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American Journal of Clinical Nutrition, Vol. 81, No. 5, 1168-1175, May 2005
© 2005 American Society for Clinical Nutrition

ORIGINAL RESEARCH COMMUNICATION

Effects of school milk intervention on cortical bone accretion and indicators relevant to bone metabolism in Chinese girls aged 10–12 y in Beijing1,2,3

Kun Zhu, Xueqin Du, Chris T Cowell, Heather Greenfield, Barbara Blades, Timothy A Dobbins, Qian Zhang and David R Fraser

From the Faculty of Veterinary Science (KZ, XD, HG, and DRF) and the School of Public Health (TAD), University of Sydney, Sydney, Australia; the Institute of Endocrinology and Diabetes, Children's Hospital at Westmead, Westmead, Australia (CTC and BB); and the Institute for Nutrition and Food Safety, Chinese Center for Disease Control and Prevention, Beijing, China (QZ)

2 Supported by the Australian Dairy Research and Development Corporation (now known as Dairy Australia) and the Nestlé Foundation. Murray Goulburn Cooperative Ltd developed and provided the milk supplement.

3 Address reprint requests to K Zhu, Faculty of Veterinary Science, University of Sydney, Sydney, NSW 2006, Australia. E-mail: kathyz{at}vetsci.usyd.edu.au.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: We previously reported that increased milk consumption enhances growth and bone mineral accretion in Chinese girls aged 10–12 y.

Objective: Our objective was to evaluate the effects of milk supplementation on cortical bone accretion and to study the physiologic mechanisms underlying the observed changes in bone.

Design: Chinese girls aged 10 y were randomly assigned into calcium-fortified milk (Ca milk), calcium and vitamin D–fortified milk (CaD milk), and control groups according to their schools in a 24-mo school milk intervention trial. Periosteal and medullary diameters of metacarpal bone were measured at baseline and 24 mo in the Ca milk (n = 177), CaD milk (n = 210), and control (n = 219) groups. Insulin-like growth factor I (IGF-I), parathyroid hormone (PTH), bone alkaline phosphatase (BAP), osteocalcin, and deoxypyridinoline concentrations were measured at baseline and at 12 and 24 mo in the Ca milk (n = 43), CaD milk (n = 44), and control (n = 41) groups.

Results: After adjustment for pubertal status and clustering by school, 24-mo supplementation led to greater increases in periosteal diameter (1.2%) and cortical thickness (5.7%) and to smaller gains in medullary diameter (6.7%) than did the control (P < 0.05). The CaD milk group had lower serum BAP at 12 mo (19.9%) and lower serum PTH at 12 (46.2%) and 24 (16.4%) mo than did the control group (P < 0.05). The effect of milk supplementation on increasing IGF-I concentrations at 24 mo (16.7–23.3%) was significant in individual analyses but not after adjustment for clustering by school.

Conclusions: Milk supplementation showed positive effects on periosteal and endosteal apposition of cortical bone.

Key Words: Fortified milk • Chinese girls • cortical bone accretion • bone alkaline phosphatase • insulin-like growth factor I


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The achievement of optimal peak bone mass is important for the prevention of osteoporosis in later life. One of the main focuses of lifestyle modification for peak bone mass is dietary calcium intake. Prospective studies have shown positive effects of dietary supplementation with calcium and dairy products on bone mineral acquisition based on measurements of bone mineral content (BMC) or areal bone mineral density (BMD) in children and adolescents from different countries with background calcium intakes ranging from 280 to 980 mg/d (1–11). The effects of milk or calcium intervention on periosteal and endocortical apposition of cortical bone have not been studied in children and adolescents, even though periosteal apposition and cortical thickness play an important role in building up bone structural strength during growth (12, 13).

