Effect of Intravenous Iron on Intracellular Reactive Oxygen Species

Methods

PBMC Isolation

Peripheral blood mononuclear cells (PBMCs) from healthy blood donor buffy coat were separated from 10 mL of whole blood on Ficoll-Hypaque density gradients, washed twice in phosphate buffered saline (PBS) (pH 7.4), and centrifuged again at 1500 × g and 4°C. Isolated PBMCs were cultured in RPMI-1640 medium supplemented with 10% heat inactivated human AB serum (Mediatech Inc, Manassas VA), 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine (Omega Scientific Inc, Tarzana CA) at 37°C at 5% CO₂ (Excella Eco-170, New Brunswick Scientific, Edison, NJ).

Reactive Oxygen Species (ROS) Detection

Intracellular oxidative stress was measured by fluorescent probe, dichlorofluorescein. Briefly, PBMCs were isolated from healthy volunteers, plated onto 96-well plates, and treated with phytohemagglutinin (PHA). The cells were treated for 2, 4, 8 and 24 hours with 100 μg/mL concentrations of IV iron agents Iron Dextran (ID) (INFeD®, Watson Pharma, Inc, Morristown, NJ), Iron Sucrose (IS) (Venofer®, American Regent, Shirley, NY) and Sodium Ferric Gluconate (SFG) (Ferrlecit®, Watson Pharma, Inc, Morristown, NJ). Untreated PBMCs analyzed at 24 hours served as controls and experiments were performed in triplicate. Cells were washed with Krebs-Ringer buffer and subsequently incubated in Dulbecco's modified essential medium containing 100 μM Dichlorofluorescein-diacetate (DCFH-DA) and 1% fetal bovine serum in 5% CO₂ and 95% air at 37°C. One hour later, DCFH-DA was removed, and cells were washed with Krebs-Ringer buffer. Fluorescence was measured in a fluorescent plate reader (Spectra Max Gemini XS, Molecular Devices Sunnyvale, CA) with the temperature maintained at 37°C.

Lymphocyte Subpopulation Analysis

PBMCs from healthy volunteers were stimulated with IV iron agents SFG, IS, and ID for 72 hours at concentrations of 10, 25, and 100 μg/mL. The PBMCs were then stained with fluorescein conjugated monoclonal antibodies and quantified by flow cytometry (BD FACS Scan (Becton Dickinson, BD Biosciences, San Jose, CA) and CellQuest Pro Version 5.2 (Rockville, MD) to assess the proportion of T helper (CD4⁺), cytotoxic T (CD8⁺), NK (CD56⁺), macrophages (CD16⁺) and B cell (CD40⁺) subpopulations among the viable cells. Untreated PBMCs served as controls and experiments were performed in duplicate.

Statistics

A two-way analysis of variance (ANOVA) was used to compare values among the treatments. Post hoc multiple-comparison test with a Bonferroni (parametric-equal variance) test was done to determine significant differences among the groups. A Student's t-test was performed to determine significance between individual treatments.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Cite this: Effect of different Intravenous Iron Preparations on Lymphocyte Intracellular Reactive Oxygen Species Generation and Subpopulation Survival - Medscape - Aug 20, 2010.

Commenting is limited to medical professionals. To comment please Log-in.

Comments on Medscape are moderated and should be professional in tone and on topic. You must declare any conflicts of interest related to your comments and responses. Please see our Commenting Guide for further information. We reserve the right to remove posts at our sole discretion.

My Alerts