Supplemental Materials: DOI:10.1515/cclm-2015-0757

Supplemental Table 1. Hepcidin and iron indices of identified G71D heterozygotes compared to wild type individuals in the Nijmegen Biomedical Study (1).

No. / Hepcidin
(nmol/L) 1 / Ferritin
(µg/L) / TSAT
(%) / Iron
(µmol/L) / Hepcidin/ferritin ratio (pmol/µg)
G71D heterozygotes / 1 / 0.18 / 18 / 18.7 / 14 / 10.0
2 / 9.6 / 214 / 24.1 / 14 / 44.7
3 / 6.2 / 267 / 46.4 / 26 / 23.2
4 / 5.5 / 321 / 31.1 / 19 / 16.7
5 / 2.5 / 43 / 33.9 / 21 / 59.1
6 / 4.5 / 151 / 34.8 / 16 / 29.8
7 / 16.3 / 224 / 23.4 / 15 / 72.7
8 / 12.0 / 123 / 28.3 / 17 / 97.5
Median (IQ) / 5.8 (3.0-11.4) / 183 (63-256) / 29.7 (23.6-34.6) / 17 (14-21) / 37.2 (18.3-69.3)*
Wild type (G71G) (n=1923) / Median (IQ) / 7.6 (4.1-12.4) / 121 (61-206) / 28.8 (22.6-35.6) / 17 (14-21) / 60.4 (39.1-92.5)

1 Serum hepcidin (total) was measured by c-ELISA;* Statistically significant difference from wild type individuals (p<0.05).

TSAT, transferrin saturation; IQ, interquartile range; SD, standard deviation. Mann-Whitney testing showed no statistical difference for ferritin, TSAT and iron between G71D heterozygotes and wildtype individuals.However, after multiplying the hepcidin levels measured in the G71D heterozygotes by the factor 2.6 (as extrapolated from the difference in peak height between serum/plasma WT hepcidin and hepcidin+58 peaks observed in MS spectra of 3 G71D heterozygotes, IDs C-E, resulting in median hepcidin of 11.4 and median hepcidin/ferritin ratio of 96.7) to correct for hepcidin+58 not being detected by c-ELISA, hepcidin and thehepcidin/ferritin ratio were significantly (p = 0.022 and p= 0.040, respectively) higher in G71D heterozygotes. Median (IQ) levels are presented since values of hepcidin, ferritin, TSAT and iron were not normally distributed in the general population.

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Supplemental Figure 1. Time-of-flight mass spectrometry profiles of hepcidin in plasma of a patient (ID C) with chronic kidney disease harboring the HAMP G71D variant, confirming that the antibodies used in the c-ELISA do not bind hep-25+58.A. Extraction of hepcidin by weak-cation-exchange beads 2. Hepcidin-24 was used as an internal standard, which overlaps with the naturally occurring hepcidin-24 isoform in this CKD plasma sample; B. Immune capture of hepcidin by anti-hepcidin antibodies bound to protein-A-sepharose beads, as described previously 3; C. Blank, protein-A-sepharose beads without anti-hepcidin antibodies added to the plasma sample to extract hepcidin. Peaks in panel B and C indicated by * represent plasma proteins that are bound aspecifically to the protein-A-sepharose beads. H, hepcidin.

This figure confirms that, whereas the WCX particles clearly extract both the WT and the mutant hepcidin isoforms (A), the anti-hepcidin antibodies only specifically extract the WT hepcidin isoforms (B, C), which cannot be attributed to non-specific binding to the beads themselves (C). The polyclonal antibodies coated to the beads, which were identical to those used in the c-ELISA, are expected to have a higher probability of binding the hepcidin+58 forms than monoclonal antibodies. However, it cannot be excluded that other ELISA assays employing antibodies with different epitope recognition do measure the hepcidin+58 isoforms.

Supplemental Figure 1

Supplemental references

1.Galesloot TE, Vermeulen SH, Geurts-Moespot AJ, Klaver SM, Kroot JJ, van Tienoven D, et al. serum hepcidin: reference ranges and biochemical correlates in the general population. Blood. 2011;117(25):e218-25.

2. Laarakkers CM, Wiegerinck ET, Klaver S, Kolodziejczyk M, Gille H, Hohlbaum AM, et al. Improved mass spectrometry assay for plasma hepcidin: detection and characterization of a novel hepcidin isoform. PLoS One. 2013;8(10):e75518.

3. Kroot JJ, Laarakkers CM, Geurts-Moespot AJ, Grebenchtchikov N, Pickkers P, van Ede AE, et al. Immunochemical and mass-spectrometry-based serum hepcidin assays for iron metabolism disorders. Clin Chem. 2010;56(12):1570-9

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