1. Introduction
Osteoporosis is a severe global public health concern, particularly in women [1,2,3]. Low bone mass acquisition in childhood is an important risk factor for osteoporosis at later ages [4,5,6]. Maximizing peak bone mass before skeletal maturity by improving modifiable factors, such as diet and physical activity [7], will delay the time for osteoporosis in the middle or elderly ages [8].
A previous meta-analysis comprising 19 randomized controlled trials (RCT) performed on 2859 children with an average age of 10 years determined that calcium supplementation (800 mg/d) for 1–2 years increased the bone mineral density (BMD) of the total body (and arms) (SD 0.14, 95%CI: 0.01 to 0.27) in children with a basal dietary calcium intake of approximately 750 mg/d [9]. In addition, a recent systematic review also reported that milk intake equivalent to 250–1200 mg/d of calcium for 12–24 months in adolescents aged 9–18 years significantly increased their bone mineral content (BMC) [10]. However, most of the subjects in the above studies were focused on ages 7–18 years [5,9,10], but little is known about the effects of calcium and dairy supplementation on bone health in children aged 4–6 years [4,11].One RCT examined the effects of physical activity and calcium supplementation (1000 mg/d) on BMC in 3–5-year-old children with considerably high baseline calcium intake (946 mg/d) in the United States [12]. However, no study has examined the effects of calcium or milk supplementation on bone mass gain in 4–6-year-old children in Asian populations. Considering the recommended nutrient intake (RNI) of calcium varied significantly with age in childhood, more evidence would thus be needed for preschool children to develop their RNI, particularly in Chinese children with quite a different diet and calcium intake from Western children.
In young children, calcium-enriched foods are limited if they do not regularly consume dairy foods in China [13,14]. According to the China Health and Nutrition Survey (CHNS), the average milk consumption per day has increased significantly from 3.9 g/day in 1991 to 26.1 g/day in 2006 in Chinese children and adolescents [15]. However, milk consumption was much lower than 350–500 g/day, as recommended in the Chinese Dietary Guidelines for Preschool Children (2022) [16]. The mean calcium intake in Chinese children aged 4–6 years ranged between 225 mg/d and 242 mg/d from 1991 to 2009 based on the data from CHNS [17]. Calcium intake was even lower in rural or undeveloped regions (about 210 mg/d) in China [17], which was much lower than that of those in Western countries [18], and was only about 26% of China’s recommendations (800 mg/d) for 4–6 years children in 2013 [19]. Furthermore, the BMD and BMC in national-wide children are much lower than those of the large cities in China [20,21,22], suggesting the bone mineral deposition is much less than their genetically determined potential, possibly due to relatively poorer nutrition in children of developing regions. Since regular milk cannot provide sufficient calcium and micronutrients for children in rural areas, particularly in Northwest China, typically with a poor economic status, formula milk would be a better choice than regular milk. To the authors’ knowledge, no study has yet examined the effect of milk supplementation on bone health in young children in China. Thus, this clinical trial aimed to evaluate the effects of 60 g of formula milk powder supplementation on bone health in Chinese children aged 4–6 years in a rural region.
2. Subjects and Methods
2.1. Study Participants
The participants were year two or year three children from two kindergartens in Huining County, Gansu Province, Northwest China, who participated in the trial from September 2021 to September 2022. Eligible participants were healthy children aged 4–6 years old. They were willing to drink milk but consumed less for economic reasons. We excluded those who had the following conditions: milk/dairy allergy or intolerance, requiring hospitalization or long-term medication (>1 month), taking supplements of calcium, vitamin D, or other dietary supplements for more than 3 months, and milk consumption of more than 600 mL per week in the past two years. Of the 351 children initially screened, 267 completed and met the screening questionnaire. Finally, 196 (103 girls and 93 boys) children were enrolled in the current trial (Figure 1).
All legal guardians of all participants provided informed consent. The study was performed in accordance with the declaration of Helsinki, and the ethics committee of the School of Public Health of Sun Yat-Sen University approved the study. The clinical trial registration number of this research was ChiCTR2100051044.
2.2. Study Design and Intervention Programs
This study was a 12-month, cluster-randomized controlled trial. One hundred ninety-six children were randomly assigned to the formula milk and control groups by a lottery method according to their natural kindergarten class, matched for the year grade. In the milk group, each child was given 60 g/d of formula milk powder (blended into 300 mL of milk) targeted for 3–6 years children (QQ Star Milk Powder, Yili Industrial Group Co., Ltd., Hohhot, China). The daily dose contained 720 mg of calcium and 4.5 μg of vitamin D (Table S1). Briefly, the formula milk was given as a part of breakfast or an extra meal after lunch. The consumption was recorded daily by kindergarten teachers or their guardians. For the control group, 20–40 g of bread was given per day to each participant during the days in kindergarten.
2.3. Bone Mineral Status Measurements
The primary outcome indicators were BMD and BMC, measured at the left forearm and calcaneus by an EXA-3000 peripheral dual-energy X-ray bone densitometer obtained from OsteoSys (Seoul, Korea) at 0 (baseline), 6, and 12 months post-intervention. The specific test site for the forearm is the ulna and radius in the distal 1/3 of the left forearm, while that for calcaneus is the central area of the left calcaneus bone at approximately 2 cm2. For quality control, the final scans at all skeletal sites were analyzed based on a reference of the baseline scan to minimize localization errors in positioning the region of interest. Each site had two readings, and the mean value was used for further analysis. Two specialists conducted all the scanning. Among 58 children, the CVs of BMD and BMC were less than 2% at the measured sites for two consecutive measurements with repositioning on the same day.
2.4. Anthropometric Examinations
Body height and weight were assessed at baseline and at 3, 6, 9, and 12 months post-intervention. Body height was determined to the nearest 0.1 cm and body weight to the nearest 0.5 kg, with the participants wearing light clothing and no shoes. In addition, the measurements of height were controlled to be taken within 2 h on the same day for quality control.
2.5. Biomarker Determinations
Samples of overnight fasting blood were collected at baseline and at 6 and 12 months post-intervention. Serum and plasma samples were separated and stored at −80 °C until the analyses for the following biomarkers. Bone formation biomarkers (bone-specific alkaline phosphatase [BAP] and osteocalcin), bone resorption marker (β-C-terminal telopeptides [β-CTx]), parathyroid hormone (PTH), and insulin-like growth factor 1 (IGF-1) were determined by the electrochemiluminescence immunoassay methods (Roche Diagnostics GmbH, Mannheim, Germany) [13,23]. Tartrate-resistant acid phosphatase-5b (Trap-5b) was determined using an enzyme-linked immunosorbent assay method (Meimian, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) [24]. Concentrations of alkaline phosphatase (ALP) were quantitatively measured by using an automated biochemistry analyzer and the corresponding commercial kit (Roche Diagnostics GmbH, Mannheim, Germany), as previously reported [25]. In addition, 25-hydroxy vitamin D2 (25-OH-D2) and 25-hydroxy vitamin D3 (25-OH-D3) were measured by liquid chromatography-mass spectrometry (LC-MS) by KingMed Diagnostics (Guangzhou, China) according to standard laboratory operating procedures [26], and total 25-hydroxy vitamin D (25-OH-D) was then calculated by summing the 25-OH-D2 and 25-OH-D3. For quality control, 10% of the samples for each indicator need to be repeated. The CVs of these biomarkers were 2.7–8.0%.
