is withdrawn and, therefore, be translated into a decreased risk of osteoporosis in later life.
No sustained effect was observed in some previous follow-up studies (24-26). This led to an assumption that the effect of calcium supplementation on bone mineral acquisition reflected a transient reduction in bone remodeling and that there was little benefit from calcium supplementation on bone mineral acquisition. If this assumption proves to be correct, a short-term increase in calcium intake in children would provide no protection against osteoporosis. However, in the study by Bonjour et al (27), a statistically significant increase in the mean BMD of the 6 skeletal sites between calcium-supplemented and control subjects was maintained 3.5 y after the end of supplementation. A significant difference in favor of the supplemented group was also seen with respect to mean BMC and mean bone area. The participants of that study were prepubertal girls with a mean age of 8 y.
Unlike the results of previous studies, the results of that study suggested that calcium supplementation may increase bone mass not only by inhibiting the process of remodeling but also by stimulating bone modeling. In a former study by our group, the effect of calcium supplementation on bone mass accretion during adolescence was evaluated in a double-blind, placebo-controlled, calcium-supplementation study (1000 mg calcium carbonate/d) (28). One hundred postmenarcheal girls with low calcium intakes (<800 mg/d) completed a 1-y intervention trial. At the end of the study, the supplemented group displayed a higher accretion of total-body BMD (TBBMD) than did the control group (3.8 ± 0.3% compared with 3.10 ± 0.29% in the placebo group; P = 0.04). The purpose of the present study was to evaluate the long-term effect of the calcium-supplementation trial on bone mass by investigating whether the difference in bone accumulation between the groups persisted 3.5 y after the withdrawal of the calcium supplement.
SUBJECTS AND METHODS
In the present study, there were 2 study periods: an intervention study (study A: a 1-y double-blind, placebo-controlled, calcium-supplementation study; 28) and a follow-up study conducted 3.5 ± 0.7 y after the termination of the intervention study (study B). The characteristics of the participants and the methods used in study A are detailed in the previous publication (28). The important aspects are described below.
Study sample
One hundred girls (49 calcium-supplemented and 51 placebo) completed study A. Ninety-seven girls who had successfully completed the trial agreed to take part in the follow-up study, which was conducted 3.5 ± 0.7 y after the termination of the intervention study. One of the girls was excluded from the follow-up study because she was pregnant during the period between the 2 studies. The final cohort in study B consisted of 96 participants: 49 girls in the original calcium-supplemented group and 47 girls in the placebo group. The Hospital Review Board approved the study protocol, and informed consent was obtained from the participants.
During the intervention study, the calcium-supplemented group received 1000 mg elemental Ca/d in the form of chewable calcium carbonate tablets (Tevasidan; Teva Pharmaceuticals Industry, Petah-Tiqva, Israel). The control group received identically shaped placebo tablets provided by the same manufacturer. Calcium supplement use was assessed monthly by trained dietitians.
Assessment of dietary intake
Individual dietary intake was assessed with a semiquantitative food-frequency questionnaire, which included all known commercial dairy products and all typical foods eaten by teenagers: fast food, snacks, etc. Nutritional analysis was performed by using a database created from Israeli food-composition tables from the Ministry of Health, information from local food manufacturers, and foreign tables of food composition. The same food-frequency questionnaire was used in study A and study B.
Assessment of influencing factors
Lifestyle habits and medical status were assessed with a questionnaire. The aim was to evaluate the different possible influencing factors on bone mass during the period between the 2 studies, such as bone fractures, medication use, smoking, and physical activity.
Weight and height
The weight of the girls was measured while they were wearing minimal clothing and no shoes. The weight was determined to the nearest 0.10 kg. Standing height was recorded to the nearest 0.10 cm. Weight and height were evaluated with the use of the same equipment and by the same operator throughout both study periods. Weight and height measurements were performed at baseline, 6 mo, and 12 mo in study A and 3.5 y after the intervention study was discontinued in study B.
