Albertine Donker

Chapter 7 232 Since we aimed to cover the different key periods of human growth and development for both sexes, we divided our study population in subgroups for age and sex; infancy and toddler stage (0- <2 years), early childhood (2-<6 years), middle childhood (6 -<12 years) and adolescence (12-17 years). 35 Because of limited numbers, infants and toddlers were merged into one group. Body mass index was determined and interpreted according to international standards, which describe age-dependent cut off points for underweight, normal weight, overweight and obesity. 36 The study was conducted according to the principles of the Declaration of Helsinki and approved by the Local Ethics Committee of the Máxima Medical Center, Veldhoven, the Netherlands (MMC). For all participants oral and written informed consent was obtained. Laboratory Methods We measured levels of the bioactive form of hepcidin: (hepcidin-25 2 ) by weak cation exchange chromatography followed by time of flight mass spectrometry (WCX -MS- TOF) as described before. 37 Our hepcidin assay was recently standardized using 2 nd reference material (RM) that was value assigned by a provisional primary RM. 22 For additional information on the laboratory methods see the Supplemental Files. Statistical Analysis Descriptive statistics were reported as medians, 2.5 th -97.5 th percentiles and/or ranges for continuous variables and as absolute frequencies and percentages for categorical variables, using original untransformed values. Normality of distributions of the continuous variables was visually checked using histograms. A logarithmic transformation was applied to normalize the non-normal distributions of serum hepcidin, ferritin, CRP and hepcidin/ferritin ratio and TSAT/ hepcidin ratio. For hepcidin values below the lower detection limit of 0.5 nM, imputation was performed with a random value between 0 and 0.5 nM.