2.6. Assessments of Dietary Intake and Physical Activity
Demographic information of all participants (e.g., sex, age, gestational age at birth, birth weight, breastfeeding time after birth, dairy products intake, the history of disease and medication, physical activity, and sleeping) and their parents (e.g., age, height, weight, and education level) were obtained by using face-to-face questionnaire interviews at the enrollment. In addition, the participants were required to complete a 3-day (including one weekend day) food diary, noting adverse reactions and side effects, physical activity, and sleeping time with their guardians’ assistance at each visit at 3, 6, 9, and 12 months [27]. Photographs of food portions were supplied to help to estimate food intake, and further analysis was performed using the average values of the 3-day food diaries. Moreover, the exercise (e.g., activity steps, walking distance, and running distance), heart rate, and sleeping time for seven consecutive days were monitored by health bracelets (Xiaomi NFC5, Guangdong, China).
2.7. Statistical Analysis
The sample size was calculated based on an 18-month intervention trial on the effects of calcium supplementation (300 mg) on forearm bone health in Chinese children aged seven years old [28], in which the percentage mean (SD) difference in the changes of BMC at the forearm between intervention and control group was 2.5 (4.5%) g/cm2. Seventy-four children per group were provided 85% power to detect a 2.5% difference in BMD change between the milk supplement group and control group at α = 0.05. Eighty-two participants were required if a 10% dropout rate was considered. Finally, 96 and 100 participants (103 girls and 93 boys) were recruited into the intervention and control groups.
Participants who completed all visits for BMD/BMC tests were included in the final analyses. Continuous variables were presented as means ± SDs or medians and interquartile (IQR), and categorical variables were expressed as numbers (percentages). Baseline characteristics were compared using Student’s t-test for normally distributed data, Kruskal–Wallis H rank sum tests for skewed distributed data, and Chi-square tests for categorical data. For the effects of milk intervention, the primary analyses compared the mean difference of 12-month changes in BMD and BMC at the left forearm and calcaneus using Student’s t-test. Additional analyses included comparisons of the differences between the formula milk and control groups in the study indices, including absolute changes and percentage changes in BMD, BMC, bone metabolism biomarkers, height, and weight at each visit using Student’s t-test or Kruskal–Wallis H rank sum tests for the univariate analyses and using analysis of covariance (ANCOVA) after adjusting potential covariates. The ANCOVA was adjusted for factors related to demographics, economics, baseline values for each indicator, dietary intake, physical activity, and other outcome health indicators. Continuous variables with skewed distribution were transformed to normality before the ANCOVA. All statistical tests were two-sided. A p-value less than 0.05 was defined as a significant difference. SPSS version 25 (IBM Statistics, IBM Corporation, New York, NY, USA) and R 4.1.2 were used for the analyses.
3. Results
3.1. Subjects’ Characteristics and Intervention Adherence
Of the 196 children enrolled, 22 withdrew during the study period, and the final group sizes for the milk and control groups were 83 and 91, respectively. The reasons for their withdrawal are presented in Figure 1. There were no significant differences between the milk and control groups in dropout rates (13.54% vs. 9.0%, p = 0.435). The baseline characteristics were all similar in the two groups, as depicted in Table 1 (p > 0.05). The overall compliance in the milk group was 92.3%, equivalent to an intake of 665 mg of calcium from formula powder.
3.2. Dietary Intake and Physical Activity during the Study Period
Table 2 and Table 3 display the mean daily intake of food categories and nutrients in milk and control groups during the intervention. The milk group showed a higher mean dairy consumption than the control group (median, 300 mL vs. 0 mL, p < 0.001) based on the estimations of four 3-day food diaries during the 12-month study period. However, there was no significant difference in the intake of other major food categories (e.g., cereals, beans, animal foods, vegetables, fruits, edible fungus, nuts, and beverages) between the milk and control groups (Table 2). As shown in Table 3, the milk group had significantly increased the median daily intakes of protein (42.5 g vs. 34.0 g), vitamin D (VD) (5.4 µg vs. 1.0 µg), and calcium (839 mg vs. 196 mg) compared with the control group (all p < 0.001). In addition, marked increases in other nutrients (such as vitamin A, vitamin B2, folic acid, potassium, magnesium, and zinc) intake were also observed in the milk group (all p < 0.05). On the other hand, there were no significant differences in the median values of walking steps (9628 vs. 9463), running distance (1341 m vs. 1239 m), and sleeping time (7.7 h vs. 7.5 h) per day between the control and supplementation groups according to the three 7-day health bracelets monitors (all p > 0.05) (Table 4).
3.3. Effects of Formula Milk Intervention on Bone Health
Table S2 shows no significant differences in baseline values of BMD and BMC at the left forearm and calcaneus between the milk and control groups (p > 0.05). However, after the 12-month intervention, bone mineral acquisition at the left forearm was significantly greater in the milk group than in the control group in both univariate and multivariate analyses (Figure 2, Table S2). In the univariate model, the mean (±SD) increments in BMD and BMC between the milk and control groups were significantly higher in the 6th and 12th month (all p < 0.0001). The mean percentage differences in bone acquisition in the milk group compared to the control group were 3.77% and 6.66% for BMD and 4.55% and 5.76% for BMC at 6 and 12 months (all p < 0.0001). For BMD and BMC changes at the left calcaneus, a similar trend (but less significant) was observed in the milk (vs. control) group. The mean percentage differences in bone acquisition in the milk group compared to the control group were 2.83% for BMD (p = 0.013) and 2.38% for BMC (p = 0.024) in the 6th month. However, there was no significant difference between the groups in the left calcaneus BMD and BMC in the 12th month (all p > 0.05). Moreover, the statistical differences were not significantly altered after adjusting the potential confounders in the multivariate model.
3.4. Effects of Formula Milk Intervention on Height and Weight
Figure 3 shows that the percentage increases in height are 0.34%, 0.45%, and 0.42% higher in the milk group than in the control group after a 3-, 6-, and 9-month intervention, respectively (p < 0.05). However, the beneficial difference did not remain significant at the end of the trial (p > 0.05). There was no significant difference in the weight gain between the two study groups at each follow-up visit during the whole intervention period (p > 0.05).