Evaluation of bone status
Bone mineral mass values determined at the beginning of the calcium-supplementation trial were used as baseline values in the current follow-up study. Bone mineral content (BMC; in g) and BMD (in g/cm2) as estimates of bone mass were determined by dual-energy X-ray absorptiometry (Lunar DPX scanner; Lunar Corp, Madison, WI). Bone mineral measurements included a total body scan and 2 skeletal sites: lumbar spine (L2-L4) and femoral neck. The precision error in vivo was 0.6%, 0.9%, and 1.5%, respectively, for the spine scans (L2-L4) at slow, medium, and fast speeds, whereas the error was 1.2% and 1.5–2%, respectively, for the femur scans at slow and medium speeds. The precision of total-body BMD was 0.5% in vitro and in vivo (29, 30). The CV of the BMD measurement at these sites (as determined in healthy young adults) is between 1% and 1.6%. The scans were acquired by using the appropriate scan mode for the patient's weight. Bone mineral measurements were performed with the same machine and by the same operator throughout both study periods. Densitometric evaluations were performed in study A at baseline, 6 mo, and 12 mo, and in study B 3.5 y after the intervention was discontinued.
Statistical analysis
Comparisons of percentage increases and absolute increases in bone measurements between the calcium-supplemented and the control groups were tested by using the Mann-Whitney U test because of the nonnormal distribution of the data. When the baseline data were controlled for, a regression analysis was performed by using the ranks of the dependent variables. For other variables, a two-tailed Student's t test was used for a comparison of means. A multivariate analysis using linear regression was used to test the potential effects of different variables on the prediction of percentage gains in BMD and BMC in the different skeletal areas. The differences in the anthropometric and osteodensitometric variables were analyzed both in terms of the intention-to-treat cohort (accounting for all subjects who entered the follow-up study) and an active-treatment cohort (which included the subjects who participated in study B and consumed 75% of the prescribed supplement during study A). The intention-to-treat cohort consisted of 96 participants: 49 in the calcium-supplemented group and 47 in the placebo group. The active-treatment cohort consisted of 45 participants: 17 in the calcium-supplemented group and 28 in the placebo group. All results are given as means ± SEMs. The level of significance for all tests was P 0.05. Statistical analysis was performed by using SPSS/PC, version 11.5 (SPSS Inc, Chicago).
RESULTS
Characteristics of the group at inclusion in the intervention study
The characteristics of the study and control groups at inclusion in the intervention study (study A) are shown in Table 1 and Table 2. At inclusion in study A, there were no significant differences between the study and control groups for age, body mass index (BMI; in kg/m2), height, body weight, time since menarche, calcium intake, energy intake, and bone mineral measures of the lumbar spine (L2-L4), femoral neck, and total body.
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TABLE 1. Characteristics of the 96 girls at baseline, at the end of 1 y of calcium supplementation, and 3.5 y after discontinuation of the intervention1
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Table 2. Densitometric values of the 96 girls at baseline, at the end of 1 y of calcium supplementation, and 3.5 y after discontinuation of the intervention1
Characteristics of the group at the follow-up study
The characteristics of the study and control groups at the follow-up study (study B) are shown in Tables 1 and 2. At the inclusion in study B, there were no significant differences between the study and control groups for age, BMI, height, body weight, time since menarche, calcium intake, and bone mineral measures of the lumbar spine (L2-L4), femoral neck, and total body. Only energy intake was significantly different between groups, with the placebo group having a higher energy intake than the calcium-supplemented group.
Compliance during the intervention study
One hundred twelve participants entered the calcium-supplementation trial (study A); only 100 subjects (49 calcium-supplemented and 51 placebo) completed the trial. Ninety-six girls who had successfully completed the trial took part in the follow-up study (49 calcium-supplemented and 47 placebo). During the calcium-supplementation trial, compliance was evaluated monthly by pill count. The mean compliance rate of the entire cohort was 67.33 ± 2.5% during study A. There was no significant difference between the compliance rate of the calcium-supplemented group and the control group.
