Albertine Donker

Standardized Pediatric Hepcidin Values 259 7 Effect of blood sampling time on serum hepcidin levels, hepcidin/ferritin ratios and TSAT/hepcidin ratios Since hepcidin concentrations in serum have been reported to follow a clear circadian rhythm in adults 1,4 and also in children, 5 we assessed the possible influence of time of blood sampling, by dividing sampling time in three categories: between 7:30 AM and 12 PM, between 12 and 3 PM and between 3 and 6 PM. For 47% of subjects, blood sampling was performed between 7:30 AM and 12 PM, for 38% between 12 and 3 PM, and for 14% between 3 PM and 6 PM. Blood sampling time was dissimilar over the age groups; children > 6 years underwent sampling more often later during the day (Chi Square p 0.057) ( Supplemental Figure 1 ). Multivariate analyses including age, sampling time and other clinical and biochemical parameters showed increasing hepcidin concentrations during the day in both males and females, especially after 3 PM (males: β= 0.37; 95 % confidence interval [CI] 0.16– 0.58 ( p 0.001), females: β= 0.31; 95% CI 0.00– 0.61 ( p 0.048)) ( Table 4 ). This corroborates with the above-mentioned data on hepcidin in children. 5 The time of blood sampling was also an independent correlate for both the hepcidin/ferritin and TSAT/hepcidin ratios ( Supplemental Table 6, Supplemental Table 7 ). The hepcidin/ferritin ratio significantly increased after 3 PM in both males and females (males: β= 0.42; 95 % confidence interval [CI] 0.21– 0.63 ( p 0.000), females: β= 0.32; 95% CI 0.02– 0.62 ( p 0.034)). Conversely, the TSAT/hepcidin ratio significantly decreased in males and tended to decrease in females after 3 PM (males: β= -0.41; 95 % CI -0.63– -0.19 ( p 0.000), females: β= -0.26; 95% CI -0.58– 0.05 ( p 0.103)). The variable effect of sampling time on the TSAT/hepcidin ratio might be explained by the fact that both TSAT and hepcidin increase during the day. 6 One might argue that the above-mentioned correlations between sampling time and serum hepcidin levels might be overestimated because of the hepcidin-increasing effect of fasting. 7 However, since only prolonged fasting of up to 66 hour 7 has been described to result in an increment of hepcidin production, we expect that the effect of the pre-operative fasting period of 4-6 hours on the hepcidin data is negligible in our study subjects. This is consistent with our previously reported observations that demonstrate ferritin sets the basal hepcidin concentration and suggest innate diurnal rhythm rather than dietary intake mediates the daily hepcidin variations. 4