3.5. Effects of Formula Milk Intervention on Bone Metabolism Biomarkers
As presented in Figure 4 and Table S3, the biomarkers of bone resorption and formation had no significant difference in their changes between the two groups throughout the trial except for osteocalcin in the 6th month and ALP in the 12th month. Compared to the control group, the milk group showed significant changes in osteocalcin level (−7.59%, p = 0.012) in the 6th month and ALP level (−10.44%, p< 0.001) in the 12th month. In addition, milk supplementation significantly improved serum 25(OH)D (+5.54%, p = 0.001), PTH (−15.22%, p = 0.003), and IGF-1 (+8.36%, p = 0.014) compared to the control group in the 6th month in March 2022. However, there were no significant changes in 25(OH)D (p = 0.083), PTH (p = 0.066), and IGF-1 (p = 0.070) levels remained at the end (September 2022) of the study.
4. Discussion
The results of this cluster randomized controlled trial indicated that 60 g/d formula milk supplementation over 6 and 12 months significantly increased bone mass acquisition. In addition, milk supplementation for 3–9 months could marginally increase the height gain. The increases in 25(OH)D and IGF-1 and the decrease in PTH may support (at least in part) the beneficial changes in bone acquisition.
4.1. Formula Milk/Calcium Supplementation and Bone Mineral Acquisition
The magnitude of the 60 g/d of milk supplementation effect was considerable, with a significant increase in left forearm BMD of 6.66% and in BMC of 5.76% after 12 months in children aged 4–6 years. Our findings were consistent with those in previous studies, demonstrating that increased calcium [9,29,30,31,32,33,34,35,36,37,38,39,40] or dairy products [11,21,41] intake increases bone mineral status. For example, a randomized, double-blind, placebo-controlled study in 160 Gambian 8–12 years children accustomed to a low-calcium diet (338 ± 142 mg/d) revealed that calcium supplementation (1000 mg/d) for 12 months significantly enhanced the BMC and BMD in the distal radius (8.4% and 7.0%) and midshaft (3.0% and 4.5%) (all p < 0.05) [18]. Another 18-month randomized trial on adolescent girls (mean age 12 y) showed that calcium supplementation (792 mg/d) had significantly greater BMD and BMC gains at the total body and upper limb (all p < 0.05) [42]. In a 2-year milk intervention trial in a school conducted on Beijing girls aged ten, a pronounced BMD increase in the total body (3.2%–5.3%) was observed compared to the control group [13]. Thus, these supported the beneficial effect of calcium supplementation on bone mineral gains in childhood populations. Our findings highlighted the importance of adequate milk consumption in rural young children with less calcium-enriched foods in China.
The milk-attributable gains in BMD and BMC were much more prominent in the non-weight-bearing forearm than in the weight-bearing forearm in this study. A meta-analysis involving 2859 children showed that an average dose of 800 mg/d of calcium supplementation also showed more benefit to BMD at the non-weight-bearing areas (total body and upper limb) than the weight-bearing sites (femoral neck and lumbar spine) in children with a mean age of 10 years [9]. A less significant beneficial effect of milk supplementation on BMD and BMC gains at the calcaneus (weight-bearing site) was found in this population. In accordance with our results, a randomized and placebo-controlled intervention study reported a significant effect of calcium supplementation on BMC gain at the radius-ulna but not the weight-bearing site (tibia-fibula) [7]. It might be because bone acquisition in the weight-bearing sites was more susceptible to gravity effects [18], which could easily mask the effects provided by environmental factors (e.g., diet). On the other hand, it has been reported that the combination of high doses of calcium and exercise showed more significant bone acquisition than that of either exercise or calcium alone at the weight-loaded sites of the hip and femoral [43]. Therefore, the effects of environmental factors (e.g., diet) on weight-bearing calcaneus bones has yet to be validated in studies with large samples, longer follow-up times, and a combination of calcium and exercise intervention in preschool children.
4.2. Formula Milk/Calcium Supplementation and Bone Metabolism Biomarkers
The benefit of milk intervention on bone gains was supported by bone-related biomarkers, such as increased serum 25(OH)D (mainly D3) and IGF-1, as well as reduced PTH, as did in a previous study [44]. The results indicated that supplemental milk had no apparent changes in most bone resorption and formation biomarkers. Previous studies showed that VD supplementation and higher circulating VD were associated with better BMD in Children [45]. Sunlight irradiation could significantly affect the circulating 25(OH)D level [46]. We observed a decreased serum 25(OH)D concentration in both groups in the early Spring (March 2022) compared to the baseline values in September 2021 due to the reduced sunlight irradiation in a long winter. Therefore, it suggested that daily supplementation of 4.5 µg vitamin D was insufficient to prevent the decrease in circulating 25(OH)D in the winter/spring in young children in Northwest China. However, there was a less decreased 25(OH)D level in the formula milk group (vs. control). Furthermore, a similar 25(OH)D concentration was found at the end of the intervention in September 2022 in both groups and to those at the baseline, suggesting the intervention dosage (4.5 µg vitamin D) could not provide additional benefit to the circulating 25(OH)D in the summer/autumn in the study population.
Parathyroid hormone (PTH) is essential in bone metabolism, especially during pediatric growth. It has been reported that PTH concentrations increased with age during childhood. A peak PTH concentration was observed in the 10–14 years with the most rapid bone growth [47]. Our study also suggested an increasing trend in PTH with bone growth at 4–6 years of age. It has been reported that calcium supplementation could decrease bone remodeling by inhibiting PTH secretion. At the same time, an increase in BMD is the consequence of space-filling for remodeling in adults [48,49]. In the present study, relatively lower increases in plasma PTH concentration were observed in the formula milk group compared to the controls, suggesting that the above mechanism was similar in children [38]. A previous meta-analysis of randomized controlled trials also reported that increased dairy intake could elevate the IGF-1 level [50]. In addition, it has been demonstrated that periosteal bone apposition might be stimulated by IGF-1, contributing to a slightly larger skeletal envelope in the milk group [51]. Therefore, the changes in IGF-1 in the milk supplementation group may support the benefits of height and bone growth.
4.3. Nutritional Factors in Formula Milk and Bone Health
The effect of formula milk powder may be attributed to milk-contained nutrients. A daily dose of the milk contains 720 mg of calcium and 4.5 mg of VD, resulting in an extra intake of 643 mg of calcium and 4.4 μg VD in the milk group compared to the controls with deficient intakes of calcium (196 mg/d) and VD (1.0 μg/d). The remarkable improvement of BMD and BMC in the milk intervention group indicated that the habitual calcium and/or VD intake was much less than their requirements for optimal bone acquisition. It highlighted the importance of adequate milk consumption in young children. In addition, formula milk provides a relatively rich source of high-quality protein and other nutrients (e.g., vitamin K, folic acid, potassium, magnesium, and zinc) that could help improve bone health in children and adolescents [52,53].
