At the end of 24 h, all urine provided by each child was pooled, total volume measured and then processed as described for the 2 h urine collection. The samples were analysed for markers of vitamin D, calcium and phosphate metabolism, and of renal and Fluorouracil hepatic function using commercially available methods according to the manufacturers’
instructions. EDTA-plasma was used for the analysis of intact PTH and C-terminal FGF23; LiHep-plasma was used for other analyses. PTH was measured by immunoradiometric assay (DiaSorin Ltd, Wokingham, Berks, UK) and FGF23 was analysed using a 2nd generation C-terminal, two-site enzyme-linked immunosorbant assay (Immutopics Inc., San Clemente, CA). For FGF23 the manufacturer’s upper
limit of the reference range of 125 RU/ml was used as a cut-off of normality. Plasma 25OHD and 1,25(OH)2D were measured by radioimmunoassay (DiaSorin, Stillwater, MN, USA and IDS, Tyne Selleckchem AZD5363 and Wear, UK respectively). For 25OHD, < 25 nmol/l was taken as an indicator of increased risk of vitamin D deficiency rickets [11]. Cyclic AMP (cAMP) was measured using the tetramethylbenzide method (R&D Systems-ELISA). The following colorimetric methods (Koni Analyser 20i, Finland) were used to determine plasma analytes: total calcium (TCa), arsenazo III; P, ammonium molybdate: creatinine (Cr), Jaffe; albumin, bromocresol purple; TALP, p-nitrophenol;
magnesium (Mg), xylidyl blue I; cystatin C (Cys C), immunoprecipitation; bilirubin, diazo coupling; and aspartase transaminase (AST), enzymatic. Acidified urine was used to determine urinary (u) uCa, uP, uCr, ucAMP and uMg employing the same colorimetric methods as for plasma. Standards used in urinary assays were acidified prior to use. Urinary concentrations were expressed in moles per unit time. Assay accuracy and precision were monitored across the working range of the assays using reference materials provided by external quality assurance schemes (NEQAS, Department of Clinical Biochemistry, Royal Bortezomib order Infirmary, Edinburgh, UK: DEQAS, Endocrine/Oncology Laboratory, Charing Cross Hospital, London, UK) or purchased commercially (Roche Human Control, Roche Diagnostic Ltd, Lewes, East Sussex, 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. Statistical analysis including multiple regression, 2-sample Student’s t-tests and chi-square tests was performed using DataDesk 6.1.1 (Data Description Inc, Ithaca, NY); p ≤ 0.05 was considered statistically significant.