Calcium Supplementation and Increases in Bone Mineral Density in Children

Subjects and Protocol

Healthy, white identical twins 6 to 14 years old were recruited from a registry of twins maintained by the Department of Medical and Molecular Genetics at the Indiana University School of Medicine and from local schools. The study protocol was approved by the institutional review board, and informed consent was obtained from the children and their parents. We screened 71 pairs of twins by administering a food-frequency questionnaire, excluding only 1 pair; the base-line dietary calcium intake of this pair was greater than 1200 mg per day, the upper limit for entry. Of the 70 pairs who entered the study, 45 completed it. The pairs who did not complete the study were compared with those who did.

On their initial visit to the home, members of the study staff showed the families how to record food consumption and obtained blood samples to determine the twins' zygosity; all pairs were confirmed as being monozygotic. The families completed three-day food records before the initial visit of the twins to the outpatient clinic of the General Clinical Research Center for a physical examination and anthropometric measurements. The food records were reviewed with the children and parents by a research assistant or registered dietitian before the records were analyzed; the details of the analysis are reported elsewhere.⁷ Twenty-two twin pairs were prepubertal throughout the study, 4 pairs were postpubertal at base line, and the remaining 19 pairs underwent puberty during the study. Overnight urine samples were collected for measurement of calcium and creatinine before the clinic visit. The bone mass of the radius, spine, and hip was measured. After these procedures were completed, one twin in each pair was randomly assigned to receive 1000 mg of calcium daily as calcium citrate malate, and the other twin to receive a placebo identical in taste and appearance to the calcium supplement. Each twin took four 250-mg tablets daily, two in the morning and two in the evening. The families received no special instructions regarding diet or physical activity. The twins' levels of physical activity were monitored at six-month intervals by questionnaire.⁸ None of the children smoked or consumed alcoholic beverages.

Members of the study staff visited the home monthly to deliver a new supply of pills and collect those left over from the previous month, a one-day food record, and a recent overnight urine sample. The subjects returned to the clinic after six months and after one and two years for measurement of radial bone mass. After three years they returned for measurement of bone mass in the radius, spine, and hip and for anthropometric measurements.

Height was measured with a wall-mounted stadiometer, and weight with an electronic scale. Calcium and creatinine were measured in each overnight urine sample according to standard methods. ^{9 , 10} The ratio of urinary calcium to urinary creatinine (the concentration of each was expressed in millimoles per liter) was used to monitor safety throughout the study; in only one pair of twins did the ratios determined in any two sequential samples exceed the upper limit of the normal range established for the population at base line (≤0.17). After the three years, 41 pairs returned for measurement of serum concentrations of osteocalcin¹¹ and tartrate-resistant acid phosphatase¹² and calcium-44 enrichment three hours after the administration of this stable isotope¹³ with a standard breakfast containing 250 mg of calcium.¹⁴

Measurements of Bone Density

Radial bone mass was measured with a Lunar SP-2 absorptiometer (Lunar Radiation, Madison, Wis.). Forearm length was measured from the radial styloid to the olecranon, and points were marked at 1/10 (distal radius) and 1/3 (midshaft radius) of this distance and scanned. If possible, eight lines were measured in the distal region, from the 1/10 point and then distally at 1.5-mm intervals. Four lines were measured at the proximal site. The coefficient of variation for repeated measurements made within one week in individual adults was 1.3 percent.

Bone mineral density was expressed as the amount of mineral (in grams) divided by the area scanned (in square centimeters). Bone mass was measured at the femoral neck, Ward's triangle (located in the femoral neck), and the greater-trochanter region of the hip and from the second to the fourth lumbar vertebrae, with a Lunar DP-3 absorptiometer. The gadolinium-153 source was replaced every six months. The coefficient of variation in adults ranged from 1.4 percent (spine) to 2.4 percent (Ward's triangle).

Calcium Supplementation

Calcium citrate malate was prepared as previously described.¹⁵ This form of calcium has been shown to be well absorbed in children¹⁶ and young adults¹⁵ and to retard bone loss in older postmenopausal women.¹⁷ Calcium citrate malate (FruitCal) is commercially available to fortify fruit juices (Citrus Hill Plus Calcium and Speas Farm Parents' Choice; Procter & Gamble, Cincinnati). All tablets used (both placebo and calcium citrate malate) were provided by Procter & Gamble.