4.4. Strengths and Limitations
This study has several strengths. First, the increased final study sizes (final/planned: 174/148) would improve the statistical power of this trial. The high adherence (92.3%) to the milk supplementation in the milk group reduced the likelihood of false negatives for the primary study outcome. Next, the consumption of dairy products did not significantly increase in the control group, showing no obvious contamination of the intervention. Finally, the comparability in baseline characteristics and physical activities, non-dairy foods, and sleeping time during the intervention period between the two groups would attenuate potential confounding bias.
Some limitations should be noted. Firstly, we could not exclude the possibility of introducing information bias due to no placebo control and double-blind measures. However, we reduced the potential by using objective outcomes, duplicated reading for BMD and BMC, and determining the laboratory indices without knowing the group status. Secondly, the cluster randomization method was less efficient in balancing the confounding factors than the individual-based randomized technique. However, the two groups had no significant differences in the main baseline characteristics. Lastly, the sample size of the trial was calculated based on the difference (SD) in changes of BMC at the upper limb, and we might not have sufficient statistical power to detect the differences in the other indices (e.g., bone biomarkers, BMD/BMC at the calcaneus).
5. Conclusions
In conclusion, the study demonstrates that supplementing 60g/d of formula milk powder with fortified calcium and VD over 6 and 12 months enhances bone mass acquisition at the left forearm in Chinese preschool children. The marginal increase of the height gain during the 3–9 months of milk intervention needed further replication in long-term intervention studies. The increase in 25(OH)D and IGF-1 and the decrease in PTH may support (at least in part) the beneficial changes in bone acquisition. Our findings highlight the importance of adequate milk consumption in young children in a rural region in China.
Conceptualization, Y.-M.C., Z.-F.Z. and L.-P.J.; formal analysis, B.-Y.L.; investigation, B.-Y.L., J.-L.M., Z.-F.Z., J.-Y.H., W.-H.Y., X.-F.B., H.-T.F. and I.M.-Y.S.; resources, Z.-F.Z., W.-H.Y., H.-T.F. and I.M.-Y.S.; writing—original draft preparation, B.-Y.L. and Y.-M.C.; writing—review and editing, Y.-M.C., Z.-F.Z., J.-Y.H., W.-H.Y. and X.-F.B.; project administration, Y.-M.C., Z.-F.Z. and L.-P.J.; funding acquisition, Z.-F.Z., J.-Y.H. and Y.-M.C. All authors have read and agreed to the published version of the manuscript.
The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of the School of Public Health of Sun Yat-sen University (No. 2021(067), 29 June 2021).
Informed consent was obtained from all subjects involved in the study.
Not applicable.
We thank all study participants of the study. We also thank all other members involved in the study.
The formula milk powder (QQ Star Milk Powder) used in the study was produced and provided by Inner Mongolia Yili Industrial Group Co., Ltd., China. Yu-ming Chen from Sun Yat-sen University received a research grant (Grant B) supported by Inner Mongolia Dairy Technology Research Institute Co. Ltd. (IMDTRI), Hohhot, China, to complete this experiment as the principal investigator (PI). The fund was a part of the support from the Huhhot Science & Technology Plan (No.2020-Ke Ji Xing Meng-National Innovation Center-10 projects) (Grant A) received by Zi-fu Zhao at IMDTR. Li-peng Jing from Lanzhou University received a research grant (Grant C) from Sun Yat-sen University as a part of Grant B to complete the trial fieldwork under the PI’s supervision. Jing-yu Hao, Zi-fu Zhao, and Ignatius Man-Yau Szeto are employees of IMDTR and Inner Mongolia Yili Industrial Group Co., Ltd., Hohhot, China (IGYIGC). Wen-hui Ye and Xue-fei Bai are employees of IGYIGC. All of them were responsible for developing and producing the formula milk powder. They also conducted regular audits for the trial implementation as per the study protocol developed by the PI.
Footnotes
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
Enlarge this image.Figure 2. Mean (95%CI) change in BMD and BMC at the left forearm and calcaneus over 6 and 12 months compared to baseline. p values are for the differences between groups by Student’s t-test. Percentages are incremental gains of BMD and BMC. (a,b) mean (95%CI) change in BMD (a) and BMC (b) at the left forearm; (c,d), mean (95%CI) change in BMD (c) and BMC (d) at the left calcaneus; BMD, bone mineral density; BMC, bone mineral content.
Enlarge this image.Figure 3. Mean (95%CI) change in height (a) and weight (b) over 6 and 12 months compared to baseline. p values are for the differences between groups by Student’s t-test. Percentages are incremental gains in height and weight.
Enlarge this image.Figure 4. Mean (95%CI) change in bone metabolism biomarkers over 6 and 12 months compared to baseline. (a–h) mean (95%CI) changes in β-CTx (a), Trap-5b (b), ALP (c), 25(OH)D (d), PTH (e), IGF-1 (f), BAP (g), and osteocalcin (h), respectively. p values are for the differences between groups by Student’s t-test. Percentages are incremental gains of bone metabolism biomarkers. β-CTx, β-C-terminal telopeptides; Trap-5b, tartrate-resistant acid phosphatase-5b; ALP, alkaline phosphatase; 25(OH)D, 25-hydroxy-vitamin-D; PTH, parathyroid hormone; IGF-1, insulin-like growth factor 1; BAP, bone-specific alkaline phosphatase.
Baseline characteristics of study participants and their parents.
