Sensitivity analysis of hearing loss among those with and without iron deficiency anemia as defined by lab values

This table shows the conductive, sensorineural, and mixed hearing loss sensitivity analysis values among those with and without iron deficiency anemia. Participants are males and females ages 4 to 21 years. Analysis performed by two-tailed chi-squared test; <i>p</i> values are given. <div><br><div>Schieffer, K. M., Connor, J. R., Pawelczyk, J. A., & Sekhar, D. L. (2017). The relationship between iron deficiency anemia and sensorineural hearing loss in the pediatric and adolescent population.<i> American Journal of Audiology, 26</i>, 155–162. <br></div></div

Total iron content in media and the content of H-ferritin in primary peritoneal macrophages after exposure to the iron formulations.

<p>Rat peritoneal macrophages were obtained by peritoneal lavage. The cells were exposed to 0.1 mg/ml of elemental iron for each of the iron formulations for 24 hours. The media was then monitored for presence of total iron by removing 50uL aliquots from the media over 9 hours. The top figure (A) shows total iron in the media was highest (indicating decreased uptake) for iron sucrose and sodium ferric gluconate (b = not statistically significantly different) at all time points. The other compounds were similar (a) until 7 hours when iron uptake was assessed greater for iron isomaltoside and ferumoxytol (c). There is almost no loss of iron from the media over the time monitored for iron gluconate or iron sucrose. At the end of the experiment, t macrophages were lysed and the amount of H-ferritin present in the cytosol was determined (bottom figure, B). A representative immunoblot for H-ferritin is shown. The bands appear at 21 kDa. The data in the graphs are the mean±SEM of three separate experiments. The asterisk (*) indicates a difference from the control group at p<0.05.</p

Effects of the iron compounds on markers of oxidative stress in HK-2 cells.

<p>HK-2 cells were seeded in T-75 culture flask (5 x 10⁶ cells/flask). After 48 h of incubation, the medium was removed and replaced with Keratinocyte-serum free medium. The cells were exposed to the different iron compounds in doses ranging from 0.1 to 0.8 mg/ml for 24 hours. After 24 hours, cells were harvested and lysed for lipid peroxidation analysis (top figure, A) or assayed for oxidative modification to the proteins using the carbonyl assay (bottom figure, B). Data represents the mean±SEM from three independent experiments and are compared to the control for statistical significance using 2- way ANOVA and post-hoc testing. *p<0.05.</p

Oxidative stress within cytoplasm and mitochondria of rat macrophages.

<p>Rat peritoneal macrophages were obtained by peritoneal lavage. The cells were exposed to 0.1 mg/ml of elemental iron for each of the iron formulations for 24 hours. After 24 hours, the cells with iron were incubated with CellROX Deep Red Reagent (top figure, A) or CellROX Green Reagent (bottom figure, B) for thirty minutes. Following incubation, the cells were washed with PBS. Fluorescence was quantified on a fluorescent plate reader at excitation/emission wavelengths of 640/665 nm for cytoplasm study or 485/520 nm for mitochondria. All data shown are means ± SEM of three independent observations in separate cell culture wells. The data shows there were no significant changes in oxidative stress following the addition of the iron compounds compared to the control (untreated).</p

The cytotoxic effect of the iron compounds on rat primary peritoneal macrophages.

<p>Rat peritoneal macrophages were obtained by peritoneal lavage. The cells were exposed to the different concentrations of iron compounds for 24 hours. Lactate dehydrogenase activity in media and in cell lysates was measured (top figure, A). In a separate experiment the MTT substrate was added to the media during the last four hours (bottom figure, B). Results are means±SEM of three experiments, each conducted in triplicate. Data represents the mean±SEM from three independent experiments and are compared to control for statistical significance using ANOVA and post-hoc testing. *p<0.05.</p

Effects of the iron compounds on markers of oxidative stress in HK-2 cells.

<p>HK-2 cells were seeded in T-75 culture flask (5 x 10⁶ cells/flask). After 48 h of incubation, the medium was removed and replaced with Keratinocyte-serum free medium. The cells were exposed to the different iron compounds in doses ranging from 0.1 to 0.8 mg/ml for 24 hours. After 24 hours, cells were harvested and lysed for lipid peroxidation analysis (top figure, A) or assayed for oxidative modification to the proteins using the carbonyl assay (bottom figure, B). Data represents the mean±SEM from three independent experiments and are compared to the control for statistical significance using 2- way ANOVA and post-hoc testing. *p<0.05.</p

Cytokine secretion by primary peritoneal macrophages.

<p>Equal concentrations of 0.1 mg/ml elemental iron of the different iron compounds was added to the media. After 24 hours, the cells were rinsed and the media was changed to 2% fetal bovine serum in which the cells resided for another 24hour. The media was then collected and assayed for the different cytokines (A) Interleukin 1B, (B) Interleukin 6, (C) Tumor necrosis factor. All data shown are means ± SEM of three independent observations in separate cell culture wells. Differences were only seen with exposure to TNFα and only ferric carboxymaltose and iron dextran increases reached statistical significance *p<0.05 compared with control.</p

Effects of exposure of the iron compounds to Rat Renal Cortical Homogenates on expression of oxidatively modified proteins.

<p>The top figure (A) shows the results for 30 minutes of exposure and in the bottom figure (B), the results for 60 minutes of exposure. Data represents the mean±SEM from three independent experiments and are compared to control for statistical significance using ANOVA and post-hoc testing. *p<0.05.</p

Cytotoxicity profile of six iron compounds on HK-2 cells.

<p>The cells were exposed to the different iron compounds in doses ranging from 0.1 to 0.8 mg/ml for 24 hours. During the final 4 hours, MTT was added (top figure, A). The MTT assay was carried out to establish cell viability. In a separate set of experiments from those used for the MTT assays, cells were exposed to the different iron compounds at the ranges indicated for 24 hours. In a separate set of experiments, plasma membrane integrity was evaluated by LDH assay. The amount of lactate dehydrogenase (LDH) released from the cells was compared to the control group (bottom figure, B).. Data represents the mean±SEM from five independent experiments. *: compared to the control p<0.05.</p

Ferroportin expression in Macrophages.

<p>Rat peritoneal macrophages were obtained as described in the methods. The cells were harvested after a 6-hour exposure to 0.1 mg/ml elemental iron from the different iron compounds in the presence or absence of 500nM hepcidin. The data show that exposure to all of the compounds is associated with an increase in ferroportin expression (band at 63 kDa not shown). The iron compound induced ferroportin expression was blunted by the presence of hepcidin. The expression levels were normalized to β-actin.</p