Measurement of biochemical markers of bone turnover can provide an estimate of the rate of bone formation and resorption (14). Parathyroid hormone (PTH) is a regulator of mineral homeostasis and bone remodeling (15). Insulin-like growth factor I (IGF-I) promotes longitudinal bone growth and bone formation (16, 17), and high intakes of milk but not of meat were reported to increase serum IGF-I concentrations in children (18). Measurement of these indicators relevant to bone metabolism and development during intervention trials could provide useful information about the physiologic mechanisms by which foods and nutrients affect bone growth. Reduced rates of bone remodeling have been reported to be associated with increased bone mineral accretion in calcium supplementation trials in children (11, 19) but not in the milk supplementation trial with English adolescent girls (9). However, the latter study indicated that some of the anabolic effects of milk on bone may be mediated by increased IGF-I concentrations in supplemented subjects, which may have enhanced periosteal bone apposition and led to a slightly larger total-body bone area.

In a school milk intervention study in Beijing girls aged 10 y at baseline (20), we showed that after 24 mo, the 2 groups that received dietary milk supplement (milk fortified with calcium with or without vitamin D) had increases significantly greater than the control group in total body size–adjusted BMC (by 1.2–2.4%), total-body BMD (by 3.2–5.3%), and height (by 0.6–0.7%). The group receiving milk fortified with vitamin D also had a significantly improved vitamin D status than did the group receiving milk alone or the control group (plasma 25-hydroxyvitamin D concentrations for vitamin D–fortified milk group: 47.6 ± 23.4 nmol/L; milk alone group, 17.9 ± 9.0 nmol/L; control group, 19.4 ± 10.2 nmol/L; P < 0.0005). The aim of this report is to analyze further the effects of increased milk consumption on periosteal and endocortical apposition of cortical bone through measurements of metacarpal morphometry and to study the physiologic mechanisms for higher growth and bone mineral accretion rate and any effect on cortical bone gain by measurements of indicators relevant to bone metabolism.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects and study design
A total of 757 urban Beijing girls aged 10 y participated in a 24-mo school milk intervention trial. The girls were from 9 randomly selected schools in one district in urban Beijing. For administrative reasons, local authority approval was given for only 9 schools to participate in this trial. Subjects were randomly assigned into 3 groups according to their schools, thus ensuring that the schools in the different groups had comparable socioeconomic circumstances. For both ethical and practical reasons it was not possible to randomize within schools so that different milk supplements or no milk would have been provided to different students in the same class. There were 238 girls in the calcium-fortified milk (Ca milk) group who received 330 mL calcium-fortified ultra high temperature (UHT) milk per school day, 260 girls in the calcium and vitamin D–fortified milk (CaD milk) group who received 330 mL calcium and vitamin D–fortified UHT milk per school day, and 259 girls in the control group who received no supplementary milk and consumed their habitual diet during the study period. A subsample of 150 subjects (50 from each group) at the start of the trial and a subsample of 240 subjects (80 from each group) after 12 and 24 mo of dietary intervention were further randomly drawn by a systematic sampling procedure for biochemistry measurements. More subjects were selected at 12 and 24 mo because cross-sectional comparisons were also planned for these 2 time points (data not shown). Calculated sample sizes for the intervention trial and the subsequent biochemical analyses were 525 and 43 subjects, respectively, with the power of 90% and 5% risk of a type I error, to detect a difference of 0.02 ± 0.1 g/cm2 in BMD and a difference of 70 ± 100 ng/mL in plasma IGF-I concentration (21). BMD was used to determine the appropriate sample size, as more published data for this variable were available at the time the study was designed. However, considering the evidence of previously published supplementation trials with children and adolescents which have shown supplementation effects with samples sizes of 24–130 in each group subject to enough dosage (>300 mg Ca/d) and duration (>18 mo) (1–6), the sample size for the trial was determined as 250 for each group, as a compromise between the calculated sample size of 525 and the actual sample sizes (24–130) for the studies cited. The sample size for the biochemical analyses was determined as 50 in each group, assuming a 10% dropout rate.

