SELDI-TOF MS profiles obtained in the different experiments.

<p>SELDI-TOF MS profiles of (A) hepidin-24 spiked urine sample showing next to the expected hepcidin forms also methionine oxidized (Ox) forms of Hepc24 and Hepc25; (B and C) different patient sera and urines, respectively, spiked with Hepc24 (5 nM into urines and 10 nM into sera). Note, the influence of the serum and urine matrices on the peak height of the Hepc24 spiked to patient samples; (D and E), blank serum and urine samples spiked with both Hepc24 and Hepc25 (7.5 nM of both hepcidin forms into urine and 10 nM into serum). Note that the method appears to be more sensitive for Hepc25 than for Hepc24, with an average peak intensity ratio Hepc24/Hepc25 of 0.693. This is probably due to the absence of a negatively charged aspartic acid residue in Hepc24, which negatively affects its binding on the IMAC-Cu²⁺ protein chip surface. The hepcidin isoforms Hepc20, Hepc22 (only in urine), Hepc24 (synthetic analogue) and Hepc25 are indicated by arrows.</p

Correlation of serum hepcidin levels with iron indices in controls with rigorously defined normal iron status.

<p>Data are correlation coefficients by Pearson correlation. STfR, soluble transferrin receptor; TS, transferrin saturation. Hepcidin and ferritin values were log transformed prior to correlation analysis ^*:P<0.05; ^**: P<0.01.</p

Effect of the use of an internal standard on the hepcidin-25 concentrations in urine and serum of selected clinical populations.

<p>Hepc25 concentrations were calculated in nM based on the known concentration of spiked hepc24 in serum (A) and urine (B) samples. For serum, hepc24 intensities were corrected for the background intensity of unspiked samples (hepc24-bl). Note that for both urine and serum specimens the LLOD depends on the individual sample matrix and therefore varies between samples. The LLOD was determined in the 25 human serum and urine samples by using the background intensities at m/z 2400, 2515, 2846 for serum and at m/z 2299, 2510, 2910, for urine samples, respectively. The detection limit was defined as the mean+2 SD of these measurements and found be 2.0 peak intensity for serum and 1.76 peak intensity for urine. The lower level of detection (LLOD) in nM of each individual sample was determined by incorporating the sample specific hepc24 peak intensity value and these mean LLOD values in peak int for hepc25 peak at 2789 m/z in the formulas 1 and 2 for urine and serum (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002706#s4" target="_blank">Material and Methods</a> section). Ctrl, control; LPS, volunteers injected with polysaccharide (6 h after injection); IDA, iron deficiency anemia; TM, thalassemia major in various stages of disease; HH, C282Y homozygous hereditary hemochromatosis patients of various stages of disease; ◊, hepcidin concentration; Ж, sample specific LLOD, the hepcidin concentration of the sample is then</p

Correlation between serum hepcidin and ferritin.

<p>Serum hepcidin levels in 23 healthy volunteers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002706#pone-0002706-t002" target="_blank">Table 2</a>), as determined by our updated MS method, were correlated with their ferritin levels. Values were log transformed prior to correlation analysis. Pearson correlation: 0.6804 (p = 0.0004).</p

Fractional excretion of hepcidin.

<p>The fractional excretion (FE) is calculated by: urine hepcidin (nM)×serum creat (µM)×100/serum hepcidin (nM)×urine creat (mM)×1000. n, the number of pairs for which all data needed to calculate the FE were available. Ctrl, controls; LPS, volunteers injected with polysaccharide (6 h after injection); TM, thalassemia major in various stages of disease; HH, C282Y homozygous hereditary hemochromatosis patients at various stages of disease; tubular reabsorbtion (%) = 100- FE (%).</p

Comparison of hepcidin results between recent studies.

<p>Data are presented in ranges as data from the current study do not allow calculation of means and SD's and the raw data of previous studies are only partially available.</p><p>LLOD; Lower limit of detection; n.d., not determined; n.a., not applicable; n.p., not provided.</p>*<p>exact value depend on the individual urine and the creatinine concentration.</p>#<p>exact value depend on the individual serum.</p>ˆ<p>conversion factors: nM to µg is 2.789; from nM/mmol creat to ng/mg creat is 23.44.</p>$<p>in a rigorously defined group of healthy controls (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002706#pone-0002706-t002" target="_blank">Table 2</a>) these figures are: n = 23, range 0.86–12.43 nM.</p

Characteristics of healthy control subjects.

<p>Data are means with 95% C.I. and p-values of males <i>vs</i> females by t-test. TS, transferrin saturation; sTfR, soluble transferrin receptor.</p><p>Body iron stores were calculated on the basis of the logarithm of the concentrations in micrograms of serum transferrin receptor/serum ferritin (TfR/ferritin ratio) and expressed as milligram per kilogram body weight, as follows: body iron (mg/kg) = −[log(TfR/Ferritin ratio) −2.8229]/0.1207 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002706#pone.0002706-Cook1" target="_blank">[30]</a>. Positive values represent the iron surplus in stores, while negative values represent iron deficit in tissues.</p