| Characteristic | Control |
Formula |
p a |
|---|---|---|---|
| Children Characteristic | |||
| Age at study entry days, year | 4.8 ± 0.7 | 4.6 ± 0.8 | 0.087 |
| Birth weight, Kg | 3.2 ± 0.5 | 3.3 ± 0.4 | 0.175 |
| Breastfeeding time, months | 12.2 ± 5.8 | 12.4 ± 5.2 | 0.834 |
| Sex (Female) | 50 (50.0) | 53 (55.2) | 0.465 |
| Gestational age at birth, weeks | |||
| <37 | 6 (6.0) | 5 (5.2) | 0.970 |
| 37~42 | 90 (90.0) | 87 (90.6) | |
| >42 | 4 (4.0) | 4 (4.2) | |
| Feeding patterns after 6 months of life | |||
| Exclusive breastfeeding | 76 (76.0) | 71 (74.0) | 0.488 |
| Partial breastfeeding | 18 (18.0) | 22 (22.9) | |
| Non-breastfeeding | 6 (6.0) | 3 (3.1) | |
| Nutritional supplements use (Yes) | 36 (36.0) | 40 (41.7) | 0.465 |
| Dairy products intake, times/week | |||
| 0~1 | 43 (43.0) | 35 (36.5) | 0.383 |
| >1 | 57 (57.0) | 61 (63.5) | |
| Maternal Characteristic | |||
| Mother’s age, year | 27.69 ± 4.46 | 28.06 ± 4.12 | 0.544 |
| Father’s age, year | 30.06 ± 5.15 | 29.95 ± 4.62 | 0.873 |
| Mother’s BMI, kg/m2 | 22.11 ± 2.84 | 22.54 ± 3.18 | 0.324 |
| Father’s BMI, kg/m2 | 23.01 ± 3.28 | 23.10 ± 3.79 | 0.867 |
| Mother’s educational achievement | |||
| Secondary school or below | 50 (50.0) | 44 (45.8) | 0.721 |
| High school | 19 (18.4) | 17 (17.7) | |
| Junior college or above | 31 (33.7) | 35 (36.5) | |
| Father’s educational achievement | 0.358 | ||
| Secondary school or below | 48 (48.0) | 37 (38.5) | |
| High school | 21 (21.0) | 21 (21.9) | |
| Junior college or above | 31 (31.0) | 38 (39.6) |
a Chi-square test for categorical and Student’s t-test for continuous variables. Nutritional supplements use: taking supplements of calcium, vitamin D, or other dietary supplements for more than three months before study entry; dairy products intake: the average frequency of milk consumption in the previous month before study entry; BMI, body mass index, weight/height2 (in kg/m2).
Comparison of daily intake of food categories in the formula and control groups during the intervention period.
| Daily Intake | Control (n = 91) |
Formula (n = 83) |
p * | ||||
|---|---|---|---|---|---|---|---|
| Cereals, g | 511 | 443 | 600 | 508 | 440 | 577 | 0.799 |
| Beans, g | 0.0 | 0.0 | 11.9 | 0.0 | 0.0 | 7.6 | 0.208 |
| Animal foods, g | |||||||
| Livestock | 31.7 | 0.0 | 51.5 | 31.7 | 2.0 | 47.6 | 0.796 |
| Poultry | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.936 |
| Eggs | 23.8 | 0.0 | 47.6 | 23.8 | 0.0 | 47.6 | 0.427 |
| Aquaculture, g | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.333 |
| Dairy, mL | 0 | 0 | 54 | 300 | 300 | 327 | <0.001 |
| Vegetables, g | |||||||
| Leafy vegetables | 35.7 | 15.9 | 55.5 | 31.7 | 13.9 | 47.6 | 0.134 |
| Other vegetables | 99.1 | 61.5 | 144.7 | 87.2 | 47.6 | 150.3 | 0.567 |
| Total | 139 | 91 | 201 | 123 | 79 | 178 | 0.199 |
| Fruits, g | 276 | 155 | 369 | 293 | 175 | 426 | 0.181 |
| Edible Fungus, g | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.482 |
| Nuts, g | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.584 |
| Beverages, mL | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.708 |
*, Kruskal–Wallis H test; IQR, interquartile range.
Comparison of daily intake of nutrients in the formula and control groups during the intervention period.
| Daily Intake | Control (n = 91) |
Formula (n = 83) |
p * | ||||
|---|---|---|---|---|---|---|---|
| Energy intake, Kcal | 978 | 877 | 1203 | 1159 | 1012 | 1364 | <0.001 |
| Protein, g | 34.0 | 27.3 | 41.5 | 42.5 | 35.3 | 50.2 | <0.001 |
| Fat, g | 16.5 | 10.1 | 23.9 | 23.5 | 17.6 | 31.4 | <0.001 |
| Carbohydrates, g | 186 | 163 | 222 | 189 | 170 | 214 | 0.548 |
| Vitamin A, µgRE | 375 | 263 | 490 | 437 | 339 | 568 | 0.012 |
| Vitamin D, µg | 1.0 | 0.5 | 1.5 | 5.4 | 5.1 | 5.8 | <0.001 |
| Vitamin K, µg | 89 | 66 | 118 | 71 | 50 | 112 | 0.026 |
| Vitamin B1, µg | 0.3 | 0.2 | 0.4 | 0.3 | 0.3 | 0.4 | 0.017 |
| Vitamin B2, µg | 0.5 | 0.4 | 0.6 | 0.7 | 0.6 | 0.9 | <0.001 |
| Vitamin C, mg | 77 | 53 | 108 | 81 | 65 | 95 | 0.456 |
| Folic acid, µg | 99 | 81 | 128 | 135 | 114 | 154 | <0.001 |
| Calcium, mg | 196 | 140 | 274 | 839 | 783 | 906 | <0.001 |
| Potassium, mg | 1005 | 779 | 1243 | 1346 | 1187 | 1593 | <0.001 |
| Magnesium, mg | 153 | 126 | 187 | 175 | 151 | 207 | 0.001 |
| Iron, mg | 9.4 | 7.8 | 11.1 | 9.1 | 7.8 | 10.8 | 0.595 |
| Zinc, mg | 5.1 | 4.1 | 6.2 | 5.6 | 4.7 | 6.4 | 0.016 |
| Selenium, mg | 24 | 19 | 31 | 28 | 23 | 33 | 0.051 |
*, Kruskal–Wallis H test; IQR, interquartile range.
Comparison of daily physical activity and sleeping time in the formula and control groups during the intervention period.
| Variables | Control (n = 91) |
Formula (n = 83) |
p * | ||||
|---|---|---|---|---|---|---|---|
| Activity steps, steps | 9463 | 7581 | 11063 | 9628 | 8197 | 11520 | 0.247 |
| Walking distance, m | 6223 | 5048 | 7504 | 5763 | 4768 | 7081 | 0.320 |
| Running distance, m | 1239 | 939 | 1530 | 1341 | 950 | 1688 | 0.323 |
| Energy expenditure, kcal | 197 | 156 | 235 | 182 | 118 | 227 | 0.143 |
| Deep sleeping time, h | 1.9 | 1.7 | 2 | 2 | 1.7 | 2.1 | 0.205 |
| Shallow sleeping time h | 5.6 | 5.3 | 5.9 | 5.7 | 5.4 | 6.1 | 0.170 |
| Total sleeping time, h | 7.5 | 7.1 | 7.9 | 7.7 | 7.2 | 8 | 0.087 |
*, Kruskal–Wallis H test; IQR, interquartile range.