The UHT milk for this project was specially formulated by Murray Goulburn Co-operative Co Ltd (Brunswick, Australia) to comply with both Chinese and Australian food regulations. The milk was fortified with a milk calcium salt (NatraCal) to give a total calcium content of 560 mg (the equivalent of the calcium in 500 mL regular milk) within a volume of 330 mL. This is to keep the supplementation milk within the amount readily consumed on each occasion and to avoid any problems of lactose intolerance associated with milk overload. Each carton of milk (330 mL) also contained 10 g fat and 10 g protein; for milk used in the CaD milk group, 5–8 µg vitamin D was added. After correcting for weekends and holidays, when no intervention milk was consumed, the average daily supplementation over the 24 mo was 144 mL milk, containing 245 mg Ca and, for the CaD milk group, 3.33 µg vitamin D. A detailed description of the composition of the milk supplements and the added milk calcium salts has been given elsewhere (20). The study was carried out with the approval of the Ethics Committees of the University of Sydney, Australia, and the Institute of Nutrition and Food Hygiene of the Chinese Academy of Preventive Medicine (now the Chinese Center for Disease Control and Prevention). A consent form in Chinese was signed by the parents of all the study participants.

Metacarpal morphometry and bone age
Posteroanterior X-ray radiographs of the nondominant hand and wrist were taken at baseline and 24 mo with the use of a Toshiba KXO-152 X-ray apparatus (Tokyo) at an average setting of 5 mA and 46 kV. The film focus distance was 110 cm, and the central beam was focused on the wrist. The radiation dose was 0.01 mSv per exposure. Complete X-ray radiographs at baseline and 24 mo were obtained from 606 girls (Ca milk group, n = 177; CaD milk group, n = 210; and control group, n = 219). Periosteal diameter (outer width) and medullary diameter (inner width) of the midpoint of the second metacarpal and the length of the second metacarpal were measured by one examiner with a digital caliper (Mitutoyo, Kawasaki, Japan). The intraobserver reliability was assessed by repeating the measurements of 28 hand radiographs at different times over 1 mo. The CVs were <1% for outer width, <2% for inner width, and <1% for length. The combined cortical thickness (CCT) was calculated as periosteal diameter –medullary diameter. Bone age (to the nearest 0.1 y) was determined from these radiographs by assessing the development stages of metacarpals, phalanges, and carpals according to the Chinese standard (22).

Biochemical analysis
Samples of overnight fasting blood (drawn between 0630 and 0900) and urine (second morning void, 0730–0900) were obtained at baseline and at 12 and 24 mo in the subsample. For each subject, repeat sample collection was at the same time of day, to minimize the effects of diurnal variation. Blood samples were collected in untreated tubes for serum and in lithium-heparin tubes for plasma. Urine samples were collected in light-protected containers at room temperature without preservatives. An aliquot of each sample was frozen within 4 h after the end of the collection period and stored at –20 °C. Frozen samples were transferred in dry ice (–78.5 °C) by air from Beijing to Sydney on each occasion and then stored at –70 °C until analysis. Urine samples were protected from light during assay as required by assay instructions. Serum and plasma samples were handled as for other routine analytic procedures.