Effect of calcium supplementation on bone mass
The aim of this research was to determine the net gain in bone mass accumulation since the time of inclusion in the calcium-supplementation study. Consequently, the term percentage accretion in this article refers to the percentage accretion in BMD or BMC since the beginning of the intervention study (study A) (28). The analyses were conducted for 2 different cohorts: the intention-to-treat cohort, which included all of the 96 participants in the follow-up study, and the active-treatment cohort, which included the subjects who participated in the follow-up study and consumed 75% of the prescribed supplement during the intervention study.
Bone mineral density: percentage accretion
At the end of study A, the calcium-supplemented girls had a higher accretion of TBBMD than did the control group (3.8 ± 0.3% compared with 3.1 ± 0.29%; P = 0.04) (Table 3). In the follow-up study (study B), TBBMD accretion tended to be higher in the calcium-supplemented group; however, statistical significance was shown only for the active-treatment cohort, who had compliance rates of 75% during study A (7.26 ± 0.84% compared with 5.51 ± 0.59% in the placebo group; P = 0.05) (Table 3). In study A, the percentage accretion of BMD in the lumbar spine (L2-L4) was higher in the calcium-supplemented group than in the placebo group (3.66 ± 0.35% compared with 2.93 ± 0.45%; P = 0.05) (Table 3). In study B, the percentage accretion of BMD in the lumbar spine (L2-L4) also tended to be higher in the calcium-supplemented group than in the placebo group, but the differences between the groups were not statistically significant, even when the active-treatment cohort was included in the analysis (Table 3). The percentage accretion of BMD in the femoral neck tended to be higher in the calcium-supplemented group than in the placebo group in both the initial and current studies, but the differences between the groups were not statistically significant, even when the active-treatment cohort was included in the analysis (Table 3).
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TABLE 3. Percentage gain in bone mineral density (BMD) from baseline to the end of each of the 2 studies1
Bone mineral content: percentage accretion
The accretion of BMC in the femoral neck and lumbar spine tended to be higher in the calcium-supplemented group than in the placebo group in both initial and current studies, but the differences between the groups were not statistically significant, even in analyzing the active-treatment cohort. Total-body BMC accretion tended to be higher in the placebo group than in the calcium-supplemented group in both initial and current studies; this trend was reversed in analyzing the active-treatment cohort (Table 4).
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TABLE 4. Percentage gain in bone mineral content (BMC) from baseline to the end of each of the 2 studies1
Regression model
A multivariate analysis using linear regression was conducted to determine the most important parameters affecting accretion of TBBMD in the active-treatment cohort from the beginning of study A to study B. The model included parameters from the period between the studies, such as physical activity, calcium intake, smoking, use of contraceptives, weight change, and height change, as well as baseline data at study A: age, time since menarche, TBBMD, attribution to a group, height, and weight. In the final regression model, attribution to the calcium-supplemented group and time since inclusion in the initial study were statistically significant positive predictors of TBBMD accretion, whereas the age and weight at inclusion in the initial study were statistically significant negative predictors (Table 5).
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TABLE 5. Regression analysis predicting accretion of total-body bone mineral density in the active-treatment cohort from baseline to 3.5 y after discontinuation of the intervention
Influencing factors
In determining the possible different effects on bone density during the period between the initial study and the follow-up study, no significant differences between the study and the control groups were found in the active-treatment cohort for weight change, BMI change, smoking, use of contraceptives and other medications, physical activity, and time since the initial study (data not shown). The same results were shown when the different effects on bone density in the intention-to-treat cohort were analyzed.
DISCUSSION
In recent years, several calcium-supplementation trials in children and adolescents were conducted to evaluate whether a positive effect on bone mass is sustained after discontinuation of calcium supplementation, which is ultimately translated into higher peak bone mass (24-27). Most clinical trials were unable to detect any differences in bone mass between treatment and control groups after supplementation ceased (24-26). This led to the assumption that the effect of calcium supplementation on bone mineral gain appears to reflect a transient reduction in bone turnover rate. If this observation is confirmed, it will minimize the importance of consistency in maintaining an adequate calcium intake to decrease the risk of fractures in later life. Unlike those previous studies, Bonjour et al (27) reported a significant difference in mean BMD in 6 skeletal sites between the treatment and control groups, which remained for 3.5 y after the end of the supplementation. Moreover, a significant difference in favor of the supplemented group was also seen with respect to mean BMC and mean bone area. The participants of that study were prepubertal girls with a mean age of 8 y.
