Urinary Hepcidin Levels in Iron-Deficient and Iron-Supplemented Piglets Correlate with Hepcidin Hepatic mRNA and Serum Levels and with Body Iron Status
Fig 1
WCX-TOF MS analysis of urine porcine hepcidin.
(A) Urine and plasma pig Hepcidin-25 quantification by mass spectrometry. Hepcidin-25 measurements in piglet plasma were performed by peptide enrichment through weak cation exchange chromatography coupled to time-of-flight mass spectrometry (WCX-TOF MS) [15]. The spectra (1–4) illustrate the appearance of hepcidin-25 in plasma on day 3, 14, 21 and 28 upon iron dextran injection to piglets on day 3 after birth. Spectra 5 and 6 show hepcidin-25 in the urine of piglets on day 28. Spectrum 5 corresponds to piglet injected with iron dextran on day 3 and spectrum 6 corresponds to non-supplemented, control piglet. IS—internal standard. (B) Immune capture of pig hepcidin-25 and isoform hepcidin-20 in piglet urine. Pig urine was incubated with anti-hepcidin overnight at 4°C, and subsequently hepcidin was measured by WCX-TOF MS. Hepcidin was almost not detectable in the urine sample incubated with anti-hepcidin antibody (upper spectrum); in the spectrum below (pig urine without anti-hepcidin antibody) both a high hepcidin-25 peak and a hepcidin-20 peak (mass 2152 Da) are present. (C) Correlation of pig hepcidin-25 in plasma with pig hepcidin-25 in urine measured with WCX-TOF-MS in piglets injected with iron dextran. Data from 4 measurements of urine and 4 measurements of plasma samples from day 28 were used for the statistical calculations. Spearman correlation r 0.9063, p = 0.0067.