Serum bone alkaline phosphatase (BAP) was assayed by single antibody immunoassay (Alkphase-B; Metra Biosystems Inc, Mountain View, CA). The intraassay CV was 5.0% and the interassay CV was 5.2% at 72 U/L. Plasma osteocalcin was evaluated by a two-site enzyme-linked immunosorbent assay (N-MID Osteocalcin; Osteometer BioTech, Copenhagen). The intraassay CV was 5.4% and the interassay CV was 6.5% at 17 ng/mL. Urinary free deoxypyridinoline was measured by competitive immunoassay (Immulite Pyrilinks-D; Diagnostic Products Corporation, Los Angeles). The intraassay CV was 4.0% and the interassay CV was 4.7% at 97 nmol/L. To correct for variations in urinary flow, deoxypyridinoline results were normalized to the urinary creatinine concentration and expressed as a ratio to urinary creatinine excretion (in nmol/mmol). Urinary creatinine was measured by Beckman Clinical Systems (Synchron CX5; Beckman Coulter Inc, Fullerton, CA) by using an enzymic method. Serum PTH was quantified by immunometric assay (Immulite Intact PTH; Diagnostic Products Corporation, Los Angeles). The intraassay CV was 5.4% and the interassay CV was 5.0% at 86 pg/mL. Plasma IGF-I was measured by double antibody radioimmunoassay (Bioclone, Marrickville, NSW, Australia). This method includes a simple extraction step with acid-ethanol in which IGF-I was separated from its binding protein in plasma. The intraassay CV was 3.4% and the interassay CV was 4.3% at 263 ng/mL.

Other measurements
Health history of the subjects and family members and family socioeconomic status were obtained by a general information questionnaire at baseline. The following assessments were made at baseline and at 12 and 24 mo: body weight by an electronic scale (Thinner, Fairfield, WI), height and sitting height by body and sitting height measures (TG-III, Beijing, China), BMC, bone area, and BMD of total body and forearm by dual-energy X-ray absorptiometry (XR-36; Norland Medical Systems Inc, Fort Atkinson, WI), dietary intakes by 7-d unweighed food record (24-h recall diary for 7 d) at baseline and 3-d food record at 12 and 24 mo (number of days were reduced at 12 and 24 mo because of subject fatigue), and pubertal stage of breast and pubic hair development according to Tanner's definitions of the 5 stages of puberty (23). Date of menarche was recorded.

Statistical analysis
Descriptive statistics are reported as means ± SDs, and differences as mean (95% CI) for all variables, unless otherwise indicated. Baseline values among the 3 groups were compared by using one-factor analysis of variance (ANOVA) and chi-square test when appropriate. A two-factor repeated-measures ANOVA was used to test time x supplementation interactions. Post hoc analysis was carried out with Tukey's honestly significant difference test. In case of significant time x supplementation interaction, the within-group comparison of values at 12 and 24 mo with baseline was carried out by one-factor repeated-measures ANOVA with a Bonferroni-corrected P value of 0.025 for measurements made at 3 time points.

To allow for clustering by school, adjusted analyses were conducted by using the linear mixed model, with school defined as a random effect (24). As outcome variables displayed a skewed distribution, they were transformed by using a log transformation. Estimates of the strength of clustering within school are provided by the intracluster correlation coefficient. Outcomes were analyzed by adjusting for the baseline value, intervention group, and potential confounding variables such as Tanner stage, menarcheal status, and bone age. As the adjusted analyses are based on the natural log-transformed outcome variables, the differences between each of the intervention groups and the control group were calculated as percentage differences. For metacarpal morphometry measurements, when no significant difference between the 2 interventions was observed, intervention groups were pooled and compared with the control group to assess the combined effect of the interventions. The supplementation effects independent of potential confounding factors were also analyzed at the individual level, with a multiple regression model with backward elimination (11, 25, 26). The significance level for test statistics was set at P < 0.05. All data were analyzed with SPSS (version 10.0; SPSS Inc, Chicago) and SAS (SAS for WINDOWS version 8.2; SAS Institute Inc, Chicago).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Six hundred ninety-eight (92.2%) of the original 757 subjects completed the trial. Overall compliance among subjects who completed the study was close to 100% according to school milk consumption record, and no subject withdrew from the study because of adverse effects from milk consumption. Data for 606 girls with complete hand X-ray radiographs and 128 girls with complete biochemical analysis data are presented in this paper. No significant differences were observed in baseline measured characteristics (anthropometry, dietary intakes, pubertal development, and bone mineral measurements) between girls who left the study and girls who completed it and between girls whose hand X-ray or biochemical analysis data are not available and girls for whom these data are available.