Investigators at Indiana University School of Medicine had no knowledge of any twin's treatment assignment; this information could be obtained, but it was not needed.

Statistical Analysis

Descriptive statistics were calculated for the dietary intakes and physical characteristics of the twin pairs. We performed t-tests to determine whether the characteristics of the subjects who dropped out of the study differed from those of the subjects who completed the study, and to test for the effect of sex on the response to the supplement. Paired t-tests were used to evaluate the differences between the calcium-supplement and placebo groups. Before the skeletal response to supplementation was analyzed, changes from base line were calculated to adjust for possible base-line differences; percentages of changes were used because they appeared to be distributed more normally than absolute changes. These changes were calculated separately for each of the six skeletal sites and for the mean of all sites, and 95 percent confidence intervals for the differences were determined.

Individual compliance was estimated from monthly pill counts, and the average daily calcium intake was calculated from these estimates of compliance and the monthly dietary records. The effect of the difference between twins in calcium intake on the difference in bone change was assessed by linear regression analysis.

Table 1. Table 1. Base-Line Characteristics of the 70 Pairs of Twins Who Entered the Study, According to Sex.*

Table 2. Table 2. Base-Line Bone Mass and Density of the Skeletal Sites, Dietary Calcium Intake, and Physical Activity in the 45 Twin Pairs Who Completed the Three Years of the Study.*

The characteristics of the 140 children who entered the study are shown in Table 1. The mean ages of the girls and the boys were similar, but the pubertal development of the girls was more advanced, as expected. The intake of all nutrients was slightly greater in the boys. The base-line bone mineral content, bone mineral density, and bone width (or area, depending on the site) of the twins in the calcium-supplement and placebo groups who finished the study were similar (Table 2). There were also no significant differences between the groups in height, weight, intake of calcium or other nutrients, or level of physical activity.

Most withdrawals occurred during the first year of the trial, and boys dropped out of the study more often than girls. Twenty-one pairs left the trial because they did not wish to continue to take the pills. Two pairs moved from the area, and one pair was dropped from the study because the urinary calcium:creatinine ratio in two consecutive samples from each twin was more than 0.17. No subject withdrew from the study because of side effects. One of the 46 pairs who finished the trial was excluded from the analysis because an orthopedic disorder (accelerated growth of the right femur) developed in one twin; including this pair would have increased the differences favoring the supplementation group. Thus, the results in 45 pairs of twins were analyzed. There were no significant differences in the base-line bone mass between the subjects who dropped out and those who completed the trial. Since many withdrawals occurred during the latter part of the first year, it was possible to compare the responses recorded at six months in the subjects who withdrew and those who remained. Those who left the trial had a slightly but not significantly better response to supplementation than those who remained (mean [±SD] increase in bone mineral density in subjects leaving vs. subjects remaining: midshaft radius, 0.017±0.029 vs. 0.010±0.021 g per square centimeter; distal radius, 0.006±0.021 vs. 0.003±0.013 g per square centimeter).

The calcium intake of the calcium-supplement and placebo groups was calculated from the monthly food records. The additional intake of calcium from the supplement as determined from the records of compliance averaged 719 mg per day during the three-year study period. Compliance was best during the first six months of the study, with an additional intake of 794 mg per day, and declined during the second six months, with 732 mg per day; the additional intake was 694 mg per day at the end of two years and 658 mg per day at the end of the study. Each of the latter three values is significantly lower than the value for the preceding period. However, declining compliance was not associated with differences in the effects of calcium. The dietary calcium intake during the study was virtually identical in the two groups (calcium-supplement group vs. placebo group, 894 vs. 908 mg per day), as would be expected given the study design, and did not vary with age. The calcium-supplement group received an average of 1612 mg of calcium per day during the three years, as compared with 908 mg per day in the placebo group.

Table 3. Table 3. Changes in Bone Mineral Density and Bone Area in the 45 Pairs Who Completed the Study.