Supplementary Materials
The following supporting information can be downloaded at:
References
1. Song, S.; Guo, Y.; Yang, Y.; Fu, D. Advances in pathogenesis and therapeutic strategies for osteoporosis. Pharmacol. Ther.; 2022; 237, 108168. [DOI: https://dx.doi.org/10.1016/j.pharmthera.2022.108168] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/35283172]
2. Khosla, S.; Hofbauer, L.C. Osteoporosis treatment: Recent developments and ongoing challenges. Lancet Diabetes Endocrinol.; 2017; 5, pp. 898-907. [DOI: https://dx.doi.org/10.1016/S2213-8587(17)30188-2] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/28689769]
3. Zhao, J.G.; Zeng, X.T.; Wang, J.; Liu, L. Association Between Calcium or Vitamin D Supplementation and Fracture Incidence in Community-Dwelling Older Adults: A Systematic Review and Meta-analysis. JAMA; 2017; 318, pp. 2466-2482. [DOI: https://dx.doi.org/10.1001/jama.2017.19344]
4. Van den Heuvel, E.; Steijns, J. Dairy products and bone health: How strong is the scientific evidence?. Nutr. Res. Rev.; 2018; 31, pp. 164-178. [DOI: https://dx.doi.org/10.1017/S095442241800001X] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/29560832]
5. Ma, D.F.; Zheng, W.; Ding, M.; Zhang, Y.M.; Wang, P.Y. Milk intake increases bone mineral content through inhibiting bone resorption: Meta-analysis of randomized controlled trials. e-SPEN J.; 2013; 8, pp. e1-e7. [DOI: https://dx.doi.org/10.1016/j.clnme.2012.10.005]
6. Chevalley, T.; Bonjour, J.P.; Ferrari, S.; Rizzoli, R. The influence of pubertal timing on bone mass acquisition: A predetermined trajectory detectable five years before menarche. J. Clin. Endocrinol. Metab.; 2009; 94, pp. 3424-3431. [DOI: https://dx.doi.org/10.1210/jc.2009-0241]
7. Cameron, M.A.; Paton, L.M.; Nowson, C.A.; Margerison, C.; Frame, M.; Wark, J.D. The effect of calcium supplementation on bone density in premenarcheal females: A co-twin approach. J. Clin. Endocrinol. Metab.; 2004; 89, pp. 4916-4922. [DOI: https://dx.doi.org/10.1210/jc.2003-031985]
8. Rizzoli, R.; Biver, E.; Brennan-Speranza, T.C. Nutritional intake and bone health. Lancet Diabetes Endocrinol.; 2021; 9, pp. 606-621. [DOI: https://dx.doi.org/10.1016/S2213-8587(21)00119-4]
9. Winzenberg, T.; Shaw, K.; Fryer, J.; Jones, G. Effects of calcium supplementation on bone density in healthy children: Meta-analysis of randomised controlled trials. BMJ; 2006; 333, 775. [DOI: https://dx.doi.org/10.1136/bmj.38950.561400.55]
10. Huncharek, M.; Muscat, J.; Kupelnick, B. Impact of dairy products and dietary calcium on bone-mineral content in children: Results of a meta-analysis. Bone; 2008; 43, pp. 312-321. [DOI: https://dx.doi.org/10.1016/j.bone.2008.02.022]
11. Matsuyama, M.; Harb, T.; David, M.; Davies, P.S.; Hill, R.J. Effect of fortified milk on growth and nutritional status in young children: A systematic review and meta-analysis. Public Health Nutr.; 2017; 20, pp. 1214-1225. [DOI: https://dx.doi.org/10.1017/S1368980016003189] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/27938461]
12. Specker, B.; Binkley, T. Randomized trial of physical activity and calcium supplementation on bone mineral content in 3- to 5-year-old children. J. Bone Miner. Res.; 2003; 18, pp. 885-892. [DOI: https://dx.doi.org/10.1359/jbmr.2003.18.5.885] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/12733728]
13. Du, X.; Zhu, K.; Trube, A.; Zhang, Q.; Ma, G.; Hu, X.; Fraser, D.R.; Greenfield, H. School-milk intervention trial enhances growth and bone mineral accretion in Chinese girls aged 10-12 years in Beijing. Br. J. Nutr.; 2004; 92, pp. 159-168. [DOI: https://dx.doi.org/10.1079/BJN20041118] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/15230999]
14. Zhu, K.; Zhang, Q.; Foo, L.H.; Trube, A.; Ma, G.; Hu, X.; Du, X.; Cowell, C.T.; Fraser, D.R.; Greenfield, H. Growth, bone mass, and vitamin D status of Chinese adolescent girls 3 y after withdrawal of milk supplementation. Am. J. Clin. Nutr.; 2006; 83, pp. 714-721. [DOI: https://dx.doi.org/10.1093/ajcn.83.3.714] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/16522922]
15. Du, W.W.; Wang, H.J.; Wang, Z.H.; Zhai, F.Y.; Zhang, B. Trend of milk consumption among Chinese children and adolescents aged 7 to 17 years old in 9 provinces from 1991 to 2006. Zhonghua Liu Xing Bing Xue Za Zhi; 2010; 31, pp. 1349-1352.
16. Maternal and Child Nutrition Branch of Chinese Nutrition Society. Dietary Guidelines for Preschool Children (2022); Chinese Nutrition Society: Beijing, China, 2022.
17. Zhang, J.; Wang, H.J.; Wang, Z.H.; Zhang, J.G.; Du, W.W.; Su, C.; Jiang, H.R.; Zhai, F.Y.; Zhang, B. Trend in dietary calcium intake among Chinese children and adolescents aged 4 to 17 years in nine provinces from 1991 to 2009. Zhonghua Liu Xing Bing Xue Za Zhi; 2013; 34, pp. 857-862.
18. Dibba, B.; Prentice, A.; Ceesay, M.; Stirling, D.M.; Cole, T.J.; Poskitt, E.M. Effect of calcium supplementation on bone mineral accretion in gambian children accustomed to a low-calcium diet. Am. J. Clin. Nutr.; 2000; 71, pp. 544-549. [DOI: https://dx.doi.org/10.1093/ajcn/71.2.544]
19. Chinese Nutrition Society. Chinese Dietary Reference Intakes (2013); China Science Publishing & Media Ltd.: Beijing, China, 2013; pp. 166-176.