The characteristics of subjects at baseline and after 24 mo of the trial are shown in Table 1Go. At baseline, except for the number of years of education of the parents, no significant differences were observed among the 3 groups in any of the variables listed. At 24 mo, subjects in all 3 groups were significantly heavier and taller than at baseline (Table 1Go) [effects of supplementation on height and weight have been discussed elsewhere (20)]. Both supplemented groups had significantly higher calcium intakes at 24 mo than their baseline calcium intakes and the calcium intake of the control group at 24 mo (Table 1Go). No significant differences were observed among the 3 groups in protein intake and percentage of subjects at each Tanner stage (breast and pubic hair) at 24 mo, but a trend was observed for a higher proportion of girls in the Ca milk and CaD milk groups to have passed through menarche at 24 mo (P = 0.065).


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  		TABLE 1 Characteristics of the supplemented and control groups at baseline and 24 mo1

 
No significant differences were observed among the 3 groups at baseline in any of the metacarpal morphometry measurements. All 3 groups of girls experienced increases in periosteal diameter, CCT, and length of the second metacarpal, whereas medullary diameter decreased in the Ca milk group, increased in the control group, and remained unchanged in the CaD milk group over the 24-mo period (Table 2Go). After 24 mo, in comparison with the control intervention, supplementation had led to significantly greater increases in periosteal diameter, CCT, and length of the second metacarpal and to smaller gains in medullary diameter after adjustment for baseline value, bone age, Tanner breast and pubic hair development stage, and menarcheal status at 24 mo and clustering by school (Table 3Go). Percentage changes in periosteal diameter were positively correlated with percentage changes in total-body BMC (r = 0.418, P < 0.001), bone area (r = 0.233, P < 0.001), and BMD (r = 0.139, P = 0.015). Percentage changes in medullary diameter were negatively correlated with those of total-body BMC (r = –0.163, P = 0.004) and BMD (r = –0.234, P < 0.001). Percentage changes in CCT were positively correlated with those of total-body BMC (r = 0.329, P < 0.001) and BMD (r = 0.251, P < 0.001).


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  		TABLE 2 Metacarpal morphometry measurements and indicators relevant to bone metabolism in the supplemented and control groups1

 

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  		TABLE 3 Effects of school milk intervention on cortical bone accretion in Beijing girls1

 
Concentrations of serum BAP, serum PTH, plasma osteocalcin, plasma IGF-I, and urinary deoxypyridinoline were not significantly different among the 3 groups at baseline (Table 2Go). Over the 24 mo, all 3 groups experienced increases in deoxypyridinoline concentrations, whereas osteocalcin concentration remained unchanged. The control group experienced increases in serum BAP concentrations. Concentrations of plasma IGF-I and serum PTH at 24 mo were significantly higher than those at baseline in all 3 groups. The CaD milk group also had concentrations significantly higher for IGF-I and significantly lower for PTH at 12 mo than at baseline (Table 2Go). Comparisons between supplemented and control groups were made for variables (BAP, IGF-I, and PTH) with significant interaction of time x supplementation (Table 4Go). After adjustment for the baseline value, Tanner breast and pubic hair development stage, menarcheal status, and clustering by school, the CaD milk group had concentrations significantly lower for serum BAP (19.9%) at 12 mo and concentrations significantly lower for serum PTH at 12 mo (46.2%) and 24 mo (16.4%) than did the control group. The effects of milk supplementation on reducing serum BAP concentrations at 12 mo in the Ca milk group and on increasing IGF-I concentrations at 24 mo in both supplemented groups were significant at the level of individual analysis but not after adjustment for clustering by school.


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  		TABLE 4 Effects of school milk intervention on indicators relevant to bone metabolism in Beijing girls1

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study showed that in Chinese girls aged 10 y at the start of the trial, 24-mo milk supplementation (fortified with calcium with or without vitamin D) was associated with a greater increase in both periosteal and endocortical apposition than in unsupplemented control subjects. These changes led to greater gain in cortical thickness in supplemented subjects.

