We measured height to the nearest 1mm using a wall mounted stadiometer and weight to the nearest 0.1kg using electronic scales. All measurements were made without shoes or outdoor clothing. Measurements were taken at every clinic visit and without prior reference to any previous measurements.
Body mass index (BMI) was calculated as the weight in kg divided by the square of the height in meters and the height, weight and BMI were corrected for age and sex by conversion to Z-scores based on Centers for Disease Control and Prevention (CDC) reference data .
Dual-energy X-ray absoptiometry (DXA) scans of the Total Body and lumbar spine were performed using a GE-Lunar Prodigy (GE Lunar Corp, Madison, WI). Daily quality assurance and quality control (spine phantom in water bath) were performed.We measured total body (including head) and body subregions (arms, legs, trunk) lean tissue and fat masses, bone mineral content (BMC), bone mineral density (BMD) and fat distribution (ratio of central to total fat; fat C/T). For estimation of the central (truncal) fat, the trunk region (which includes the pelvis) was delineated by a single assessor using standard manufacturer analysis guidelines. The assessor (JB) has shown good inter-rater reliability with a colleague . Lumbar spine (L2-L4), BMD and BMC were also determined. Raw DXA values were converted into Z-scores using DXA control data.
DXA control data measured using a Lunar DPX (GE Lunar Corp, Madison, WI) were available from 241 healthy children (105 boys), aged 4.2-12.0 years (mean 8.54±1.93 years). Parents of these children had consented for them to have a single DXA to provide reference data for research purposes. Although there was no direct comparison of the Lunar DPX and the Lunar Prodigy used for the subjects, our data from 97 children comparing another Lunar Prodigy with the Lunar DPX showed no significant differences in BMC or BMD. There were small differences in lean tissue mass (mean 28.6 vs 29 kg), and fat (mean 11.4 vs 12.3 kg) but the results were all highly correlated (all r2 > 0.98).
Blood samples were collected unmedicated after an 8 hour overnight fast. The following bone turnover markers and calciotropic hormones were measured: osteocalcin, 25-hydroxy vitamin D, parathyroid hormone (PTH), amino-terminal propeptide of type I collagen (P1NP) and C-telopeptides (CTX). In addition, appetite regulating hormones (leptin and ghrelin) and the growth hormone dependent growth factors insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3), together with fasting insulin, glucose albumin, pre-albumin, ferritin and transferrin were measured to look for changes in growth and appetite regulation and for biochemical evidence of undernutrition. Vitamin D and osteocalcin were analysed by chemiluminescence immunoassays on a Diasorin Liaisonanalyzer (Diasorin, Italy). CTX and P1NP were measured with commercial electrochemiluminescence immunoassays (Modular E170, Roche Diagnostics, Australia). Albumin was analysed using an automated colorimetric assay using a Vitros Fusion analyser (Ortho Clinical Diagnostics, Australia). Pre-albumin used an immunoturbid metric assay on an Integra (Roche Diagnostics, Australia). Ferritin was assayed using an immunometric assay on a Vitros ECI (Ortho Clinical Diagnostics, Australia). Iron deficiency was defined as ferritin <20 ug/L. Transferrin used the TRNF method, which is a quantitative turbid metric assay (Dimension Xpand, Siemens Medical Solutions Diagnostics Pty Ltd, Sydney, Australia). Glucose was analysed using routine laboratory procedures. Leptin and total ghrelin levels were analysed using radioimmunoassay (Millipore, MA) and insulin by chemiluminescence (IMMULITE 2000®, Siemens Medical Solutions Diagnostics Pty Ltd, Sydney, Australia). PTH, IGF-I and IGFBP-3 were measured by ELISA using the IMMULITE 1000® analyser (Siemens Medical Solutions Diagnostics Pty Ltd, Sydney, Australia).