20. Liu, J.T.; Wang, L.; Sun, J.H.; Liu, G.S.; Yan, W.L.; Xi, B.; Xiong, F.; Ding, W.Q.; Huang, G.M.; Heymsfield, S. et al. Bone mineral density reference standards for Chinese children aged 3-18: Cross-sectional results of the 2013–2015 China Child and Adolescent Cardiovascular Health (CCACH) Study. BMJ Open; 2017; 7, e014542. [DOI: https://dx.doi.org/10.1136/bmjopen-2016-014542]
21. Zhu, K.; Du, X.; Cowell, C.T.; Greenfield, H.; Blades, B.; Dobbins, T.A.; Zhang, Q.; Fraser, D.R. Effects of school milk intervention on cortical bone accretion and indicators relevant to bone metabolism in Chinese girls aged 10–12 y in Beijing. Am. J. Clin. Nutr.; 2005; 81, pp. 1168-1175. [DOI: https://dx.doi.org/10.1093/ajcn/81.5.1168]
22. Guo, B.; Xu, Y.; Gong, J.; Tang, Y.; Xu, H. Age trends of bone mineral density and percentile curves in healthy Chinese children and adolescents. J. Bone Miner. Metab.; 2013; 31, pp. 304-314. [DOI: https://dx.doi.org/10.1007/s00774-012-0401-1]
23. Brett, N.R.; Parks, C.A.; Lavery, P.; Agellon, S.; Vanstone, C.A.; Kaufmann, M.; Jones, G.; Maguire, J.L.; Rauch, F.; Weiler, H.A. Vitamin D status and functional health outcomes in children aged 2-8 y: A 6-mo vitamin D randomized controlled trial. Am. J. Clin. Nutr.; 2018; 107, pp. 355-364. [DOI: https://dx.doi.org/10.1093/ajcn/nqx062] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/29566192]
24. Cheng, T.; Wang, M.; Chen, Z.; Eisenberg, R.A.; Zhang, Y.; Zou, Y.; Deng, Y.; Wang, M.; Zhou, L. Tartrate-resistant acid phosphatase 5b is a potential biomarker for rheumatoid arthritis: A pilot study in Han Chinese. Chin. Med. J.; 2014; 127, pp. 2894-2899. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25131223]
25. Dinu, M.; Whittaker, A.; Pagliai, G.; Giangrandi, I.; Colombini, B.; Gori, A.M.; Fiorillo, C.; Becatti, M.; Casini, A.; Benedettelli, S. et al. A Khorasan Wheat-Based Replacement Diet Improves Risk Profile of Patients With Nonalcoholic Fatty Liver Disease (NAFLD): A Randomized Clinical Trial. J. Am. Coll. Nutr.; 2018; 37, pp. 508-514. [DOI: https://dx.doi.org/10.1080/07315724.2018.1445047] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/29652567]
26. Kaufmann, M.; Gallagher, J.C.; Peacock, M.; Schlingmann, K.P.; Konrad, M.; DeLuca, H.F.; Sigueiro, R.; Lopez, B.; Mourino, A.; Maestro, M. et al. Clinical utility of simultaneous quantitation of 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D by LC-MS/MS involving derivatization with DMEQ-TAD. J. Clin. Endocrinol. Metab.; 2014; 99, pp. 2567-2574. [DOI: https://dx.doi.org/10.1210/jc.2013-4388]
27. Zhang, Z.Q.; Ma, X.M.; Huang, Z.W.; Yang, X.G.; Chen, Y.M.; Su, Y.X. Effects of milk salt supplementation on bone mineral gain in pubertal Chinese adolescents: A 2-year randomized, double-blind, controlled, dose-response trial. Bone; 2014; 65, pp. 69-76. [DOI: https://dx.doi.org/10.1016/j.bone.2014.05.007]
28. Lee, W.T.; Leung, S.S.; Wang, S.H.; Xu, Y.C.; Zeng, W.P.; Lau, J.; Oppenheimer, S.J.; Cheng, J.C. Double-blind, controlled calcium supplementation and bone mineral accretion in children accustomed to a low-calcium diet. Am. J. Clin. Nutr.; 1994; 60, pp. 744-750. [DOI: https://dx.doi.org/10.1093/ajcn/60.5.744]
29. Closa-Monasterolo, R.; Zaragoza-Jordana, M.; Ferré, N.; Luque, V.; Grote, V.; Koletzko, B.; Verduci, E.; Vecchi, F.; Escribano, J. Adequate calcium intake during long periods improves bone mineral density in healthy children. Data from the Childhood Obesity Project. Clin. Nutr.; 2018; 37, pp. 890-896. [DOI: https://dx.doi.org/10.1016/j.clnu.2017.03.011]
30. Prentice, A.; Ginty, F.; Stear, S.J.; Jones, S.C.; Laskey, M.A.; Cole, T.J. Calcium supplementation increases stature and bone mineral mass of 16- to 18-year-old boys. J. Clin. Endocrinol. Metab.; 2005; 90, pp. 3153-3161. [DOI: https://dx.doi.org/10.1210/jc.2004-2114]
31. Bonjour, J.P.; Carrie, A.L.; Ferrari, S.; Clavien, H.; Slosman, D.; Theintz, G.; Rizzoli, R. Calcium-enriched foods and bone mass growth in prepubertal girls: A randomized, double-blind, placebo-controlled trial. J. Clin. Investig.; 1997; 99, pp. 1287-1294. [DOI: https://dx.doi.org/10.1172/JCI119287]
32. Chevalley, T.; Bonjour, J.P.; Ferrari, S.; Hans, D.; Rizzoli, R. Skeletal site selectivity in the effects of calcium supplementation on areal bone mineral density gain: A randomized, double-blind, placebo-controlled trial in prepubertal boys. J. Clin. Endocrinol. Metab.; 2005; 90, pp. 3342-3349. [DOI: https://dx.doi.org/10.1210/jc.2004-1455]
33. Rozen, G.S.; Rennert, G.; Dodiuk-Gad, R.P.; Rennert, H.S.; Ish-Shalom, N.; Diab, G.; Raz, B.; Ish-Shalom, S. Calcium supplementation provides an extended window of opportunity for bone mass accretion after menarche. Am. J. Clin. Nutr.; 2003; 78, pp. 993-998. [DOI: https://dx.doi.org/10.1093/ajcn/78.5.993] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/14594787]
34. Dodiuk-Gad, R.P.; Rozen, G.S.; Rennert, G.; Rennert, H.S.; Ish-Shalom, S. Sustained effect of short-term calcium supplementation on bone mass in adolescent girls with low calcium intake. Am. J. Clin. Nutr.; 2005; 81, pp. 168-174. [DOI: https://dx.doi.org/10.1093/ajcn/81.1.168] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/15640477]
35. Mølgaard, C.; Thomsen, B.L.; Michaelsen, K.F. Effect of habitual dietary calcium intake on calcium supplementation in 12–14-y-old girls. Am. J. Clin. Nutr.; 2004; 80, pp. 1422-1427. [DOI: https://dx.doi.org/10.1093/ajcn/80.5.1422] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/15531696]
36. Chevalley, T.; Rizzoli, R.; Hans, D.; Ferrari, S.; Bonjour, J.P. Interaction between calcium intake and menarcheal age on bone mass gain: An eight-year follow-up study from prepuberty to postmenarche. J. Clin. Endocrinol. Metab.; 2005; 90, pp. 44-51. [DOI: https://dx.doi.org/10.1210/jc.2004-1043]
