For 25OHD, < 25 nmol/l was
taken as an indicator of increased risk of vitamin D-deficiency rickets [11]. The following colorimetric methods (Koni Analyser 20i, Finland) were used to determine plasma analytes: P, ammonium molybdate; albumin, bromocresol purple; total alkaline phosphatase (TALP), p-nitrophenol and cystatin C (Cys C), immunoprecipitation. Acidified urine was used to determine urinary (u) P, Ca and creatinine (Cr) employing the same colorimetric methods as for plasma P, the arsenazo III method for uCa and the Jaffe method for uCr. Standards used in urinary assays were acidified prior to use. Assay accuracy and precision were monitored across the working range of the assays using reference materials provided by external quality assurance schemes (National-external-quality-assessment-scheme learn more (NEQAS), Department of Clinical Biochemistry, Royal Infirmary, Edinburgh, UK: Vitamin D-external-quality-assessment-scheme
(DEQAS), Endocrine/Oncology Laboratory, Charing Cross Hospital, London, UK) or purchased commercially Cyclopamine cell line (Radiometer Medical, MA, USA and Roche Human Control, Roche Diagnostics Ltd, UK) and kit controls supplied by the manufacturer. In addition, an aliquot of a pooled plasma sample was assayed in each batch to monitor possible drift over time and to provide running quality assurance for analytes where no external reference material was available. Multiple regression tests were performed using DataDesk 6.1 (Data Description Inc., NY, USA) and two-tailed Chi-square tests (without Yates’ correction) were performed using GraphPad QuickCalcs (GraphPad Software, Inc.). Normally distributed check details data are presented as mean (1SD), positively skewed distributions of data are presented as geometric mean (− 1SD, + 1SD) obtained from the antilog of mean (1SD) for the logged values. Variables with positively skewed distributions were transformed to natural logarithms before further statistical analysis. Regression analysis was used to assess the relationships between age (as a continuous variable),
group, and sex with each variable (anthropometric or biochemical). To determine differences in the relationships between variables and group (BD Index vs. BD Sibling, BD vs. LC, anaemic vs. non-anaemic or FGF23 > 125 RU/ml vs.FGF23 ≤ 125 RU/ml) an interaction term (group × independent variable) was used in the model as an independent variable. Sex was not found to be a significant factor in predicting any of the variables, and therefore was not included in the models presented in this paper. However, weight, height, BMI, 25OHD, iCa, P, TALP, Hb, FGF23, 1,25(OH)2D, PTH, uP:uCr and tubular maximal reabsorption of phosphate (TmP:GFR) were influenced by age. Age, therefore, was added as an independent variable in regression models and age-adjusted data have been used throughout the text and in the tables.