Before puberty, periosteal apposition increases bone width in both boys and girls. During puberty, most of the increase in cortical thickness is achieved by periosteal apposition in boys and by medullary contraction in girls in the upper limbs (27, 28). From age 10 to 12 y, when most of our subjects experienced early pubertal development, the cortical thickness increased by 0.41–0.67 mm. Most of the increase was due to the 0.52–0.59-mm periosteal expansion. With the milk intervention, the medullary diameter remained unchanged or decreased at 24 mo compared with baseline in the supplementation groups, whereas it increased by 0.12 mm in the control group, suggesting that the milk supplementation led to earlier medullary contraction in supplemented groups than in the control group.

Supplementation of dairy products or calcium in children and adolescents has shown positive effects of supplementation on bone mineral accretion according to BMC or areal BMD measurements (1–11). For bone sites where cortical bone is predominant, a greater BMC or areal BMD could be caused by a greater cortical thickness (from greater periosteal apposition, greater endocortical apposition, or both) or a greater volume BMD. However, the changes at the bone surfaces during dietary supplementation with calcium and dairy products have, to our knowledge, not previously been studied in children and adolescents. In a 3-y calcium supplementation study in perimenopausal women, it was found that decrease in metacarpal cortical thickness was 1% lower in the supplemented group than in the control group (29). The results of the present study showed that increases in periosteal diameter and CCT and reduction in medullary diameter are associated with increases in total-body BMC and BMD, indicating that the greater increase in total-body BMC and BMD of the supplemented groups could be explained partly by enhanced periosteal and endocortical bone apposition.

Although the increases in total-body BMD and size-adjusted BMC were 2.0% and 1.3% greater, respectively, in the CaD milk group than the Ca milk group (20), we found that there were no differences between these 2 groups in increases in periosteal diameter and CCT. Therefore, we suggest that the greater increases in BMD and size-adjusted BMC of the CaD milk group could be the consequence of a greater increase in volume BMD in this group. This finding is a further indication of the role of vitamin D in promoting the supply of calcium from the diet for bone mineralization.

IGF-I promotes longitudinal bone growth and cortical and trabecular bone formation (16, 17, 30). English adolescent girls who received milk supplementation were reported to have higher concentrations of IGF-I (9). In a study in 8-y-old boys, high intakes of milk but not of meat were reported to increase serum IGF-I concentrations (18). We suggest that the observed greater gain in height (20), in length of the second metacarpal, and in increased periosteal and endocortical apposition in the supplemented groups in the present study could have resulted from increased IGF-I concentrations in response to the increased milk intake. However, the effect of milk supplementation on increasing IGF-I concentrations at 24 mo was significant only at the level of individual analysis, and the significant difference was lost after adjustment for clustering by school because of a high intracluster correlation (0.25). It is not clear why IGF-I had a high variation between schools. Because samples from the different schools were all assayed together and each assay was well controlled, assay variability is unlikely to be a cause.