37. Wosje, K.S.; Specker, B.L. Role of calcium in bone health during childhood. Nutr. Rev.; 2000; 58, pp. 253-268. [DOI: https://dx.doi.org/10.1111/j.1753-4887.2000.tb01879.x]
38. Johnston, C.C., Jr.; Miller, J.Z.; Slemenda, C.W.; Reister, T.K.; Hui, S.; Christian, J.C.; Peacock, M. Calcium supplementation and increases in bone mineral density in children. N. Engl. J. Med.; 1992; 327, pp. 82-87. [DOI: https://dx.doi.org/10.1056/NEJM199207093270204]
39. Lee, W.T.; Leung, S.S.; Leung, D.M.; Tsang, H.S.; Lau, J.; Cheng, J.C. A randomized double-blind controlled calcium supplementation trial, and bone and height acquisition in children. Br J Nutr; 1995; 74, pp. 125-139. [DOI: https://dx.doi.org/10.1079/BJN19950112]
40. Lloyd, T.; Andon, M.B.; Rollings, N.; Martel, J.K.; Landis, J.R.; Demers, L.M.; Eggli, D.F.; Kieselhorst, K.; Kulin, H.E. Calcium supplementation and bone mineral density in adolescent girls. JAMA; 1993; 270, pp. 841-844. [DOI: https://dx.doi.org/10.1001/jama.1993.03510070063037]
41. Merrilees, M.J.; Smart, E.J.; Gilchrist, N.L.; Frampton, C.; Turner, J.G.; Hooke, E.; March, R.L.; Maguire, P. Effects of diary food supplements on bone mineral density in teenage girls. Eur. J. Nutr.; 2000; 39, pp. 256-262. [DOI: https://dx.doi.org/10.1007/s003940070004]
42. Lambert, H.L.; Eastell, R.; Karnik, K.; Russell, J.M.; Barker, M.E. Calcium supplementation and bone mineral accretion in adolescent girls: An 18-mo randomized controlled trial with 2-y follow-up. Am. J. Clin. Nutr.; 2008; 87, pp. 455-462. [DOI: https://dx.doi.org/10.1093/ajcn/87.2.455]
43. Iuliano-Burns, S.; Saxon, L.; Naughton, G.; Gibbons, K.; Bass, S.L. Regional specificity of exercise and calcium during skeletal growth in girls: A randomized controlled trial. J. Bone Miner. Res.; 2003; 18, pp. 156-162. [DOI: https://dx.doi.org/10.1359/jbmr.2003.18.1.156] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/12510818]
44. Lu, J.X.; Pan, H.; Hu, X.Q.; Huang, Z.W.; Zhang, Q. Effects of milk powder intervention on bone mineral density and indicators related to bone metabolism in Chinese adolescents. Osteoporos. Int.; 2019; 30, pp. 2231-2239. [DOI: https://dx.doi.org/10.1007/s00198-019-05105-4] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31375874]
45. Karimian, P.; Ebrahimi, H.K.; Jafarnejad, S.; Delavar, M.A. Effects of vitamin D on bone density in healthy children: A systematic review. J. Family Med. Prim. Care; 2022; 11, pp. 870-878.
46. Carswell, A.T.; Oliver, S.J.; Wentz, L.M.; Kashi, D.S.; Roberts, R.; Tang, J.C.Y.; Izard, R.M.; Jackson, S.; Allan, D.; Rhodes, L.E. et al. Influence of Vitamin D Supplementation by Sunlight or Oral D3 on Exercise Performance. Med. Sci. Sports Exerc.; 2018; 50, pp. 2555-2564. [DOI: https://dx.doi.org/10.1249/MSS.0000000000001721]
47. Vissing Landgrebe, A.; Asp Vonsild Lund, M.; Lausten-Thomsen, U.; Frithioff-Bojsoe, C.; Esmann Fonvig, C.; Lind Plesner, J.; Aas Holm, L.; Jespersen, T.; Hansen, T.; Christian Holm, J. Population-based pediatric reference values for serum parathyroid hormone, vitamin D, calcium, and phosphate in Danish/North-European white children and adolescents. Clin. Chim. Acta; 2021; 523, pp. 483-490. [DOI: https://dx.doi.org/10.1016/j.cca.2021.10.024] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/34695445]
48. Heaney, R.P. The bone-remodeling transient: Implications for the interpretation of clinical studies of bone mass change. J. Bone Miner Res.; 1994; 9, pp. 1515-1523. [DOI: https://dx.doi.org/10.1002/jbmr.5650091003] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/7817796]
49. Reid, I.R.; Ames, R.W.; Evans, M.C.; Gamble, G.D.; Sharpe, S.J. Long-term effects of calcium supplementation on bone loss and fractures in postmenopausal women: A randomized controlled trial. Am. J. Med.; 1995; 98, pp. 331-335. [DOI: https://dx.doi.org/10.1016/S0002-9343(99)80310-6]
50. Hidayat, K.; Chen, J.S.; Wang, T.C.; Liu, Y.J.; Shi, Y.J.; Su, H.W.; Liu, B.; Qin, L.Q. The Effects of Milk Supplementation on Bone Health Indices in Adults: A Meta-Analysis of Randomized Controlled Trials. Adv. Nutr.; 2022; 13, pp. 1186-1199. [DOI: https://dx.doi.org/10.1093/advances/nmab136]
51. Cadogan, J.; Eastell, R.; Jones, N.; Barker, M.E. Milk intake and bone mineral acquisition in adolescent girls: Randomised, controlled intervention trial. BMJ; 1997; 315, pp. 1255-1260. [DOI: https://dx.doi.org/10.1136/bmj.315.7118.1255]
52. Mager, D.R.; Qiao, J.; Turner, J. Vitamin D and K status influences bone mineral density and bone accrual in children and adolescents with celiac disease. Eur. J. Clin. Nutr.; 2012; 66, pp. 488-495. [DOI: https://dx.doi.org/10.1038/ejcn.2011.176]
53. Laudermilk, M.J.; Manore, M.M.; Thomson, C.A.; Houtkooper, L.B.; Farr, J.N.; Going, S.B. Vitamin C and zinc intakes are related to bone macroarchitectural structure and strength in prepubescent girls. Calcif. Tissue Int.; 2012; 91, pp. 430-439. [DOI: https://dx.doi.org/10.1007/s00223-012-9656-8] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/23076447]
© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.
Details
; Zi-Fu, Zhao 3 ; Yu-Ming, Chen 1
1 Department of Epidemiology, School of Public Health, Sun Yet-sen University, Guangzhou 510080, China
2 Institute of Epidemiology and Statistics, School of Public Health, Lanzhou University, Lanzhou 730000, China
3 Inner Mongolia Yili Industrial Group Co., Ltd., Hohhot 010110, China; Inner Mongolia Dairy Technology Research Institute Co., Ltd., Hohhot 010110, China
4 Inner Mongolia Yili Industrial Group Co., Ltd., Hohhot 010110, China