The changes in bone mineral density and bone area at the various sites measured at three years are shown in Table 3. The mean increase in bone mineral density for all six sites was 1.4 percent greater in the calcium-supplement group. The differences in the increase were greater at the midshaft radius and distal radius (2.5 percent and 3.3 percent, respectively). The differences in the increase ranged from 0.4 percent (femoral neck) to 1.8 percent (greater trochanter) at other sites. There were no consistent changes in bone area or width (Table 3).

Table 4. Table 4. Difference in Bone Mineral Density between the Calcium-Supplement and Placebo Groups, According to Pubertal Status.

The benefits of supplementation were modified by sexual maturation. The difference in the percent increase in bone mineral density between the two groups was greatest among the prepubertal twins (Table 4) at both radial sites (mean difference: midshaft radius, 5.1 percent; distal radius, 3.8 percent) and in the spine (2.8 percent); those given calcium supplements had marginally greater increases at Ward's triangle (2.9 percent) and the greater trochanter (3.5 percent). There were no significant differences in bone mineral density among the 4 twin pairs who were postpubertal or the 19 pairs who went through puberty during the study.

Figure 1. Figure 1. Mean Differences within Twin Pairs in the Change in the Bone Mineral Density of the Midshaft Radius among Prepubertal and Older Children, According to Their Time in the Study.

The differences were significant among the prepubertal children at six months and thereafter.

The changes in the bone mineral density of the midshaft radius at six months and one, two, and three years are shown in Figure 1. Among the prepubertal twins, the increase in density in those given the supplements was significantly greater at all times. In contrast, among the older twins there was little difference at any time. The results of measurement of the distal radius were similar. Most of the difference between the calcium-supplement and placebo groups was observed after six months, although the difference between them gradually increased between six months and three years among the prepubertal twins (a difference of 3.4 percent after six months and 5.1 percent after three years) (Fig. 1 and Table 4).

Height and weight increased in these growing children, but the increases were similar in both those receiving calcium and those receiving placebo, whether they were prepubertal or maturing sexually. Thus, calcium supplementation affected bone mineral density but not longitudinal growth. This dichotomy in the response to supplementation is supported by the observation that, although the calcium-supplement and placebo groups differed in terms of the increase in radial bone mass, they did not differ in terms of the increases in bone area and bone width. There was no difference between boys and girls in the responses to the calcium supplements, and the responses were independent of the level of physical activity and the base-line calcium intake.

Since compliance was monitored by pill count, it was possible to relate the differences in the increase in bone mineral density between the twins in each pair to the differences in the intake of calcium between them. As the difference in calcium intake between twins increased, the difference in the bone mineral density of the midshaft radius increased (r = 0.40; 95 percent confidence interval, 0.12 to 0.62). When the difference in intake was 750 mg or more, the difference favored the twin receiving the supplement in all but one pair. However, there was no correlation between the difference in calcium intake and the difference in the increase in bone mineral density at other sites. There was no difference in compliance between the prepubertal subjects and the older subjects (three-year average, 74 percent and 72 percent, respectively).

Serum osteocalcin was measured in 41 pairs of twins at the end of the trial. The mean (±SD) concentration in the calcium-supplement group was significantly lower than that in the placebo group (48.5±17.3 vs. 54.0±21.1 μg per liter, P = 0.008). This difference was due to larger differences in the prepubertal subjects (supplementation vs. placebo, 49.6 vs. 58.4 μg per liter; P = 0.017) and smaller differences in the older subjects. The serum concentration of tartrate-resistant acid phosphatase was slightly but not significantly lower in the calcium-supplement group. These data suggest that bone turnover was lower in the subjects receiving calcium supplements. In addition, serum calcium enrichment (i.e., dietary absorption) was significantly lower (P = 0.004) in the prepubertal but not the sexually maturing subjects who received supplements, as compared with the subjects who received placebo.

We found that calcium supplementation had a positive effect on the rate of increase in bone mineral density at several skeletal sites in growing children. By taking advantage of the unique homogeneity of monozygotic twins, we could detect small differences in response.¹⁸ Approximately six times as many unrelated children (according to comparisons of midshaft-radius values) would have been required to demonstrate a similar degree of significance in comparisons of bone mass.