Lower serum BAP concentrations were observed in the CaD milk group at 12 mo, associated with reduced PTH secretion after the increased calcium intake and improved vitamin D status. Lower serum BAP concentrations were also indicated in the Ca milk group at 12 mo at the level of individual analysis. Markers of bone turnover during growth reflect both bone modeling and remodeling (19). The present study showed that the supplementation had led to increased bone formation, as reflected by greater periosteal and endocortical apposition. Therefore, the lower BAP concentration in the supplemented groups was unlikely to have resulted from a reduced rate of bone modeling but rather from a reduced rate of bone remodeling. However, no difference was observed among the 3 groups in the other 2 biomarkers of bone turnover measured in this study, osteocalcin and deoxypyridinoline. This absence of any effect may be explained by the observation that these 2 biomarkers are related to longitudinal growth during puberty (31, 32), and a decrease in osteocalcin and deoxypyridinoline concentration resulted from the reduced rate of bone remodeling being offset by an increase in their concentrations associated with the higher growth rate in supplemented groups. The reduced rate of bone remodeling, reflected by reduced BAP concentration in supplemented groups, indicated that the phenomenon known as the "bone remodeling transient" partly mediated the effect of supplementation on bone mineral status in the present study, especially in the CaD milk group (33). Decreased rates of bone turnover were also observed in other calcium supplementation trials in children (1, 11, 19) but not in a milk supplementation trial in adolescent English girls (9). These findings suggest that the reduced rate of bone turnover and reduced PTH concentration in the CaD milk group found in the present study possibly resulted from the fortification of the milk with milk calcium salts and vitamin D.

Although our study was a randomized controlled trial and could, therefore, allow conclusions of causality to be drawn, it does have inherent limitations. Because of practical and ethical considerations and other physical constraints, only 9 schools participated in the study, and subjects were randomly assigned according to their schools. When the analysis was adjusted for the cluster design, some of the significant effects found in the randomized group analysis, including the effects of IGF-1 in both supplemented groups and BAP in the Ca milk group, became obscured because of the reduced effective sample size and power. This limited the interpretation of such findings, although they are likely to indicate the underlying biologic mechanisms. The assessment of cortical bone accretion was based on the traditional method, measurement of metacarpal morphometry. It would be ideal if the changes in periosteal and medullary diameters, cortical thickness, and bone cross-sectional area of sites of interests (such as radius) had been monitored by more advanced techniques, such as peripheral quantitative computed tomography. The second morning void urine sample was used for administrative reasons, although it is preferable to collect total 24-h urine because circadian variation in urinary deoxypyridinoline excretion has been observed (34). Estrogen concentrations were not examined in the study subjects.

Our data showed a positive effect of milk supplementation on periosteal apposition and cortical bone accretion in Chinese girls aged 10–12 y. Such effects, if maintained, could confer greater bone strength in supplemented girls. The positive effects of 24-mo of milk supplementation (fortified with calcium with or without vitamin D) on bone mineral accretion are partly mediated by greater periosteal and endocortical apposition of cortical bone and partly by reduced bone remodeling. Follow-up studies of calcium supplementation trials in children yielded inconsistent results (19, 35–38). It seems that the effects on modeling bone size may persist (37), whereas the effects on remodeling would not (19). A follow-up study, with the same primary school subjects now dispersed to >30 secondary schools, is currently under way to determine whether the effects of short-term supplementation with fortified milk are maintained after the cessation of the experimental dietary intervention.


   ACKNOWLEDGMENTS
 
We thank the staff of Beijing Xicheng Student Health Institute and the Department of School Nutrition, Institute of Nutrition and Food Safety, for their help with the field work; C Cai, J Liu, and Z Wen from Beijing No. 304 Hospital for hand X-rays and bone densitometry; J Lee from the Children's Hospital at Westmead for technical support with the biochemical analysis; and J Simpson of the School of Public Health, University of Sydney, for advice on the mixed-model analysis. We appreciate the support from all school principals and teachers involved and the cooperation from all participating students and their parents.

KZ was involved in the conception and design of the study, data collection, and data analysis and drafted the manuscript. XD was involved in the conception and design of the study and in the data collection. CTC was involved in the laboratory analysis consultation and interpretation of the study. HG and DRF were involved in the conception, design, and interpretation of the study. BB was involved in the laboratory analysis consultation. TD was involved in the data analysis with the linear mixed model, which allowed clustering by school. ZQ was involved in the data collection. All authors contributed to the writing of the manuscript. None of the authors had any financial or personal conflicts of interest with any sponsors of this research.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Received for publication March 1, 2004. Accepted for publication December 28, 2004.




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