In our study, at the end of study A (28), the calcium-supplemented group had a higher accretion of TBBMD than did the placebo group. In study B, TBBMD accretion from the time of inclusion in study A was still higher in the calcium-supplemented group. However, statistical significance was shown only for the active-treatment cohort, who had compliance rates of 75% during the intervention study (Table 3). Our findings suggest that after cessation of the calcium supplementation, the calcium-supplemented group did not show any further increases in bone mass. However, the positive effect of calcium supplementation on TBBMD accretion in study A in the active-treatment cohort did not disappear.
Another important result that supported this hypothesis was that attribution to the calcium-supplemented group in study A was found to be a statistically significant positive predictor of TBBMD accretion from baseline to study B in the active-treatment cohort. The results of our research suggest that the increase in BMD resulting from calcium supplementation for 1 y in postmenarcheal girls with low calcium intakes could be maintained >3 y after the end of the intervention. Consequently, our research tends to support the idea that calcium supplementation may provide a sustained effect on BMD and positively influence bone accretion during growth.
A possible reason for not achieving statistically significant results, as in the Bonjour et al study (27), was the difference in the pubertal stages of the participants in the 2 studies. In the study by Bonjour et al (27), the participants were premenarcheal girls with a mean age of 8 y. In our study, the participants were 1 y postmenarcheal with a mean age of 14 y. Puberty is a time of accelerated growth in which pronounced bone gain is achieved. Another possible reason was the low mean compliance rate in study A (67.33 ± 2.5%). This low compliance rate was probably a major obstacle for achieving significant results.
A great deal of effort was invested to evaluate the potential influence of other bone-modifying factors on bone mass acquisition during the period between the studies. The aim was to estimate whether the study and the control groups were exposed to the same factors that might alter bone mass and therefore affect the densitometric values. In the active-treatment and in the intention-to-treat cohorts, no significant differences were found between the study and the control groups.
Consequently the differences between the groups in bone mass accretion from the beginning of the calcium-supplementation study to the follow-up study arose from the primary distinction between the groups, ie, calcium supplementation in study A. This study suggests that 1 y of calcium supplementation in postmenarcheal girls with low calcium intakes had a sustained effect on total-body BMD, but no sustained effect was observed on the other skeletal areas. A possible explanation for this was that the permanent effect was present only in the cortical bone; therefore, the TBBMD that represents the cortical bone was the most important of the bone variables to be affected.
To the best of our knowledge, this study had the longest follow-up period for monitoring bone mass accumulation in postmenarcheal girls. It evaluated the net effect of calcium supplementation on bone mass in a population that had completed the rapid bone accretion that occurs during puberty.
The main conclusion of the study is that calcium supplementation of postmenarcheal girls with low calcium intakes for 1 y may provide a sustained effect on TBBMD accretion in girls with compliance rates of 75%. Longer-term calcium-supplementation studies in adolescents are necessary to confirm whether high calcium intakes can improve peak bone mass.
ACKNOWLEDGMENTS
SI-S designed the study, obtained the research grant support from the Chief Scientist Fund of the Israeli Ministry of Health, and supervised RPD-G's work and the writing of the manuscript. RPD-G conducted the field research and wrote the manuscript as part of the requirements for an MD thesis. GSR obtained the research grant support from the Rambam Medical Center Research Foundation, contributed to the fieldwork, and participated in the design of the study, the supervision of the research, and the writing of the manuscript. GR participated in the supervision of the research and the statistical analysis. HSR performed the statistical analyses. The authors had no financial or other interests relating to the study.
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