Effect of iron on the ability of enteric bacteria to induce cell damage.

<p>LDH-release (mean+SD) as a measure of cell damage of Caco-2 cells upon co-incubation with <i>S. typhimurium</i> (St, <i>n</i> = 5), <i>C. freundii</i> (Cf, <i>n</i> = 4), <i>E. coli</i> (Ec, <i>n</i> = 4), <i>E. faecalis</i> (Ef, <i>n</i> = 4), and <i>L. plantarum</i> (Lp, <i>n</i> = 2) pre-incubated with or without ferric citrate. The percentage LDH release compared to the control (no bacteria) was corrected for the number of bacteria in the medium (average between t = 0 and t = 2 h). Means within a group and without a common letter differ significantly, <i>P</i><0.05.</p

Effect of iron on bacterial adhesion to an epithelial monolayer.

<p>Adhesion (mean+SD) of enteric bacteria to a monolayer of Caco-2 cells is given as percentage of the inoculum. <b>A</b>: <i>S. typhimurium</i>, <i>n</i> = 8. <b>B</b>: <i>C. freundii</i>, <i>n</i> = 4. <b>C</b>: <i>E. coli</i>, <i>n</i> = 6. <b>D</b>: <i>E. faecalis</i>, <i>n</i> = 6. <b>E</b>: <i>L. plantarum</i>, <i>n</i> = 5. Means without a common letter differ, <i>P</i><0.05. Notably, adhesion data of <i>S. typhimurium</i> were derived from 4 separate experiments performed at 13, 15, 18 and 21 days post-seeding of Caco-2 cells. The fact that each experiment revealed the same trend is indicative for similar physiochemical properties of the monolayer at these time points.</p

Effect of iron on invasion of <i>S. typhimurium</i> into epithelial cells.

<p>Invasion (mean+SD) of <i>S. typhimurium</i> into Caco-2 cells, <i>n</i> = 2. Invasion after 3.5 h is given as percentage of the inoculum. The inoculum was removed after 2 hours of adhesion time. Means of 0–10 µmol/L ferric citrate were compared by one-way ANOVA.</p

Effect of iron on the ability of <i>S. typhimurium</i> to cross and deteriorate an epithelial monolayer.

<p>Effect of iron on the ability of <i>S. typhimurium</i> to cross an epithelial monolayer of Caco-2 cells and the integrity of this monolayer. <b>A</b>: The translocation is given as percentage of the inoculum (mean+SD), <i>n</i> = 2. Means without a common letter differ, <i>P</i><0.07. <b>B</b>: The integrity of the Caco-2 monolayer during <i>S. typhimurium</i> (St) and <i>L. plantarum</i> (Lp) translocation, monitored by TEER measurements.</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

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

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

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

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

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