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Project Method and Results

Study design: two-year double-blind randomised controlled intervention trial.

Subjects:
750 prepubertal Beijing girls aged 11 yr at study entry (April 1999), after exclusion of girls with any disorder of kidney, liver or bone having the potential to disturb normal bone growth and development. Three groups with 250 girls in each, from Beijing schools selected at random: (1) girls receiving 330 ml/school-day of calcium fortified UHT milk; (2) girls receiving 330 ml/school-day of the same milk fortified additionally with vitamin D; and (3) a group of unsupplemented controls.

Milk supplement:
A slightly sweetened UHT full cream milk product specially designed to conform to Australian and Chinese regulations for UHT milk, packaged in individual pink or green Tetra-Pak 330 ml cartons labelled according to Chinese regulations, developed and supplied without cost to a set of specifications provided under an agreement between Sydney University and Murray Goulburn Cooperative Ltd. Pre-trialled for acceptable taste, mouthfeel and homogeneity. Conformity to specifications monitored by chemical analysis by the company using standard dairy industry methods.

Milk supplement designed to supply the calcium equivalent of 500 ml of milk within the supplied volume of 330 ml. Nutrients from milk supplement: 9.9 g fat, 9.9 g protein, 16.5 g lactose, 4.95 g sucrose, 928 kJ and 560 mg calcium per school-day while those consuming the vitamin D fortified product received in addition from the milk 8 mg vitamin D per school-day. The calcium supplement was derived from milk salts.

All subjects, administrators and investigators blind to the identity of the milk; one coloured package product contained milk with the additional calcium; the other coloured package product contained the same milk with additional vitamin D.

Milk shipped to China, transported by road to Beijing and warehoused in Beijing in batches for two months supply. Weekly deliveries of the milk to the school (for reasons of safety and security) Institute of Nutrition and Food Hygiene. Milk was an official donation from the Australian government to the Chinese government, made and received at a launch at the Australian Embassy in Beijing. Supply, shipping and storage was all supplied by Murray Goulburn Cooperative Ltd, with the assistance of the Australian Dairy Corporation and Austrade.

Ethical approvals.
This project obtained approval from the University of Sydney Human Ethics Committee and a consent form (in Chinese) was completed by parents of participating girls (participation rate 90%). Approval was also granted by the Institute of Nutrition and Food Hygiene, and the Beijing Department of Education.

Baseline measurements.
General information including health history and socioeconomic status of the family. Vitamin D receptor (VDR) genotype of study subjects was determined at baseline, using methods provided by Garvan Institute of Medical Research, Sydney.

Exposure measurements:

  • dietary intake by 24-h recall for 3 consecutive days (2 weekdays, one weekend day) every 6 months;
  • UV exposure by use of a personal UV dosimeter attached to the outer clothes on five consecutive days at baseline, mid-trial and end-trial;
  • physical activity recorded by physical education score as assessed by teachers as part of the normal school curriculum.

Outcome measurements:

  • Anthropometry including height, sitting height, knee height and weight.
  • Pubertal stage assessed by a health professional using Tanner stages.
  • Lean body mass, fat body mass, bone mineral density (BMD) and bone mineral content (BMC) of total body, proximal and distal forearm measured by dual-energy X-ray absorptiometry on a XR-36 densitometer (Norland).
  • Plasma 25(OH)D analysed by competitive protein binding assay.
  • Plasma calcium analysed by atomic absorption.
  • Plasma osteocalcin (OC) analysed by ELISA assay (N-MIDTM Osteocalcin, Osteometer BioTech, Copenhagen, Denmark).
  • Serum bone alkaline phosphatase (BAP) analysed by ELISA assay (Alkphase-B; Metra Biosystems, Inc., Mountain View, CA.)
  • Urinary free deoxypyridinoline (Dpd) analysed by competitive immunoassay (Immulite Pyrilinks-D; Diagnostic Products Corporation, Los Angeles, CA)
  • Serum parathyroid hormone (PTH) analysed by immunometric assay (Immulite Pyrilinks-D; Diagnostic Products Corporation, Los Angeles, CA).
  • Serum insulin-like growth factor I (IGF-I) analysed by double antibody radioimmunoassay (Bioclone, Marrickville, Australia).


Results
Compliance with the trial was high at 92%. There were no differences between the three groups on any measured parameter at the commencement of the study.

The main findings in general were that supplemented girls grew about 0.8 cm taller, 1 kg heavier and accrued 32 g more bone mineral (45 g more in the vitamin D supplemented girls) compared with unsupplemented girls. Consumption of vitamin D fortified milk virtually abolished sublinical vitamin D deficiency in that group by the end of 12 months, and this abolition was maintained until the end of the two-year trial.

VDR genotyping confirmed that the prevalence of bb (considered to be protective against osteoporotic fracture in later life) was very high at >84%.

Further analyses will be done to examine the relationship of VDR genotype with bone variables and with the response to the milk supplementation.

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