The subjects given supplements received on average 719 mg more calcium per day than their twins and had an increase of about 3 percent more mineral in the radius during the three-year study period. However, sexual maturity was an important modifying factor. The prepubertal twins given supplements had nearly 4 percent greater increases in bone density in the spine and radius and 3 percent greater increases in Ward's triangle and the greater trochanter. Moreover, the differences within pairs in radial bone mineral density increased from about 3 percent in the first year to 5 percent after three years. In contrast, older children benefited little. To account for the difference in the response to calcium we considered several possibilities. First, the prepubertal subjects received more calcium per kilogram of body weight. However, we found no dose–response effect at most sites, whether we used absolute or weight-adjusted doses. Second, other pubertal changes (e.g., in the secretion of growth hormone and sex steroids) may so dominate mineral accretion that bone is either maximally stimulated or changing so rapidly that the relatively small effects of a greater daily calcium intake cannot be detected. The rapid loss of cancellous bone early in menopause, a loss that cannot be reversed by calcium supplementation,^{19 , 20} is probably an analogous condition. However, Matkovic et al.⁶ reported a positive trend in bone accretion in 20 pubertal girls with an average intake of 1640 mg of calcium per day, as compared with 9 girls of similar age with an intake of 750 mg per day. Third, it may be more difficult to detect small changes in bone mineral density when skeletal growth is rapid. Finally, the size of the study population may have been inadequate to allow the detection of small but important differences among pubertal children.

Other studies have found that persons who consume greater quantities of calcium early in life have greater bone mass later on.^{3 4 5} These studies compared subjects whose intakes were near or slightly above the recommended dietary allowance with subjects whose intakes were well below the allowance, which may explain why the magnitude of the differences in skeletal responses between those with high calcium intakes and those with low intakes was slightly greater than the magnitude of the differences found in this study. Moreover, these other results^{3 4 5} may reflect the effect of many years of differences in calcium intake, in contrast to the three years this study lasted. Other nutrients or lifestyle factors could also have been potential confounders in the retrospective studies. The co-twin model, in which both treated and control subjects reside in the same household, are the same age, and participate in the same activities, provides an inherent control for many of the variables that may confound other types of studies.

Small increases in peak bone mass in the population could lead to decreases in the rate of fractures later in life. According to prospective studies of the relation of bone mass to the incidence of subsequent fractures, a change of 1 SD in bone mass may alter the risk of fracture by as much as 100 percent.^{21 , 22} The difference in radial bone mass that we observed in prepubertal children was about 0.4 SD. If this increase continued, the decrease in the risk of fractures would be important. In the study of Matkovic et al.³ the additional bone mass (about 6 percent) in subjects living where calcium intake was high was associated with a large reduction in the risk of hip fracture.

The mechanisms by which calcium supplementation produces its effect could not be determined by our study. The significantly lower serum osteocalcin concentrations suggested that a decrease in bone turnover could be responsible. This, and the greater rates of gain in bone mineral density early in the study (at least in the radius), are consistent with a reduced rate of bone remodeling. Other conditions in which turnover is reduced are also associated with increased bone mass (e.g., in blacks as compared with whites²³).

We do not know whether the differences in the increase in bone mineral density in the twins given calcium supplements will persist. The retrospective studies that found a greater bone mass later in life among subjects who had high calcium intakes during childhood^{3 4 5} suggest that the differences do persist, although this persistence may be due to a greater lifelong calcium intake. Calcium supplementation had no effects on the general growth of the children in our study (height, weight, and bone area and width), in contrast to reports early in this century of increased height in children whose diets were supplemented with milk.^{24 , 25} However, that effect may have been due to increased protein and caloric intake, as well as calcium intake, in less well nourished children. The children we studied were well nourished, and most of those under the age of 12 years (those with the greatest response) were receiving an amount of calcium close to the recommended dietary allowance (800 mg per day).²⁶ Calcium-balance studies of children of these ages have demonstrated increasingly positive balances throughout the range of intakes in this study.²⁷ Surveys of adolescents indicate that their dietary calcium intake is considerably lower than that recommended, which is 1200 mg per day.^{28 , 29}

In conclusion, dietary supplementation with calcium alone is associated with a gain in bone mineral acquisition, especially in prepubertal children. If the gain persists, it would probably result in an increase in peak bone mass that would reduce the risk of osteoporotic fractures later in life. The results also raise the question whether the recommended allowances for calcium should be revised upward.